Thesis for doctoral degree (Ph.D.)
2016
Thesis for doctoral degree (Ph.D.) 2016
The role of the cohesin loader in genome stability:
a journey from yeast to human
The role of the cohesin loader in genome stability: a journey from yeast to human
Fosco Giordano
Fosco Giordano
From Department of Cell and Molecular Biology
Karolinska Institutet, Stockholm, Sweden
From Department of Cell and Molecular Biology
Karolinska Institutet, Stockholm, Sweden
THE ROLE OF THE COHESIN LOADER IN
GENOME STABILITY: A JOURNEY FROM
YEAST TO HUMAN
THE ROLE OF THE COHESIN LOADER IN
GENOME STABILITY: A JOURNEY FROM
YEAST TO HUMAN
Fosco Giordano
Fosco Giordano
Stockholm 2016
Stockholm 2016
All previously published papers were reproduced with permission from the publisher.
Published by Karolinska Institutet.
Printed by E-Print AB
© Fosco Giordano, 2016
ISBN 978-91-7676-437-4
All previously published papers were reproduced with permission from the publisher.
Published by Karolinska Institutet.
Printed by E-Print AB
© Fosco Giordano, 2016
ISBN 978-91-7676-437-4
The role of the cohesin loader in genome stability: a
journey from yeast to human
The role of the cohesin loader in genome stability: a
journey from yeast to human
THESIS FOR DOCTORAL DEGREE (Ph.D.)
THESIS FOR DOCTORAL DEGREE (Ph.D.)
By
By
Fosco Giordano
Fosco Giordano
Principal Supervisor:
Docent Lena Ström
Karolinska Institutet
Dep. of Cell and Molecular Biology
Opponent:
Professor Jessica Downs
University of Sussex
Genome Damage and Stability Centre
Principal Supervisor:
Docent Lena Ström
Karolinska Institutet
Dep. of Cell and Molecular Biology
Opponent:
Professor Jessica Downs
University of Sussex
Genome Damage and Stability Centre
Co-supervisor(s):
Professor Piergiorgio Percipalle
NYU, Abu Dhabi
Examination Board:
Professor Stefan Åström
Stockholm University
Dep. of Molecular Biosciences
Co-supervisor(s):
Professor Piergiorgio Percipalle
NYU, Abu Dhabi
Examination Board:
Professor Stefan Åström
Stockholm University
Dep. of Molecular Biosciences
PhD Christopher Bot
Karolinska Institutet
Dep. of Cell and Molecular Biology
Docent Teresa Frisan
Karolinska Institutet
Dep. of Cell and Molecular Biology
Docent Herwig Schüler
Karolinska Institutet
Dep. of Medical Biochemistry and Biophysics
PhD Christopher Bot
Karolinska Institutet
Dep. of Cell and Molecular Biology
Docent Teresa Frisan
Karolinska Institutet
Dep. of Cell and Molecular Biology
Docent Herwig Schüler
Karolinska Institutet
Dep. of Medical Biochemistry and Biophysics
To Giulia
To Giulia
ABSTRACT
ABSTRACT
Structural Maintenance of Chromosome (SMC) complexes, as their name suggests, have a
Structural Maintenance of Chromosome (SMC) complexes, as their name suggests, have a
central role in maintaining the higher structure of genomes, from bacteria to human, and in
central role in maintaining the higher structure of genomes, from bacteria to human, and in
doing so protecting their integrity.
doing so protecting their integrity.
Cohesin, one of three SMC complexes, is required to hold sister chromatids together until
Cohesin, one of three SMC complexes, is required to hold sister chromatids together until
anaphase, and for homologous recombination-based DNA repair. In these cellular processes,
anaphase, and for homologous recombination-based DNA repair. In these cellular processes,
a separate complex, named NIPBL/MAU2 (Scc2/4 in Saccharmomyces cerevisiae) is needed
a separate complex, named NIPBL/MAU2 (Scc2/4 in Saccharmomyces cerevisiae) is needed
to drive the loading of cohesin onto DNA.
to drive the loading of cohesin onto DNA.
This thesis focuses on the cohesin loader, in different model organisms and in the different
This thesis focuses on the cohesin loader, in different model organisms and in the different
Scc2
cellular functions in which NIPBL
is involved.
cellular functions in which NIPBLScc2 is involved.
Paper I describes the requirements for Scc2 binding at an HO-induced DNA double strand
Paper I describes the requirements for Scc2 binding at an HO-induced DNA double strand
break. ChIP-qPCR profiles show presence of Scc2 after break induction 30 kb around the
break. ChIP-qPCR profiles show presence of Scc2 after break induction 30 kb around the
break with strong binding 5 kb from the HO cut-site. Moreover, these Scc2 levels are found
break with strong binding 5 kb from the HO cut-site. Moreover, these Scc2 levels are found
to depend on the MRX complex, the Tel1 kinase and H2A phosphorylation, but unlike
to depend on the MRX complex, the Tel1 kinase and H2A phosphorylation, but unlike
cohesin not on Mec1.
cohesin not on Mec1.
Conversely Paper II, performed in human cell lines, shows a dual recruitment model for
Conversely Paper II, performed in human cell lines, shows a dual recruitment model for
NIPBL at laser and FokI endonuclease-induced DNA damage. First, NIPBL is recruited to
NIPBL at laser and FokI endonuclease-induced DNA damage. First, NIPBL is recruited to
DSB via an HP1 binding motif located in its N-terminal. On the contrary NIPBL truncations
DSB via an HP1 binding motif located in its N-terminal. On the contrary NIPBL truncations
containing the HEAT repeat rich C-terminal region, but lacking the HP1 motif, are not
containing the HEAT repeat rich C-terminal region, but lacking the HP1 motif, are not
recruited at FokI foci but localizes only at laser tracks. The latter pathway depends on the
recruited at FokI foci but localizes only at laser tracks. The latter pathway depends on the
activity of ATR/ATM kinases. Moreover a role for the ubiquitin ligases RNF8/RNF168 in
activity of ATR/ATM kinases. Moreover a role for the ubiquitin ligases RNF8/RNF168 in
the NIPBL recruitment to DNA damage is also described.
the NIPBL recruitment to DNA damage is also described.
In recent years a new function was discovered, for cohesin and its loader, in gene regulation.
In recent years a new function was discovered, for cohesin and its loader, in gene regulation.
Paper III shows that Scc2 affects both general gene expression and DNA damage dependent
Paper III shows that Scc2 affects both general gene expression and DNA damage dependent
transcription by microarray analysis. Lastly paper IV focuses on another important process
transcription by microarray analysis. Lastly paper IV focuses on another important process
in which cohesin is involved, meiosis, describing NIPBL chromosomal localization in male
in which cohesin is involved, meiosis, describing NIPBL chromosomal localization in male
and female murine germ cells, during meiotic prophase I.
and female murine germ cells, during meiotic prophase I.
LIST OF SCIENTIFIC PAPERS
I.
II.
III.
IV.
GIORDANO F., Rutishauser D., and Ström L. Requirements for DNA
LIST OF SCIENTIFIC PAPERS
I.
GIORDANO F., Rutishauser D., and Ström L. Requirements for DNA
double strand break accumulation of Scc2, Similarities and Differences
double strand break accumulation of Scc2, Similarities and Differences
with Cohesin. Manuscript.
with Cohesin. Manuscript.
Bot C., Pfeiffer A., GIORDANO F., Edara D. M., Dantuma N. P. and
II.
Bot C., Pfeiffer A., GIORDANO F., Edara D. M., Dantuma N. P. and
Ström L. Independent Mechanisms Recruit the Cohesin Loader Protein
Ström L. Independent Mechanisms Recruit the Cohesin Loader Protein
NIPBL to Sites of DNA Damage. Manuscript.
NIPBL to Sites of DNA Damage. Manuscript.
Lindgren E., Hägg S., GIORDANO F., Björkegren J., and Ström L.
III.
Lindgren E., Hägg S., GIORDANO F., Björkegren J., and Ström L.
Inactivation of the budding yeast cohesin loader Scc2 alters gene
Inactivation of the budding yeast cohesin loader Scc2 alters gene
expression both globally and in response to a single DNA double strand
expression both globally and in response to a single DNA double strand
break. Cell cycle, 2014, 12, 3645-58.
break. Cell cycle, 2014, 12, 3645-58.
Visnes T., GIORDANO F., Kuznetsova A., Suja J. A., Lander A., Anne L
IV.
Visnes T., GIORDANO F., Kuznetsova A., Suja J. A., Lander A., Anne L
Calof and Lena Ström. Localisation of the SMC loading complex
Calof and Lena Ström. Localisation of the SMC loading complex
Nipbl/Mau2 during mammalian meiotic prophase I. Chromosoma, 2014,
Nipbl/Mau2 during mammalian meiotic prophase I. Chromosoma, 2014,
123, 239-52.
123, 239-52.
CONTENTS
CONTENTS
1
1
INTRODUCTION .............................................................................................................. 1
1.1.
GENOME STABILITY .......................................................................................................... 1
1.1.
GENOME STABILITY .......................................................................................................... 1
1.2.
THE CELL CYCLE ................................................................................................................ 1
1.2.
THE CELL CYCLE ................................................................................................................ 1
1.3.
MEIOSIS ................................................................................................................................... 3
1.3.
MEIOSIS ................................................................................................................................... 3
1.4.
DNA REPAIR ........................................................................................................................... 3
1.4.
DNA REPAIR ........................................................................................................................... 3
1.4.1.
Early events in DNA damage repair ................................................................................... 3
1.4.1.
Early events in DNA damage repair ................................................................................... 3
1.4.2.
Homologous recombination................................................................................................ 4
1.4.2.
Homologous recombination................................................................................................ 4
1.4.3.
Non Homologous End Joining............................................................................................ 8
1.4.3.
Non Homologous End Joining............................................................................................ 8
1.4.4.
Other events in DNA damage repair .................................................................................. 8
1.4.4.
Other events in DNA damage repair .................................................................................. 8
1.5.
1.5.
SMC COMPLEXES ................................................................................................................ 9
The cohesin complex ........................................................................................................ 10
1.5.1.
The cohesin complex ........................................................................................................ 10
1.5.2.
The cohesin loader ............................................................................................................ 11
1.5.2.
The cohesin loader ............................................................................................................ 11
THE COHESIN CYCLE IN BUDDING YEAST .............................................................. 13
1.6.
THE COHESIN CYCLE IN BUDDING YEAST .............................................................. 13
1.6.1.
G1 cohesin loading and localization ................................................................................. 13
1.6.1.
G1 cohesin loading and localization ................................................................................. 13
1.6.2.
S phase and cohesion establishment ................................................................................. 13
1.6.2.
S phase and cohesion establishment ................................................................................. 13
1.6.3.
Cohesin removal ............................................................................................................... 14
1.6.3.
Cohesin removal ............................................................................................................... 14
1.7.
COHESIN CYCLE IN METAZOAN ................................................................................. 14
1.7.
COHESIN CYCLE IN METAZOAN ................................................................................. 14
1.7.1.
Cohesin loading ................................................................................................................. 14
1.7.1.
Cohesin loading ................................................................................................................. 14
1.7.2.
Cohesion establishment .................................................................................................... 15
1.7.2.
Cohesion establishment .................................................................................................... 15
1.7.3.
Cohesin removal ............................................................................................................... 15
1.7.3.
Cohesin removal ............................................................................................................... 15
1.8.
COHESIN & DNA DAMAGE REPAIR ............................................................................. 15
1.8.
COHESIN & DNA DAMAGE REPAIR ............................................................................. 15
1.8.1.
Budding yeast DNA damage response ............................................................................. 16
1.8.1.
Budding yeast DNA damage response ............................................................................. 16
1.8.2.
Metazoan cohesin and DNA damage ............................................................................... 16
1.8.2.
Metazoan cohesin and DNA damage ............................................................................... 16
1.9.
COHESIN AND MEIOSIS ................................................................................................... 17
1.9.
COHESIN AND MEIOSIS ................................................................................................... 17
1.10.
COHESION AND BEYOND ............................................................................................. 18
1.10.
COHESION AND BEYOND ............................................................................................. 18
METHODOLOGY ........................................................................................................... 21
2.1
2
MODEL ORGANISMS ......................................................................................................... 21
METHODOLOGY ........................................................................................................... 21
2.1
MODEL ORGANISMS ......................................................................................................... 21
2.1.1
Saccharomyces cerevisiae ................................................................................................. 21
2.1.1
Saccharomyces cerevisiae ................................................................................................. 21
2.1.2
Mus musculus ..................................................................................................................... 21
2.1.2
Mus musculus ..................................................................................................................... 21
2.1.3
Human cell culture ............................................................................................................. 21
2.1.3
Human cell culture ............................................................................................................. 21
2.2
3
SMC COMPLEXES ................................................................................................................ 9
1.5.1.
1.6.
2
INTRODUCTION .............................................................................................................. 1
IMMUNOFLUORESCENCE OF TESTICULAR AND OVARIAN NUCLEAR
2.2
IMMUNOFLUORESCENCE OF TESTICULAR AND OVARIAN NUCLEAR
SPREADS ........................................................................................................................................... 22
SPREADS ........................................................................................................................................... 22
2.3
MICROARRAY ANALYSIS ................................................................................................ 22
2.3
MICROARRAY ANALYSIS ................................................................................................ 22
2.4
CHROMATIN IMMUNOPRECIPITATION .................................................................... 23
2.4
CHROMATIN IMMUNOPRECIPITATION .................................................................... 23
2.5
DNA DAMAGE INDUCTION .............................................................................................. 24
2.5
DNA DAMAGE INDUCTION .............................................................................................. 24
RESULTS AND DISCUSSION ...................................................................................... 27
3
RESULTS AND DISCUSSION ...................................................................................... 27
4
3.1
PAPER I .................................................................................................................................. 27
3.1
PAPER I .................................................................................................................................. 27
3.2
PAPER II ................................................................................................................................. 30
3.2
PAPER II ................................................................................................................................. 30
3.3
PAPER III ............................................................................................................................... 33
3.3
PAPER III ............................................................................................................................... 33
3.4
PAPER IV ............................................................................................................................... 36
3.4
PAPER IV ............................................................................................................................... 36
FUTURE PERSPECTIVE .............................................................................................. 39
4.1
4
HOW DOES THE LOADER WORK ................................................................................. 39
Scc4
FUTURE PERSPECTIVE .............................................................................................. 39
4.1
HOW DOES THE LOADER WORK ................................................................................. 39
4.2
THE MYSTERY PROTEIN: MAU2
.............................................................................. 40
4.2
THE MYSTERY PROTEIN: MAU2Scc4.............................................................................. 40
4.3
REMODELLING, TRANSCRIPTION AND COHESIN LOADING ............................ 41
4.3
REMODELLING, TRANSCRIPTION AND COHESIN LOADING ............................ 41
4.4
FINAL REMARKS ................................................................................................................ 42
4.4
FINAL REMARKS ................................................................................................................ 42
5
ACKNOWLEDGEMENTS ............................................................................................ 43
5
ACKNOWLEDGEMENTS ............................................................................................ 43
6
REFERENCES ................................................................................................................. 45
6
REFERENCES ................................................................................................................. 45
LIST OF ABBREVIATIONS
LIST OF ABBREVIATIONS
53BP1
p53 binding protein 1
53BP1
p53 binding protein 1
ATM
Ataxia telangiectasia mutated
ATM
Ataxia telangiectasia mutated
ATR
Ataxia telangiectasia and Rad3-related
ATR
Ataxia telangiectasia and Rad3-related
bp/kb
base pair/ kilobase pair
bp/kb
base pair/ kilobase pair
BRCA 1,2
Breast cancer antigen 1,2
BRCA 1,2
Breast cancer antigen 1,2
BrdU
Bromodeoxyuridine
BrdU
Bromodeoxyuridine
CAR
Cohesin associated region
CAR
Cohesin associated region
CdLS
Cornelia de Lange syndrome
CdLS
Cornelia de Lange syndrome
ChIP
Chromatin immunoprecipitation
ChIP
Chromatin immunoprecipitation
CTCF
CCCTC-binding factor required for transcriptional regulation
CTCF
CCCTC-binding factor required for transcriptional regulation
CHK1,2
Checkpoint kinase 1,2
CHK1,2
Checkpoint kinase 1,2
CDK
Cyclin-dependent kinase
CDK
Cyclin-dependent kinase
CSD
chromoshadow domain
CSD
chromoshadow domain
DI-Cohesion
Damage-induced cohesion
DI-Cohesion
Damage-induced cohesion
DNA
Deoxyribonucleic acid
DNA
Deoxyribonucleic acid
DNA lig4
DNA ligase 4
DNA lig4
DNA ligase 4
DNA PKcs
DNA dependent protein kinase catalytic subunit
DNA PKcs
DNA dependent protein kinase catalytic subunit
Dnl4
DNA ligase 4
Dnl4
DNA ligase 4
DSB
double strand break
DSB
double strand break
Eco1
Establishment of cohesion 1
Eco1
Establishment of cohesion 1
ESCO1,2
Establishment of cohesion 1,2
ESCO1,2
Establishment of cohesion 1,2
FACS
Fluorescence-activated cell sorting
FACS
Fluorescence-activated cell sorting
HEAT
Huntingtin, Elongation factor 3, protein phosphatase 2A and
HEAT
Huntingtin, Elongation factor 3, protein phosphatase 2A and
Tor1
Tor1
H2A(X)
Histone 2A(X)
H2A(X)
Histone 2A(X)
HR
Homologous recombination
HR
Homologous recombination
Lif1
Ligase interacting factor 1
Lif1
Ligase interacting factor 1
Mec1
Mitosis entry checkpoint 1
Mec1
Mitosis entry checkpoint 1
Mre11
Meiotic recombination 11 homolog A
Mre11
Meiotic recombination 11 homolog A
MRN/MRX
MRE11 RAD50 NBS1/Mre11 Rad50 Xrs2
MRN/MRX
MRE11 RAD50 NBS1/Mre11 Rad50 Xrs2
NBS1
Nijmegen breakage syndrome defective 1
NBS1
Nijmegen breakage syndrome defective 1
Nej1
Nonhomologous end joining defective 1
Nej1
Nonhomologous end joining defective 1
NHEJ
Nonhomologous end joining
NHEJ
Nonhomologous end joining
NIPBL
Nipped-B-like
NIPBL
Nipped-B-like
ORF
open reading frame
ORF
open reading frame
PCNA
Proliferating cell nuclear antigen
PCNA
Proliferating cell nuclear antigen
PDS5
Precocious dissociation of sisters 5
PDS5
Precocious dissociation of sisters 5
PFGE
Pulse-field gel electrophoresis
PFGE
Pulse-field gel electrophoresis
PLK1
Polo-like kinase 1
PLK1
Polo-like kinase 1
qPCR
Quantitative polymerase chain reaction
qPCR
Quantitative polymerase chain reaction
RAD
Radiation sensitive
RAD
Radiation sensitive
RSC
Remodel the Structure of Chromatin
RSC
Remodel the Structure of Chromatin
SA
Stromal antigen
SA
Stromal antigen
Sae2
Sumo activating enzyme subunit 2
Sae2
Sumo activating enzyme subunit 2
SCC
Sister chromatid cohesion
SCC
Sister chromatid cohesion
Sgs1
Small growth suppressor 1
Sgs1
Small growth suppressor 1
SMC
Structural maintenance of chromosome
SMC
Structural maintenance of chromosome
ssDNA
single strand DNA
ssDNA
single strand DNA
Tel1
Telomere maintenance 1
Tel1
Telomere maintenance 1
TMP
trimethylpsoralen
TMP
trimethylpsoralen
TRP
Tetratricopeptide
TRP
Tetratricopeptide
WAPL
Wings apart like
WAPL
Wings apart like
XLF
XRCC4-like factor
XLF
XRCC4-like factor
XRCC4
X-ray repair cross-complementing protein 4
XRCC4
X-ray repair cross-complementing protein 4
Xrs2
X-ray sensitivity 2
Xrs2
X-ray sensitivity 2
!
Introduction
!
1 INTRODUCTION
!
!
Introduction
1 INTRODUCTION
1.1. GENOME STABILITY
1.1. GENOME STABILITY
Genome stability is the sum of processes that a cell employs to preserve and to deliver free of
Genome stability is the sum of processes that a cell employs to preserve and to deliver free of
error to daughter cells, its genetic information; it is a broad concept including events
error to daughter cells, its genetic information; it is a broad concept including events
connected to DNA replication, maintenance of chromosome structure during the cell cycle,
connected to DNA replication, maintenance of chromosome structure during the cell cycle,
and DNA repair.
and DNA repair.
Orthologs important for genome integrity usually exert the same function, but might carry
Orthologs important for genome integrity usually exert the same function, but might carry
different names. To avoid confusion I will refer to the metazoan gene or protein, putting the
different names. To avoid confusion I will refer to the metazoan gene or protein, putting the
Mfg
Saccharomyces cerevisiae version in superscript (i.e. MFG
). In case a protein function is
unique for a certain organism, only the name of that specific protein or gene will be used.
Saccharomyces cerevisiae version in superscript (i.e. MFGMfg). In case a protein function is
unique for a certain organism, only the name of that specific protein or gene will be used.
1.2. THE CELL CYCLE
1.2. THE CELL CYCLE
The cell cycle represents all the steps required for a single cell to grow, replicate and
The cell cycle represents all the steps required for a single cell to grow, replicate and
propagate DNA, in order to generate two daughter cells with identical genetic information.
propagate DNA, in order to generate two daughter cells with identical genetic information.
A cell cycle is composed of four different phases: a DNA replication phase called S, in which
A cell cycle is composed of four different phases: a DNA replication phase called S, in which
the genetic material is duplicated, generating two DNA molecules called sister chromatids,
the genetic material is duplicated, generating two DNA molecules called sister chromatids,
and a cell division phase called M, which comprises two major events: nuclear division or
and a cell division phase called M, which comprises two major events: nuclear division or
Mitosis, and cytokinesis. M phase is composed of sub-phases when DNA is structured and
Mitosis, and cytokinesis. M phase is composed of sub-phases when DNA is structured and
reorganized inside the cell; in prophase the genetic material is condensed in rod-like
reorganized inside the cell; in prophase the genetic material is condensed in rod-like
structures kept together by sister chromatid cohesion. Subsequently, during metaphase, DNA
structures kept together by sister chromatid cohesion. Subsequently, during metaphase, DNA
is attached to a microtubule-based structure, called the spindle, and aligned at the center of
is attached to a microtubule-based structure, called the spindle, and aligned at the center of
the cell. During anaphase, sister chromatids separate and migrate to the opposite poles of the
the cell. During anaphase, sister chromatids separate and migrate to the opposite poles of the
cell. In the last portion of mitosis (telophase) before cytokinesis, the spindle is disassembled
cell. In the last portion of mitosis (telophase) before cytokinesis, the spindle is disassembled
and DNA is de-condensed into new nuclei (Figure 1).
and DNA is de-condensed into new nuclei (Figure 1).
Between the S and M phases there are two gap phases (G1 and G2) that are needed for cells
Between the S and M phases there are two gap phases (G1 and G2) that are needed for cells
to grow, double their mass, produce new organelles and monitor if environmental and
to grow, double their mass, produce new organelles and monitor if environmental and
internal conditions are suitable for DNA replication and cell division.
internal conditions are suitable for DNA replication and cell division.
1
1
Introduction
Introduction
Meiosis&
Mitosis&
S phase
Meiosis&
Mitosis&
S phase
S phase
S phase
DNA
replication
DNA
replication
Prophase
Prophase I
Sister chromatids
condensed and kept
together by cohesin
Prophase
Prophase I
Sister chromatids
condensed and kept
together by cohesin
Formation of
Synaptonemal
complex
Metaphase
Metaphase I
Metaphase
Homologs held
together by
chiasmata
Sister chromatids
attach to the spindle
Formation of
Synaptonemal
complex
Metaphase I
Homologs held
together by
chiasmata
Sister chromatids
attach to the spindle
Anaphase
Anaphase I
Anaphase
Anaphase I
Sister
chromatids
segregate
Homologs segregate
Sister
chromatids
segregate
Homologs segregate
Two 2N daughter cells
Meiosis I
Meiosis I
Metaphase II
Metaphase II
Anaphase II
Anaphase II
Four haploid gametes
Figure 1: Scheme of the different phases of mitosis (on the left) and meiosis (on the right). Blue rings around
chromosomes represent cohesin molecules. In black and red are represented maternal and paternal
chromosomes respectively.
2
Two 2N daughter cells
Four haploid gametes
Figure 1: Scheme of the different phases of mitosis (on the left) and meiosis (on the right). Blue rings around
chromosomes represent cohesin molecules. In black and red are represented maternal and paternal
chromosomes respectively.
2
!
!
Introduction
!
1.3. MEIOSIS
!
Introduction
1.3. MEIOSIS
Meiosis is a specialized form of nuclear division that occurs in diploid eukaryotes
Meiosis is a specialized form of nuclear division that occurs in diploid eukaryotes
reproducing sexually, leading to the formation of four haploid cells, which then differentiate
reproducing sexually, leading to the formation of four haploid cells, which then differentiate
into reproductive cells called gametes. Meiosis starts with DNA replication, meiotic S phase,
into reproductive cells called gametes. Meiosis starts with DNA replication, meiotic S phase,
followed by two consecutive cell divisions called meiosis I and meiosis II (Figure 1). After
followed by two consecutive cell divisions called meiosis I and meiosis II (Figure 1). After
meiotic S phase chromosomes are present as two pairs of sister chromatids, called homologs,
meiotic S phase chromosomes are present as two pairs of sister chromatids, called homologs,
connected through non-sister linkages.
connected through non-sister linkages.
To help homolog pairing and facilitate the resolution of DSBs, a protein structure called
To help homolog pairing and facilitate the resolution of DSBs, a protein structure called
synaptonemal complex (SC) is formed. The appearance of the SC changes through prophase I
synaptonemal complex (SC) is formed. The appearance of the SC changes through prophase I
and defines four different sub-phases: leptotene, zygotene, pachytene and diplotene. Right
and defines four different sub-phases: leptotene, zygotene, pachytene and diplotene. Right
after replication, during leptotene, axial elements (AE) composed of SYCP2 and SYCP3 are
after replication, during leptotene, axial elements (AE) composed of SYCP2 and SYCP3 are
formed. During zygotene, when the homologs start to pair, transverse filaments (TF),
formed. During zygotene, when the homologs start to pair, transverse filaments (TF),
composed of SYCP1, are loaded between the AEs, forming the central element (CE). During
composed of SYCP1, are loaded between the AEs, forming the central element (CE). During
pachytene, the homologs are aligned and tied together along their entire length by the SC in a
pachytene, the homologs are aligned and tied together along their entire length by the SC in a
process called synapsis. After pachytene the AE starts to dissociate, a process that ends
process called synapsis. After pachytene the AE starts to dissociate, a process that ends
during diplotene.
during diplotene.
Resolution of the SC is tightly regulated, such that the linkage it forms between the non-sister
Resolution of the SC is tightly regulated, such that the linkage it forms between the non-sister
chromatids remains until DNA exchange between homologs, also called crossovers, have
chromatids remains until DNA exchange between homologs, also called crossovers, have
been established. The homologs are kept together by chiasmata, the visible crossing overs
been established. The homologs are kept together by chiasmata, the visible crossing overs
between chromosomes formed thanks to homologous recombination based DNA repair of
between chromosomes formed thanks to homologous recombination based DNA repair of
double strand breaks (DSB). During meiosis II, sister chromatids from each homolog are then
double strand breaks (DSB). During meiosis II, sister chromatids from each homolog are then
separated in essence through conventional mitosis (Handel, 2010).
separated in essence through conventional mitosis (Handel, 2010).
1.4. DNA REPAIR
1.4. DNA REPAIR
Cells are continuously under the risk of encountering DNA damage, from environmental
Cells are continuously under the risk of encountering DNA damage, from environmental
sources such as chemicals and ionizing radiation, or cellular processes like oxidative stress or
sources such as chemicals and ionizing radiation, or cellular processes like oxidative stress or
replication fork collapse. A number of mechanisms protect the genetic material from harmful
replication fork collapse. A number of mechanisms protect the genetic material from harmful
events, in form of mutations, deletions or rearrangements that can ultimately lead to cell
events, in form of mutations, deletions or rearrangements that can ultimately lead to cell
death.
death.
1.4.1. Early events in DNA damage repair
1.4.1. Early events in DNA damage repair
The initial steps in DNA damage repair include: recognition of the damage, checkpoint
The initial steps in DNA damage repair include: recognition of the damage, checkpoint
activation and modification of DNA ends at the break. The metazoan MRN (MRE11,
activation and modification of DNA ends at the break. The metazoan MRN (MRE11,
3
3
Introduction
Introduction
RAD50, NBS1) (De Jager, 2001) or yeast MRX (Mre11, Rad50, Xrs2) (Lisby, 2004)
Ku70/Ku80
RAD50, NBS1) (De Jager, 2001) or yeast MRX (Mre11, Rad50, Xrs2) (Lisby, 2004)
, are recruited to the site of
complex, together with, but independently of KU70/KU80Ku70/Ku80, are recruited to the site of
DNA damage early (Milne, 1996). These two complexes affect the choice of repair pathway:
DNA damage early (Milne, 1996). These two complexes affect the choice of repair pathway:
Homologous Recombination (HR) via MRNMRX or Non-Homologous End Joining (NHEJ)
Homologous Recombination (HR) via MRNMRX or Non-Homologous End Joining (NHEJ)
through KU70/KU80Ku70/Ku80. The selection of one of the two mutually exclusive repair
through KU70/KU80Ku70/Ku80. The selection of one of the two mutually exclusive repair
mechanisms mostly depends on the cell cycle phase in which the damage took place. After
mechanisms mostly depends on the cell cycle phase in which the damage took place. After
DNA replication, HR becomes not only available but also a favored choice, especially in
DNA replication, HR becomes not only available but also a favored choice, especially in
budding yeast, where the cyclin dependent kinase (CDK) promotes the switching from NHEJ
budding yeast, where the cyclin dependent kinase (CDK) promotes the switching from NHEJ
to HR (Aylon, 2004). Evidence indicates in fact an increased expression of HR factors after S
to HR (Aylon, 2004). Evidence indicates in fact an increased expression of HR factors after S
phase (Chen, 1997). In mammalian cells however, NHEJ is the mostly used pathway. Even in
phase (Chen, 1997). In mammalian cells however, NHEJ is the mostly used pathway. Even in
G2, 80% of the cells still repair DNA damage via NHEJ (Beucher, 2009; Shibata, 2011).
G2, 80% of the cells still repair DNA damage via NHEJ (Beucher, 2009; Shibata, 2011).
In human, MRN recruits the ATM kinase for HR (You, 2005) while KU70/KU80 recruits the
In human, MRN recruits the ATM kinase for HR (You, 2005) while KU70/KU80 recruits the
DNA-PKcs kinase for NHEJ (Gottlieb, 1993). In budding yeast on the other hand, the ATM
DNA-PKcs kinase for NHEJ (Gottlieb, 1993). In budding yeast on the other hand, the ATM
ortholog Tel1 is recruited by the MRX complex (Nakada, 2003) and its activity is necessary
ortholog Tel1 is recruited by the MRX complex (Nakada, 2003) and its activity is necessary
for both HR and for NHEJ (X. Zhang, 2005). Tel1 phosphorylates the histone H2A (Redon,
for both HR and for NHEJ (X. Zhang, 2005). Tel1 phosphorylates the histone H2A (Redon,
2003), while both ATM and DNA-PKcs are capable of post-translationally modify H2AX,
2003), while both ATM and DNA-PKcs are capable of post-translationally modify H2AX,
the mammals H2A histone variant (Rogakou, 1998, 1999; Stiff, 2004). Phosphorylation of
the mammals H2A histone variant (Rogakou, 1998, 1999; Stiff, 2004). Phosphorylation of
complex, together with, but independently of KU70/KU80
H2A
H2AX
spreads from the break and promotes the recruitment of additional factors for DNA
H2AXH2A spreads from the break and promotes the recruitment of additional factors for DNA
repair (Paull, 2000).
repair (Paull, 2000).
In case a cell is not capable of a rapid and efficient response to DNA lesions in order to
In case a cell is not capable of a rapid and efficient response to DNA lesions in order to
ensure enough time for proper repair, the cell cycle is arrested by activation of a DNA
ensure enough time for proper repair, the cell cycle is arrested by activation of a DNA
damage checkpoint. Three different DNA damage checkpoints are available; the G1, the
damage checkpoint. Three different DNA damage checkpoints are available; the G1, the
intra-S, and the G2/M phase checkpoints ( Paulovich, 1995; Siede, 1996; Weinert, 1988).
intra-S, and the G2/M phase checkpoints ( Paulovich, 1995; Siede, 1996; Weinert, 1988).
1.4.2. Homologous recombination
1.4.2. Homologous recombination
As mentioned before, in case an undamaged DNA template is available, cells can repair DNA
As mentioned before, in case an undamaged DNA template is available, cells can repair DNA
DSBs by HR (Figure 2 and refer to Table 1 for a list of factors involved in HR in human and
DSBs by HR (Figure 2 and refer to Table 1 for a list of factors involved in HR in human and
yeast).
yeast).
Cells commit to HR when DNA ends are subjected to initial 5´- 3´end-resection by the
MRX
Sae2
MRN
complex and the endonuclease CtIP
ssDNA,
which
becomes
Sgs1/Dna2
BLM2/DNA2
4
substrate
for
(Clerici, 2005; Sartori, 2007), creating
long-range
resection
by
EXO1Exo1
or
(Mimitou, 2008; Nimonkar, 2011). Formation of ssDNA also marks the
Cells commit to HR when DNA ends are subjected to initial 5´- 3´end-resection by the
MRNMRX complex and the endonuclease CtIPSae2 (Clerici, 2005; Sartori, 2007), creating
ssDNA,
which
becomes
Sgs1/Dna2
BLM2/DNA2
4
substrate
for
long-range
resection
by
EXO1Exo1
or
(Mimitou, 2008; Nimonkar, 2011). Formation of ssDNA also marks the
!
Introduction
!
!
!
Introduction
initiation of dissociation of CtIPSae2, ATMTel1, and MRX (while MRN stays on DNA), and
initiation of dissociation of CtIPSae2, ATMTel1, and MRX (while MRN stays on DNA), and
consequent binding of RPARPA to DNA ends which prevents their degradation.
consequent binding of RPARPA to DNA ends which prevents their degradation.
RPARPA is also necessary for the recruitment of ATRMec1 through the regulatory subunit
RPARPA is also necessary for the recruitment of ATRMec1 through the regulatory subunit
ATRIPDdc2 (Zou, 2003), and of Rad52 (yeast) or BRCA2 (metazoans) which mediate
ATRIPDdc2 (Zou, 2003), and of Rad52 (yeast) or BRCA2 (metazoans) which mediate
substitution of RPA with RAD51Rad51 (New, 1998). RAD51Rad51 is the recombinase that
substitution of RPA with RAD51Rad51 (New, 1998). RAD51Rad51 is the recombinase that
catalyze the formation of a D loop by mediating the strand invasion of one of the ssDNA
catalyze the formation of a D loop by mediating the strand invasion of one of the ssDNA
ends, followed by replication of 3´DNA ends (Shinohara, 1992). An additional factor is the
ends, followed by replication of 3´DNA ends (Shinohara, 1992). An additional factor is the
Rad54
binding to DNA and
chromatin remodelling ATPase RAD54Rad54 that stimulates RAD51Rad51 binding to DNA and
the formation of the D-loop (Clever, 1997; Swagemakers, 1998; Wolner, 2005). In yeast
the formation of the D-loop (Clever, 1997; Swagemakers, 1998; Wolner, 2005). In yeast
Rad59 facilitates Rad52 binding at break sites (Davis, 2001).
Rad59 facilitates Rad52 binding at break sites (Davis, 2001).
The final step of DSB repair is the formation of a Holliday junction (HJ), created by the
The final step of DSB repair is the formation of a Holliday junction (HJ), created by the
annealing of the remaining 3´ end with the opposite broken strand. Resolution of the HJ can
annealing of the remaining 3´ end with the opposite broken strand. Resolution of the HJ can
lead to a product, with or without cross-over, depending on the resolution method.
lead to a product, with or without cross-over, depending on the resolution method.
Until now this model of repair is the most accepted and it is often used to explain meiotic
Until now this model of repair is the most accepted and it is often used to explain meiotic
DSB recombination, on the other hand mitotic recombination has a lower level of cross-over
DSB recombination, on the other hand mitotic recombination has a lower level of cross-over
events. To explain this phenomenon two other models were formulated: the synthesis
events. To explain this phenomenon two other models were formulated: the synthesis
dependent strand annealing (SDSA) and the migrating D-loop models, which are normally
dependent strand annealing (SDSA) and the migrating D-loop models, which are normally
referred both as SDSA.
referred both as SDSA.
The first one proposes that, contrary to the DSB repair model, both 3´ends invade the
The first one proposes that, contrary to the DSB repair model, both 3´ends invade the
homologous strands, however after limited DNA synthesis both strands are displaced and
homologous strands, however after limited DNA synthesis both strands are displaced and
anneal the complementary 5´ strands followed by fill-in that results in repair with a non cross-
anneal the complementary 5´ strands followed by fill-in that results in repair with a non cross-
over product. The migrating D-loop model on the other hand proposes, similarly to the DSB
over product. The migrating D-loop model on the other hand proposes, similarly to the DSB
repair model, that a single 3´end invades the homologous duplex. A limited DNA synthesis
repair model, that a single 3´end invades the homologous duplex. A limited DNA synthesis
provides the sufficient template for repair, and the strand is then displaced and anneal to the
provides the sufficient template for repair, and the strand is then displaced and anneal to the
other 3´end. Again the consequent fill-in produces a non-crossover product (Symington,
other 3´end. Again the consequent fill-in produces a non-crossover product (Symington,
2014).
2014).
chromatin remodelling ATPase RAD54
Rad51
that stimulates RAD51
5
5
Introduction
Role in DSB repair
End resection
Adaptors
Checkpoint Signaling
Single-strand DNA coating
Single-strand annealing
Mediators
Strand invasion
Introduction
S. cerevisiae
H. Sapiens
Mre11-Rad50-Xrs2
MRE11-RAD50-NBS1
Sae2, Exo1
S. cerevisiae
H. Sapiens
Mre11-Rad50-Xrs2
MRE11-RAD50-NBS1
CtIP, EXO1
Sae2, Exo1
CtIP, EXO1
Dna2-Sgs1
DNA2-BLM
Dna2-Sgs1
DNA2-BLM
Rad9
53BP1,
Rad9
53BP1,
-
MDC1 –BRCA1
-
MDC1 –BRCA1
Tel1
ATM
Tel1
ATM
Mec1-Ddc2
ATR-ATRIP
Mec1-Ddc2
ATR-ATRIP
Rfa1 – Rfa2- Rfa3 (RPA)
RPA1 – RPA2 – RPA3 (RPA)
Rfa1 – Rfa2- Rfa3 (RPA)
RPA1 – RPA2 – RPA3 (RPA)
Rad52
RAD52
Rad52
RAD52
Rad59
-
Rad59
-
-
BRCA2
-
BRCA2
Rad52
-
Rad52
-
Rad51
RAD51
Rad51
RAD51
Rad54
RAD54A, RAD54B
Rad54
RAD54A, RAD54B
Table 1: List of different factors involved in HR, classified according to their functions in DSB repair, in
budding yeast and corresponding orthologs in human.
6
Role in DSB repair
End resection
Adaptors
Checkpoint Signaling
Single-strand DNA coating
Single-strand annealing
Mediators
Strand invasion
Table 1: List of different factors involved in HR, classified according to their functions in DSB repair, in
budding yeast and corresponding orthologs in human.
6
!
Introduction
!
!
A
A
B
B
P
P
P
P
P
P
P
P
P
C
C
D
D
E
E
Introduction
!
P
P
P
P
P
P
P
ATMTel1
MRNMRX
CtIPSae2
!!EXO1
ATMTel1
MRNMRX
CtIPSae2
!!EXO1
RPA
RAD52
RAD51
ATRMec1
RPA
RAD52
RAD51
ATRMec1
BLM-DNA2Sgs1/Dna2
P
Phosphorylated H2A(X)
BLM-DNA2Sgs1/Dna2
P
Phosphorylated H2A(X)
Figure 2: Scheme of Homologous Recombination: (A) DNA damage, recruitment of MRNMRX complex,
ATMTel1 and CtIPSae2 (B) H2A(X) phosphorylation and short range resection by MRNMRX and CtIPSae2. (C)
EXO1 and BLM-DNA2Sgs1/Dna2 depedent long-range resection; RPA and ATRMec1 recruitment. (D)
Recruitment of RAD52 and RAD51. (E) Formation of a D loop followed by creation of a Holliday junction
and repair.
Figure 2: Scheme of Homologous Recombination: (A) DNA damage, recruitment of MRNMRX complex,
ATMTel1 and CtIPSae2 (B) H2A(X) phosphorylation and short range resection by MRNMRX and CtIPSae2. (C)
EXO1 and BLM-DNA2Sgs1/Dna2 depedent long-range resection; RPA and ATRMec1 recruitment. (D)
Recruitment of RAD52 and RAD51. (E) Formation of a D loop followed by creation of a Holliday junction
and repair.
7
7
Introduction
Introduction
1.4.3. Non Homologous End Joining
1.4.3. Non Homologous End Joining
Classical NHEJ is the process of ligation of DNA ends at a DSB, and considered a rapid
Classical NHEJ is the process of ligation of DNA ends at a DSB, and considered a rapid
pathway for the cell to deal with the repair of the same. However, it is also considered an
pathway for the cell to deal with the repair of the same. However, it is also considered an
error prone mechanism. In fact DNA ends need to be processed if they are not compatible for
error prone mechanism. In fact DNA ends need to be processed if they are not compatible for
ligation, thus the loss of short sequences is quite common (Lieber, 2010). Additional factors
ligation, thus the loss of short sequences is quite common (Lieber, 2010). Additional factors
of NHEJ are recruited by the initial binding of the Ku complex; first the DNA end processing
of NHEJ are recruited by the initial binding of the Ku complex; first the DNA end processing
complex Artemis-DNA-PKcs binds KU70/80 (in budding yeast Mec1 or Tel1 substitute for
complex Artemis-DNA-PKcs binds KU70/80 (in budding yeast Mec1 or Tel1 substitute for
Dnl4
PKcs), then the break is repaired by DNA ligase IV together with its cofactors XRCC4
PKcs), then the break is repaired by DNA ligase IV together with its cofactors XRCC4Dnl4
and XLFLif1, assisted by Nej1 in yeast (Lieber, 2010).
and XLFLif1, assisted by Nej1 in yeast (Lieber, 2010).
1.4.4. Other events in DNA damage repair
1.4.4. Other events in DNA damage repair
Aside from the actual recognition of DNA damage and joining of the broken ends, additional
Aside from the actual recognition of DNA damage and joining of the broken ends, additional
events are essential in order to ensure proper repair.
events are essential in order to ensure proper repair.
One is modification of chromatin; H2AXH2A phosphorylation was previously mentioned as
One is modification of chromatin; H2AXH2A phosphorylation was previously mentioned as
one of the most important modifications related to DNA damage. Both histone bodies and
one of the most important modifications related to DNA damage. Both histone bodies and
tails are however subjected to multiple post-translational modifications (phosphorylation,
tails are however subjected to multiple post-translational modifications (phosphorylation,
acetylation, ubiquitination, sumoylation and methylation). The role of these modifications is
acetylation, ubiquitination, sumoylation and methylation). The role of these modifications is
to recruit factors at different stages of the repair process. Of the many examples that can be
to recruit factors at different stages of the repair process. Of the many examples that can be
described, relevant for this thesis, is MDC1, which by sensing ubiquitinated histone H1
described, relevant for this thesis, is MDC1, which by sensing ubiquitinated histone H1
(Thorslund, 2015), recruits RNF8 that in turn recruits RNF168. H2A and H2AX are then
(Thorslund, 2015), recruits RNF8 that in turn recruits RNF168. H2A and H2AX are then
poly-ubiquitinated by the RNF168 E3 ubiquitin ligase, which in turn promotes the
poly-ubiquitinated by the RNF168 E3 ubiquitin ligase, which in turn promotes the
recruitment of other repair factors, such as 53BP1 and BRCA1 (Doil, 2009; Huen, 2007;
recruitment of other repair factors, such as 53BP1 and BRCA1 (Doil, 2009; Huen, 2007;
Kolas, 2007; Stewart, 2009).
Kolas, 2007; Stewart, 2009).
Another event in DNA damage is change of transcription of two classes of genes. The first
Another event in DNA damage is change of transcription of two classes of genes. The first
one is composed of transcripts whose products are directly involved in the repair process. The
one is composed of transcripts whose products are directly involved in the repair process. The
second class includes genes encoding proteins related to DNA metabolism. The specific
second class includes genes encoding proteins related to DNA metabolism. The specific
genes whose expression is changed depends on the type of DNA damage.
genes whose expression is changed depends on the type of DNA damage.
The change in gene expression due to DNA damage requires a complex transduction
The change in gene expression due to DNA damage requires a complex transduction
pathway. The loss of DNA integrity activates various sensors, depending on the type of lesion
pathway. The loss of DNA integrity activates various sensors, depending on the type of lesion
and cell cycle phase. The signal derived from the sensors is amplified by the transducers,
and cell cycle phase. The signal derived from the sensors is amplified by the transducers,
often kinases, and relayed to effectors. These are likely transcription factors acting on the
often kinases, and relayed to effectors. These are likely transcription factors acting on the
promoters of the target genes (Fu, 2008).
promoters of the target genes (Fu, 2008).
8
8
!
!
Introduction
!
!
Introduction
In order to respond to a threat that can impair cell survival, the expression of multiple other
In order to respond to a threat that can impair cell survival, the expression of multiple other
genes is either induced or repressed in a mechanism called environmental stress response
genes is either induced or repressed in a mechanism called environmental stress response
(ESR). Repressed genes are involved in protein synthesis, likely in order for the cell to
(ESR). Repressed genes are involved in protein synthesis, likely in order for the cell to
preserve energy. Induced genes on the other hand are related to cellular functions spanning
preserve energy. Induced genes on the other hand are related to cellular functions spanning
from oxidation-reduction, maintenance of protein stability and balancing of osmolarity
from oxidation-reduction, maintenance of protein stability and balancing of osmolarity
(Gasch, 2001, 2002).
(Gasch, 2001, 2002).
1.5. SMC COMPLEXES
1.5. SMC COMPLEXES
SMC (structural maintenance of chromosome) proteins are a conserved family of proteins,
SMC (structural maintenance of chromosome) proteins are a conserved family of proteins,
present from bacteria to humans and with central roles in regulating genome stability by
present from bacteria to humans and with central roles in regulating genome stability by
maintaining chromosome structure during mitosis and meiosis, and having additional
maintaining chromosome structure during mitosis and meiosis, and having additional
functions in gene regulation and DNA repair.
functions in gene regulation and DNA repair.
SMC proteins are characterized by a typical structure; two nucleotide binding motifs, named
SMC proteins are characterized by a typical structure; two nucleotide binding motifs, named
Walker A and Walker B, are located at the N- and C- terminals respectively. The two protein
Walker A and Walker B, are located at the N- and C- terminals respectively. The two protein
ends interact, forming the HEAD domain, thanks to an anti-parallel folding of the peptide
ends interact, forming the HEAD domain, thanks to an anti-parallel folding of the peptide
chain into a structure called coiled-coil motif. Opposite to the HEAD domain is the HINGE,
chain into a structure called coiled-coil motif. Opposite to the HEAD domain is the HINGE,
through which two SMC monomers interact with each other (Figure 3) (M. Hirano, 2002;
through which two SMC monomers interact with each other (Figure 3) (M. Hirano, 2002;
Melby, 1998).
Melby, 1998).
While bacteria contain only one homodimeric SMC complex (Melby, 1998), eukaryotes
While bacteria contain only one homodimeric SMC complex (Melby, 1998), eukaryotes
possess three heterodimeric complexes, composed of six different SMC proteins. Cohesin,
possess three heterodimeric complexes, composed of six different SMC proteins. Cohesin,
formed by SMC1 and SMC3, is involved in sister chromatid cohesion and DNA repair,
formed by SMC1 and SMC3, is involved in sister chromatid cohesion and DNA repair,
Condensin (SMC2 and SMC4) mainly promotes DNA condensation, and the SMC5/6
Condensin (SMC2 and SMC4) mainly promotes DNA condensation, and the SMC5/6
complex has been suggested to resolve DNA topological structures derived from DNA
complex has been suggested to resolve DNA topological structures derived from DNA
replication stress and is also involved in DNA repair (Guacci, 1997; T. Hirano, 1994, 1997;
replication stress and is also involved in DNA repair (Guacci, 1997; T. Hirano, 1994, 1997;
Kegel, 2011; Lehmann, 1995; Michaelis, 1997).
Kegel, 2011; Lehmann, 1995; Michaelis, 1997).
9
9
Introduction
Introduction
Walker%A%
A%
Walker%B%
Hinge%
N
Walker%A%
A%
C%
Walker%B%
Hinge%
N
B%
C%
B%
Nse5%
Smc3%
Smc1%
Nse5%
Nse6%
Smc5%
Smc2%
Smc6%
Smc4%
Smc3%
Smc1%
Nse2%
Brn1%
Nse3%
Nse4%
Scc1%
Scc3%
Wpl1%
Pds5%
Ycs4% Ycg1%
Figure 3: Representation of the different domains in an unfolded SMC protein. The N- and C-terminals
interact due to the protein folding at the Hinge domain (A). The three S. cerevisiae SMC complexes with core
and accessory proteins (B).
Smc2%
Smc4%
Nse3%
Nse4%
Brn1%
Ycs4% Ycg1%
Figure 3: Representation of the different domains in an unfolded SMC protein. The N- and C-terminals
interact due to the protein folding at the Hinge domain (A). The three S. cerevisiae SMC complexes with core
and accessory proteins (B).
1.5.1. The cohesin complex
1.5.1. The cohesin complex
Cohesin is a multi subunit complex composed of, in addition to the two already mentioned
Smc3
and SMC3
Nse1%
Nse1%
Scc3%
Wpl1%
Pds5%
SMC proteins SMC1
Smc6%
Nse2%
Scc1%
Smc1
Nse6%
Smc5%
Scc1
, RAD21
, a member of the kleisin family and
Cohesin is a multi subunit complex composed of, in addition to the two already mentioned
SMC proteins SMC1Smc1 and SMC3Smc3, RAD21Scc1, a member of the kleisin family and
either SA1 or SA2 in metazoans, orthologs of yeast Scc3 (Table 2).
either SA1 or SA2 in metazoans, orthologs of yeast Scc3 (Table 2).
RAD21Scc1 binds the HEAD domains of SMC1Smc1 with its C-terminus, and SMC3Smc3 with
RAD21Scc1 binds the HEAD domains of SMC1Smc1 with its C-terminus, and SMC3Smc3 with
its N-terminal portion, creating a tripartite ring (Haering, 2002, 2008). As for the other SMC
its N-terminal portion, creating a tripartite ring (Haering, 2002, 2008). As for the other SMC
complexes (Figure 3), accessory proteins are associated with cohesin. These are called
complexes (Figure 3), accessory proteins are associated with cohesin. These are called
Pds5
and interact both with each other, and with the large HEAT repeat
PDS5Pds5 and WAPLwpl1 and interact both with each other, and with the large HEAT repeat
protein SA1/2Scc3. Moreover, PDS5Pds5 interacts with cohesin via Rad21Scc1 (Hartman, 2000;
protein SA1/2Scc3. Moreover, PDS5Pds5 interacts with cohesin via Rad21Scc1 (Hartman, 2000;
Kueng, 2006; Panizza, 2000). However, Wpl1 does not bind cohesin in a stoichiometric
Kueng, 2006; Panizza, 2000). However, Wpl1 does not bind cohesin in a stoichiometric
manner, in fact only some cohesin complexes contain Wpl1 (Chan, 2012). Associated with
manner, in fact only some cohesin complexes contain Wpl1 (Chan, 2012). Associated with
cohesin in vertebrates is an additional component called sororin. Unlike other cohesin
cohesin in vertebrates is an additional component called sororin. Unlike other cohesin
accessory proteins its binding appears to be cell cycle dependent (Nishiyama, 2010; Rankin,
accessory proteins its binding appears to be cell cycle dependent (Nishiyama, 2010; Rankin,
2005; Schmitz, 2007). To accommodate DNA the cohesin “ring” needs to be opened, and the
2005; Schmitz, 2007). To accommodate DNA the cohesin “ring” needs to be opened, and the
PDS5
wpl1
and WAPL
Smc1
(Gruber,
proposed “entry gate” is situated between the hinges of SMC1Smc1 and SMC3Smc3 (Gruber,
2006) (Figure 3). Cohesin can also be removed from chromosomes, not only through
2006) (Figure 3). Cohesin can also be removed from chromosomes, not only through
proposed “entry gate” is situated between the hinges of SMC1
Scc1
Rad21
and SMC3
degradation (further discussed below), but also via an exit gate located between
SMC3Smc3 and Rad21Scc1 (Buheitel, 2013; Chan, 2012).
10
Smc3
Rad21Scc1 degradation (further discussed below), but also via an exit gate located between
SMC3Smc3 and Rad21Scc1 (Buheitel, 2013; Chan, 2012).
10
!
Introduction
!
!
Introduction
!
Function
S. cerevisiae
H. Sapiens/M. musculus
Function
S. cerevisiae
H. Sapiens/M. musculus
Cohesion
Smc3
SMC3
Cohesion
Smc3
SMC3
Smc1
SMC1α (SMC1β)
Smc1
SMC1α (SMC1β)
Scc1 (Rec8)
RAD21, (RAD21L, REC8)
Scc1 (Rec8)
RAD21, (RAD21L, REC8)
Scc3
SA1, SA2 (SA3)
Scc3
SA1, SA2 (SA3)
Scc2
NIPBL
Scc2
NIPBL
Scc4
MAU2
Scc4
MAU2
Establishment
Eco1
ESCO1, ESCO2
Establishment
Eco1
ESCO1, ESCO2
Maintenance
Pds5
PDS5A, PDS5B
Maintenance
Pds5
PDS5A, PDS5B
Wpl1
WAPL
Wpl1
WAPL
-
Sororin
-
Sororin
Esp1
Separase
Esp1
Separase
Pds1
Securin
Pds1
Securin
Loading
Dissolution
Loading
Dissolution
Table 2: List of cohesin subunits and accessory proteins in budding yeast and corresponding orthologs in
human, divided on function. In brackets meiosis specific subunits.
1.5.2. The cohesin loader
Table 2: List of cohesin subunits and accessory proteins in budding yeast and corresponding orthologs in
human, divided on function. In brackets meiosis specific subunits.
1.5.2. The cohesin loader
Cohesin is loaded onto DNA by a separate complex, an heterodimer first discovered in
Cohesin is loaded onto DNA by a separate complex, an heterodimer first discovered in
budding yeast (Ciosk, 2000), but present in all eukaryotes investigated (Gillespie, 2004;
budding yeast (Ciosk, 2000), but present in all eukaryotes investigated (Gillespie, 2004;
Krantz, 2004; Rollins, 2004; Seitan, 2006; Takahashi, 2004; Tonkin, 2004; Watrin, 2006).
Krantz, 2004; Rollins, 2004; Seitan, 2006; Takahashi, 2004; Tonkin, 2004; Watrin, 2006).
The Saccharomyces cerevisiae cohesin loader Scc2/4, and the human NIPBL/MAU2, share a
The Saccharomyces cerevisiae cohesin loader Scc2/4, and the human NIPBL/MAU2, share a
Scc2
is a large HEAT repeat protein (Neuwald, 2000) while
certain degree of similarity. NIPBLScc2 is a large HEAT repeat protein (Neuwald, 2000) while
MAU2Scc4 is a tetratricopeptide repeat (TRP) protein; two different kinds of repeats with a
MAU2Scc4 is a tetratricopeptide repeat (TRP) protein; two different kinds of repeats with a
common feature of protein-protein interaction. However unlike cohesin, the protein sequence
common feature of protein-protein interaction. However unlike cohesin, the protein sequence
of both the subunits of the loader are poorly conserved between yeast and metazoan.
of both the subunits of the loader are poorly conserved between yeast and metazoan.
certain degree of similarity. NIPBL
11
11
Introduction
Introduction
It is not clear how the loader exerts its function, previous reports have shown that the ATPase
It is not clear how the loader exerts its function, previous reports have shown that the ATPase
activity of cohesin is important for its DNA association (Arumugam, 2003) and in vitro
activity of cohesin is important for its DNA association (Arumugam, 2003) and in vitro
Scc2
have shown that the loader affects cohesin
studies on Schizosaccharomyces pombe Mis4Scc2 have shown that the loader affects cohesin
ATP hydrolysis (Arumugam, 2003; Murayama, 2014). Moreover it appears that the HEAT
ATP hydrolysis (Arumugam, 2003; Murayama, 2014). Moreover it appears that the HEAT
repeats are necessary for Scc2 recruitment of cohesin (Takahashi, 2008).
repeats are necessary for Scc2 recruitment of cohesin (Takahashi, 2008).
Little is known about the structure of NIPBLScc2, neither which region of the protein is
Little is known about the structure of NIPBLScc2, neither which region of the protein is
required for DNA binding, nor what domains are involved in cohesin interaction. Work in
required for DNA binding, nor what domains are involved in cohesin interaction. Work in
Xenopus laevi however shows that the first 500 aminoacids of NIPBL bound to MAU2 are
Xenopus laevi however shows that the first 500 aminoacids of NIPBL bound to MAU2 are
capable of binding DNA (Takahashi, 2008). This could mean that the N-terminal of NIPBL is
capable of binding DNA (Takahashi, 2008). This could mean that the N-terminal of NIPBL is
sufficient for DNA interaction, or MAU2 is, or a combination of the two.
sufficient for DNA interaction, or MAU2 is, or a combination of the two.
Still in vitro studies on MAU2Scc4 have shown that it is not required for the binding of Scc2 to
Still in vitro studies on MAU2Scc4 have shown that it is not required for the binding of Scc2 to
naked DNA, but has been hypothesized to be necessary for in vivo chromatin interactions
naked DNA, but has been hypothesized to be necessary for in vivo chromatin interactions
(Murayama, 2014). Recently, two independent studies have managed to obtain crystals of
(Murayama, 2014). Recently, two independent studies have managed to obtain crystals of
Scc4, which appears organized in three different domains, forming a hydrophobic channel
Scc4, which appears organized in three different domains, forming a hydrophobic channel
that wraps the unstructured N-terminal of Scc2, in an anti-parallel orientation. A conserved
that wraps the unstructured N-terminal of Scc2, in an anti-parallel orientation. A conserved
patch on the surface of Scc4 is required for the recruitment of the loading complex at
patch on the surface of Scc4 is required for the recruitment of the loading complex at
centromere regions in vivo (Chao, 2015; Hinshaw, 2015).
centromere regions in vivo (Chao, 2015; Hinshaw, 2015).
The human ortholog of Scc2 was named Nipped-B Like (NIPBL) after the Drosophila
The human ortholog of Scc2 was named Nipped-B Like (NIPBL) after the Drosophila
melanogaster version of the cohesin loader Nipped-B (Rollins, 2004), and was discovered as
melanogaster version of the cohesin loader Nipped-B (Rollins, 2004), and was discovered as
one of the causes of a developmental disorder called Cornelia de Lange Syndrome (Krantz,
one of the causes of a developmental disorder called Cornelia de Lange Syndrome (Krantz,
2004; Tonkin, 2004). NIPBL is more than twice the size of the budding yeast version, thus it
2004; Tonkin, 2004). NIPBL is more than twice the size of the budding yeast version, thus it
is possible to imagine that the metazoan Scc2 ortholog likely possesses new functions or
is possible to imagine that the metazoan Scc2 ortholog likely possesses new functions or
forms of regulation not present in S. cerevisiae. One example is related to the fact that two
forms of regulation not present in S. cerevisiae. One example is related to the fact that two
different transcripts of NIPBL have been observed, encoding for two protein isoforms,
different transcripts of NIPBL have been observed, encoding for two protein isoforms,
NIPBL A and NIPBL B (Tonkin, 2004). To date no specific function has been associated to
NIPBL A and NIPBL B (Tonkin, 2004). To date no specific function has been associated to
either splice variant. An other example can be the NIPBL PxVxL motif known to bind the
either splice variant. An other example can be the NIPBL PxVxL motif known to bind the
chromoshadow domain (CSD) of HP1, a protein not present in budding yeast, which is
chromoshadow domain (CSD) of HP1, a protein not present in budding yeast, which is
known to interact with methylated histone H3, and be involved in gene silencing (Lechner,
known to interact with methylated histone H3, and be involved in gene silencing (Lechner,
2005).
2005).
studies on Schizosaccharomyces pombe Mis4
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Introduction
!
1.6. THE COHESIN CYCLE IN BUDDING YEAST
!
Introduction
1.6. THE COHESIN CYCLE IN BUDDING YEAST
1.6.1. G1 cohesin loading and localization
1.6.1. G1 cohesin loading and localization
In budding yeast cohesin is loaded onto DNA from late G1 phase, by the cohesin loader
In budding yeast cohesin is loaded onto DNA from late G1 phase, by the cohesin loader
(Ciosk, 2000). Multiple studies show an accumulation of the complex around centromeres
(Ciosk, 2000). Multiple studies show an accumulation of the complex around centromeres
and in pericentromeric regions (Glynn, 2004; Lengronne, 2004; Weber, 2004). On
and in pericentromeric regions (Glynn, 2004; Lengronne, 2004; Weber, 2004). On
chromosomes arms cohesin localizes at AT rich regions, mostly in intergenic regions with
chromosomes arms cohesin localizes at AT rich regions, mostly in intergenic regions with
converging transcription, even though no DNA binding motif has been linked to the complex
converging transcription, even though no DNA binding motif has been linked to the complex
(Lengronne, 2004).
(Lengronne, 2004).
The Scc2/4 complex is also enriched at centromeres, and this binding requires the
The Scc2/4 complex is also enriched at centromeres, and this binding requires the
kinetochore Ctf19 subcomplex (Eckert, 2007; Fernius, 2009; Ng, 2009). On chromosome
kinetochore Ctf19 subcomplex (Eckert, 2007; Fernius, 2009; Ng, 2009). On chromosome
arms however, it does not co-localize with cohesin but resides mostly at sites of high
arms however, it does not co-localize with cohesin but resides mostly at sites of high
transcription (Hu, 2011; Lengronne, 2004). Due to this, a model has been proposed according
transcription (Hu, 2011; Lengronne, 2004). Due to this, a model has been proposed according
to which cohesin is shifted to its terminal binding sites by the transcription machinery, and
to which cohesin is shifted to its terminal binding sites by the transcription machinery, and
the loading and translocation are ATP dependent events that require the ATPase function of
the loading and translocation are ATP dependent events that require the ATPase function of
cohesin (Arumugam, 2003; Hu, 2011).
cohesin (Arumugam, 2003; Hu, 2011).
It appears that Scc1 expression is necessary to trigger Scc2/4 DNA binding, at least at
It appears that Scc1 expression is necessary to trigger Scc2/4 DNA binding, at least at
centromeres where cohesin and the loader co-localize, as demonstrated by ectopic expression
centromeres where cohesin and the loader co-localize, as demonstrated by ectopic expression
of Scc1 in early G1 phase (Fernius, 2013).
of Scc1 in early G1 phase (Fernius, 2013).
Furthermore, a recent study has proposed that the nucleosome remodeling complex RSC
Furthermore, a recent study has proposed that the nucleosome remodeling complex RSC
participates in cohesin loading by recruiting Scc2/4 to nucleosome-free regions (Lopez-Serra,
participates in cohesin loading by recruiting Scc2/4 to nucleosome-free regions (Lopez-Serra,
2014).
2014).
1.6.2. S phase and cohesion establishment
1.6.2. S phase and cohesion establishment
As previously mentioned cohesin loading is a dynamic event where the complex keeps
As previously mentioned cohesin loading is a dynamic event where the complex keeps
dissociating from DNA. For its primary function, to maintain the sister chromatids paired
dissociating from DNA. For its primary function, to maintain the sister chromatids paired
until cell division, that was first discovered in Saccharomyces cerevisiae, loading is however
until cell division, that was first discovered in Saccharomyces cerevisiae, loading is however
not sufficient (Guacci, 1997; Michaelis, 1997). In order to keep sister chromatids together
not sufficient (Guacci, 1997; Michaelis, 1997). In order to keep sister chromatids together
until anaphase, cohesin molecules must become cohesive, a process that requires acetylation
until anaphase, cohesin molecules must become cohesive, a process that requires acetylation
on residues K112/113 of Smc3 by the acetyltransferase Eco1 (Ivanov, 2002; Rolef Ben-
on residues K112/113 of Smc3 by the acetyltransferase Eco1 (Ivanov, 2002; Rolef Ben-
Shahar, 2008; J. Zhang, 2008). These modifications counteract the anti-establishment activity
Shahar, 2008; J. Zhang, 2008). These modifications counteract the anti-establishment activity
of Wpl1, Pds5 and Scc3 (Chan, 2012; Lopez-Serra, 2013; Rowland, 2009). Once cohesion
of Wpl1, Pds5 and Scc3 (Chan, 2012; Lopez-Serra, 2013; Rowland, 2009). Once cohesion
has been established the Scc2/4 function becomes dispensable for cohesion maintenance
has been established the Scc2/4 function becomes dispensable for cohesion maintenance
(Ciosk, 2000).
(Ciosk, 2000).
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Introduction
Introduction
Early studies have shown a dependency between cohesion and DNA replication (Skibbens,
Early studies have shown a dependency between cohesion and DNA replication (Skibbens,
1999; Tóth, 1999; Uhlmann, 1998). This was strengthened by the fact that Eco1 contains a
1999; Tóth, 1999; Uhlmann, 1998). This was strengthened by the fact that Eco1 contains a
PCNA-binding motif (Lengronne, 2006; Moldovan, 2006), DNA replication affects Eco1
PCNA-binding motif (Lengronne, 2006; Moldovan, 2006), DNA replication affects Eco1
acetyltransferase activity (Rolef Ben-Shahar, 2008) and multiple replication factors are
acetyltransferase activity (Rolef Ben-Shahar, 2008) and multiple replication factors are
required for sister chromatid cohesion (Sherwood, 2010). Two models were proposed to
required for sister chromatid cohesion (Sherwood, 2010). Two models were proposed to
explain the basis for sister chromatid cohesion. The first one is the so-called “ring model”
explain the basis for sister chromatid cohesion. The first one is the so-called “ring model”
where a single cohesin molecule is capable of embracing two sisters. This hypothesis is
where a single cohesin molecule is capable of embracing two sisters. This hypothesis is
supported by the fact that the kleisin subunit closes the ring and only its degradation is
supported by the fact that the kleisin subunit closes the ring and only its degradation is
capable of releasing DNA entrapped by cohesin, both in in vivo and in vitro experiments.
capable of releasing DNA entrapped by cohesin, both in in vivo and in vitro experiments.
Moreover the cohesin complex dissociates from linearized minichromosomes (Gruber, 2003;
Moreover the cohesin complex dissociates from linearized minichromosomes (Gruber, 2003;
Haering, 2002; Ivanov, 2005; Uhlmann, 1999). The alternative proposed model is the
Haering, 2002; Ivanov, 2005; Uhlmann, 1999). The alternative proposed model is the
“handcuff model”, in which two cohesin molecules, each surrounding a single sister
“handcuff model”, in which two cohesin molecules, each surrounding a single sister
chromatid, interact (Huang, 2005; N. Zhang, 2008).
chromatid, interact (Huang, 2005; N. Zhang, 2008).
1.6.3. Cohesin removal
1.6.3. Cohesin removal
Dissolution of sister chromatid cohesion requires careful timing in order to avoid mitotic
Dissolution of sister chromatid cohesion requires careful timing in order to avoid mitotic
arrest due to checkpoint activation, or incomplete chromatid separation and consequent
arrest due to checkpoint activation, or incomplete chromatid separation and consequent
aneuploidy.
aneuploidy.
In budding yeast cohesin removal takes place at anaphase, when the protease separase (Esp1)
In budding yeast cohesin removal takes place at anaphase, when the protease separase (Esp1)
cleaves Scc1 on two specific residues (R268 and R269), thus opening the cohesin ring and
cleaves Scc1 on two specific residues (R268 and R269), thus opening the cohesin ring and
releasing the sister chromatids that are now free to move following the pulling forces of the
releasing the sister chromatids that are now free to move following the pulling forces of the
microtubules (Uhlmann, 1999). To avoid precocious cleavage by separase, its activity is
microtubules (Uhlmann, 1999). To avoid precocious cleavage by separase, its activity is
inhibited by the regulatory protein securin, which is degraded through APC/C dependent
inhibited by the regulatory protein securin, which is degraded through APC/C dependent
ubiquitination, when the spindle checkpoint is inactivated. Scc1 is then re-synthetized in the
ubiquitination, when the spindle checkpoint is inactivated. Scc1 is then re-synthetized in the
following G1 phase.
following G1 phase.
1.7. COHESIN CYCLE IN METAZOAN
1.7. COHESIN CYCLE IN METAZOAN
1.7.1. Cohesin loading
1.7.1. Cohesin loading
Metazoan cohesin loading shares with budding yeast the necessity of a loader (NIPBL-
Metazoan cohesin loading shares with budding yeast the necessity of a loader (NIPBL-
MAU2), and the requirement for ATP hydrolysis. Moreover in the same way as in S.
MAU2), and the requirement for ATP hydrolysis. Moreover in the same way as in S.
cerevisiae, two different modes of interaction with DNA, before and after S phase, that differ
cerevisiae, two different modes of interaction with DNA, before and after S phase, that differ
in stability, can be observed for cohesin, by fluorescent recovery after photobleaching
in stability, can be observed for cohesin, by fluorescent recovery after photobleaching
(FRAP) (Gerlich, 2006). However some differences can also be reported; a first discrepancy
(FRAP) (Gerlich, 2006). However some differences can also be reported; a first discrepancy
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!
Introduction
!
!
Introduction
concerns cell cycle timing of cohesin binding. In metazoan cohesin is present on chromatin
concerns cell cycle timing of cohesin binding. In metazoan cohesin is present on chromatin
from telophase to anaphase, while budding yeast cohesin loading occurs exclusively from late
from telophase to anaphase, while budding yeast cohesin loading occurs exclusively from late
G1, after Scc1 is re-synthetized. Another difference is the interaction between NIPBL,
G1, after Scc1 is re-synthetized. Another difference is the interaction between NIPBL,
cohesin and the mediator complex, required for the loading of cohesin at active promoters
cohesin and the mediator complex, required for the loading of cohesin at active promoters
(Kagey, 2010). Lastly, in both metazoans and yeast, cohesin does not persist at the loading
(Kagey, 2010). Lastly, in both metazoans and yeast, cohesin does not persist at the loading
sites but is translocated to final sites of interaction, which however differ in character. In
sites but is translocated to final sites of interaction, which however differ in character. In
higher eukaryotes they are found at DNA sequences containing CCCTC-binding factor
higher eukaryotes they are found at DNA sequences containing CCCTC-binding factor
(CTCF) motifs, and not at regions of convergent transcription as in yeast (Parelho, 2008;
(CTCF) motifs, and not at regions of convergent transcription as in yeast (Parelho, 2008;
Rubio, 2008; Stedman, 2008; Wendt, 2008).
Rubio, 2008; Stedman, 2008; Wendt, 2008).
1.7.2. Cohesion establishment
1.7.2. Cohesion establishment
As mentioned before, after S phase, cohesin can be seen interacting more stably with DNA.
As mentioned before, after S phase, cohesin can be seen interacting more stably with DNA.
Similar to budding yeast, acetylation of SMC3 is required to counteract the WAPL-PDS5
Similar to budding yeast, acetylation of SMC3 is required to counteract the WAPL-PDS5
activity (J. Zhang, 2008). In metazoans two different orthologs of Eco1 exist, ESCO1 and
activity (J. Zhang, 2008). In metazoans two different orthologs of Eco1 exist, ESCO1 and
ESCO2, both capable of acetylating SMC3, on the conserved residues K105/K106 and
ESCO2, both capable of acetylating SMC3, on the conserved residues K105/K106 and
promoting sister chromatid cohesion (Hou, 2005; J. Zhang, 2008). The additional factor
promoting sister chromatid cohesion (Hou, 2005; J. Zhang, 2008). The additional factor
sororin, present in vertebrates, interacts with acetylated cohesin to further protect the complex
sororin, present in vertebrates, interacts with acetylated cohesin to further protect the complex
from the action of WAPL (Nishiyama, 2010; Rankin, 2005; Schmitz, 2007).
from the action of WAPL (Nishiyama, 2010; Rankin, 2005; Schmitz, 2007).
1.7.3. Cohesin removal
1.7.3. Cohesin removal
In higher eukaryotes cohesin removal takes place in two separate steps. The majority of
In higher eukaryotes cohesin removal takes place in two separate steps. The majority of
cohesin is removed from chromosome arms during prophase. Moreover in contrast to
cohesin is removed from chromosome arms during prophase. Moreover in contrast to
budding yeast also the cohesin associated proteins (WAPL PDS5, sororin, NIPBL/MAU2)
budding yeast also the cohesin associated proteins (WAPL PDS5, sororin, NIPBL/MAU2)
are removed from chromosome arms. The prophase removal of cohesin is independent of
are removed from chromosome arms. The prophase removal of cohesin is independent of
kleisin cleavage, but is due to WAPL activity, and regulated by phosphorylation of SA2 by
kleisin cleavage, but is due to WAPL activity, and regulated by phosphorylation of SA2 by
PLK1 and Aurora B in what is called the “prophase pathway”. Only centromeric cohesin
PLK1 and Aurora B in what is called the “prophase pathway”. Only centromeric cohesin
remains intact till anaphase, when RAD21 is finally cleaved by separase (Giménez-Abián,
remains intact till anaphase, when RAD21 is finally cleaved by separase (Giménez-Abián,
2004; Lénárt, 2007; Sumara, 2002; Waizenegger, 2000).
2004; Lénárt, 2007; Sumara, 2002; Waizenegger, 2000).
1.8. COHESIN & DNA DAMAGE REPAIR
1.8. COHESIN & DNA DAMAGE REPAIR
Surprisingly the DNA repair function of cohesin was discovered prior to its role in sister
Surprisingly the DNA repair function of cohesin was discovered prior to its role in sister
chromatid cohesion. First, in S. pombe, Scc1 mutations were linked to UV and IR sensitivity.
chromatid cohesion. First, in S. pombe, Scc1 mutations were linked to UV and IR sensitivity.
Later the role of cohesin in DNA damage repair was also discovered in S. cerevisiae, chicken
Later the role of cohesin in DNA damage repair was also discovered in S. cerevisiae, chicken
and human (Atienza, 2005; Birkenbihl, 1992; Sonoda, 2001).
and human (Atienza, 2005; Birkenbihl, 1992; Sonoda, 2001).
15
15
Introduction
Introduction
1.8.1. Budding yeast DNA damage response
1.8.1. Budding yeast DNA damage response
In budding yeast, repair by HR requires the cohesin complex and the auxiliary factors for
In budding yeast, repair by HR requires the cohesin complex and the auxiliary factors for
loading (Scc2) and establishment (PdS5, Eco1), pointing at a role for cohesion in DNA repair
loading (Scc2) and establishment (PdS5, Eco1), pointing at a role for cohesion in DNA repair
(Sjögren, 2001).
(Sjögren, 2001).
In an unchallenged cell cycle, after DNA replication, Eco1 is degraded due to a complex
In an unchallenged cell cycle, after DNA replication, Eco1 is degraded due to a complex
phosphorylation cascade. The proposed model is that Eco1 is first modified in early S phase
phosphorylation cascade. The proposed model is that Eco1 is first modified in early S phase
by Cdk1 that primes, in late S phase, Cdc7-Dbf4 dependent phosphorylation which in turn
by Cdk1 that primes, in late S phase, Cdc7-Dbf4 dependent phosphorylation which in turn
determines Mck1 phosphorylation and consequent degradation (Lyons, 2011, 2013). For this
determines Mck1 phosphorylation and consequent degradation (Lyons, 2011, 2013). For this
reason the cohesin molecules, even though they can still be loaded onto DNA, cannot be
reason the cohesin molecules, even though they can still be loaded onto DNA, cannot be
made cohesive. In case of DNA damage, however, new cohesin is loaded and becomes
made cohesive. In case of DNA damage, however, new cohesin is loaded and becomes
cohesive, around the DSB and throughout the genome (Ström, 2004, 2007; Ünal, 2004,
cohesive, around the DSB and throughout the genome (Ström, 2004, 2007; Ünal, 2004,
2007). In order to achieve this damage-induced cohesion (DI-cohesion), Eco1 is stabilized
2007). In order to achieve this damage-induced cohesion (DI-cohesion), Eco1 is stabilized
due to Cdc7-Dbf4 inhibition (Lyons, 2013). Moreover Scc1 phosphorylation on serine 83 by
due to Cdc7-Dbf4 inhibition (Lyons, 2013). Moreover Scc1 phosphorylation on serine 83 by
Chk1, activated in turn by the Mec1 kinase, was suggested to drive acetylation by Eco1,
Chk1, activated in turn by the Mec1 kinase, was suggested to drive acetylation by Eco1,
necessary for G2 specific cohesion establishment (Heidinger-Pauli, 2008). DI-cohesion
necessary for G2 specific cohesion establishment (Heidinger-Pauli, 2008). DI-cohesion
shows some differences compared to canonical cohesion establishment. First of all it does not
shows some differences compared to canonical cohesion establishment. First of all it does not
require DNA replication since it is independent of Rad52 (Ström, 2007; Ünal, 2007).
require DNA replication since it is independent of Rad52 (Ström, 2007; Ünal, 2007).
Moreover in a DNA damage situation Eco1 acetylates different residues than the canonical
Moreover in a DNA damage situation Eco1 acetylates different residues than the canonical
Smc3 sites (K112, 113), however it is still counteracted by Wpl1 activity (Heidinger-Pauli,
Smc3 sites (K112, 113), however it is still counteracted by Wpl1 activity (Heidinger-Pauli,
2008, 2009).
2008, 2009).
Binding of cohesin around a DSB covers approximately100 kb, except for a region of 5 kb in
Binding of cohesin around a DSB covers approximately100 kb, except for a region of 5 kb in
direct vicinity of the break, and depends on the MRX complex, Tel1, Mec1 and H2A
direct vicinity of the break, and depends on the MRX complex, Tel1, Mec1 and H2A
phosphorylation (Ström, 2004; Ünal, 2004). The role of cohesin in DNA damage repair is
phosphorylation (Ström, 2004; Ünal, 2004). The role of cohesin in DNA damage repair is
still not clear but the most accredited hypothesis is that the complex keeps the DSB in close
still not clear but the most accredited hypothesis is that the complex keeps the DSB in close
proximity to the undamaged DNA substrate in order to enable repair. Even though this
proximity to the undamaged DNA substrate in order to enable repair. Even though this
concept is surely appealing for its intuitiveness it still needs to be proven.
concept is surely appealing for its intuitiveness it still needs to be proven.
1.8.2. Metazoan cohesin and DNA damage
1.8.2. Metazoan cohesin and DNA damage
Cohesin is recruited to DNA damage also in human cells, specifically in S/G2 phase, as
Cohesin is recruited to DNA damage also in human cells, specifically in S/G2 phase, as
shown in both immunofluorescence experiments with laser damage (J. S. Kim, 2002) and in
shown in both immunofluorescence experiments with laser damage (J. S. Kim, 2002) and in
ChIP experiments with I-Sce induced DSB (Potts, 2006). Moreover in human cells, in the
ChIP experiments with I-Sce induced DSB (Potts, 2006). Moreover in human cells, in the
same way as in budding yeast, cohesin likely requires the MRN complex as demonstrated by
same way as in budding yeast, cohesin likely requires the MRN complex as demonstrated by
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!
!
Introduction
!
!
Introduction
experiments carried out on cell line lacking functional MRE11 and further strengthen by the
experiments carried out on cell line lacking functional MRE11 and further strengthen by the
finding of a direct interaction between the two complexes (J. S. Kim, 2002).
finding of a direct interaction between the two complexes (J. S. Kim, 2002).
There are still no clear evidence that DI-cohesion is present also in metazoans but some data
There are still no clear evidence that DI-cohesion is present also in metazoans but some data
indicates that it might indeed be a conserved process. As a start, sororin which is required for
indicates that it might indeed be a conserved process. As a start, sororin which is required for
cohesion formation, has been reported to be needed for DNA damage repair in G2 cells
cohesion formation, has been reported to be needed for DNA damage repair in G2 cells
(Schmitz, 2007). Moreover, ChIP-seq mapping shows that, upon DNA damage induction,
(Schmitz, 2007). Moreover, ChIP-seq mapping shows that, upon DNA damage induction,
cohesin binding is reinforced at pre-existing binding sites, and ESCO1 dependent acetylation
cohesin binding is reinforced at pre-existing binding sites, and ESCO1 dependent acetylation
increases in quantitative mass spectrometry analysis (B. J. Kim, 2010).
increases in quantitative mass spectrometry analysis (B. J. Kim, 2010).
Human cohesin has been also implicated in DNA damage checkpoint activation. The intra-S-
Human cohesin has been also implicated in DNA damage checkpoint activation. The intra-S-
phase checkpoint activation depends on ATM and ATR that directly phosphorylate SMC1
phase checkpoint activation depends on ATM and ATR that directly phosphorylate SMC1
and SMC3 (Garg, 2004; Kitagawa, 2004; Luo, 2008; Yazdi, 2002). A role of cohesin in the
and SMC3 (Garg, 2004; Kitagawa, 2004; Luo, 2008; Yazdi, 2002). A role of cohesin in the
G2/M checkpoint was confirmed by the fact that depletion of RAD21, affects foci formation
G2/M checkpoint was confirmed by the fact that depletion of RAD21, affects foci formation
of 53BP1 (Watrin, 2009).
of 53BP1 (Watrin, 2009).
NIPBL is as cohesin recruited to DNA damage, but this is a much less studied process.
NIPBL is as cohesin recruited to DNA damage, but this is a much less studied process.
(discussed further below). Previous work have however shown that NIPBL recruitment to
(discussed further below). Previous work have however shown that NIPBL recruitment to
DNA damage depends on MCD1, RNF168 and HP1γ (Kong, 2014; Oka, 2011).
DNA damage depends on MCD1, RNF168 and HP1γ (Kong, 2014; Oka, 2011).
1.9. COHESIN AND MEIOSIS
1.9. COHESIN AND MEIOSIS
The meiotic prophase I poses a big challenge for cells, since homologs need to undergo
The meiotic prophase I poses a big challenge for cells, since homologs need to undergo
proper pairing and the formation of chiasmata and resolution of DSBs require perfect steric
proper pairing and the formation of chiasmata and resolution of DSBs require perfect steric
and topological control.
and topological control.
The cohesin complex is an absolute necessity for proper meiosis progression. In both yeasts
The cohesin complex is an absolute necessity for proper meiosis progression. In both yeasts
and metazoan, meiosis specific cohesin subunits are different than in somatic cells. Budding
and metazoan, meiosis specific cohesin subunits are different than in somatic cells. Budding
yeast Scc1 is substituted by Rec8 (Klein, 1999), while mammalian SMC1α, RAD21 and
yeast Scc1 is substituted by Rec8 (Klein, 1999), while mammalian SMC1α, RAD21 and
SA1/2 are complemented by SMC1β, REC8, the newly discovered RAD21L and SA3
SA1/2 are complemented by SMC1β, REC8, the newly discovered RAD21L and SA3
respectively. All these additional cohesin isoforms have specific binding patterns and non-
respectively. All these additional cohesin isoforms have specific binding patterns and non-
redundant functions compared to their mitotic paralogs (Hopkins, 2014; Lee, 2011;
redundant functions compared to their mitotic paralogs (Hopkins, 2014; Lee, 2011;
Revenkova, 2001; Xu, 2005).
Revenkova, 2001; Xu, 2005).
Observations from mouse meiosis showed Nipbl chromosomal association from zygotene
Observations from mouse meiosis showed Nipbl chromosomal association from zygotene
until late pachytene, the same stages during which Rad21 containing cohesin complexes are
until late pachytene, the same stages during which Rad21 containing cohesin complexes are
17
17
Introduction
Introduction
binding DNA. These evidences point to the possibility that meiotic cohesin loading is
binding DNA. These evidences point to the possibility that meiotic cohesin loading is
independent of replication.
independent of replication.
The cohesin complex, aside from its canonical role of keeping the sister chromatids together,
The cohesin complex, aside from its canonical role of keeping the sister chromatids together,
also supports the formation of the axial elements of the synaptonemal complex. Meiotic
also supports the formation of the axial elements of the synaptonemal complex. Meiotic
cohesin is released in two steps: first during anaphase I, separase cleaves cohesin from
cohesin is released in two steps: first during anaphase I, separase cleaves cohesin from
chromosome arms, while shugoshin protects centromeric cohesion until meiosis II, when
chromosome arms, while shugoshin protects centromeric cohesion until meiosis II, when
cohesin is finally removed from sister chromatids, allowing their segregation (Rankin, 2015).
cohesin is finally removed from sister chromatids, allowing their segregation (Rankin, 2015).
1.10. COHESION AND BEYOND
1.10. COHESION AND BEYOND
Several studies have shown that cohesin and its loader play a role in gene regulation, in
Several studies have shown that cohesin and its loader play a role in gene regulation, in
addition to being essential for correct chromosomes segregation and DNA repair.
addition to being essential for correct chromosomes segregation and DNA repair.
The first evidence for the involvement of the cohesin loader in transcription came from D.
The first evidence for the involvement of the cohesin loader in transcription came from D.
Melanogaster, where Nipped-B was found to promote long-range interactions between
Melanogaster, where Nipped-B was found to promote long-range interactions between
enhancers and promoters of the homeobox gene family (Rollins, 1999, 2004).
enhancers and promoters of the homeobox gene family (Rollins, 1999, 2004).
Other proofs came over the years from different models, in C. Elegans and X. Laevis Mau2,
Other proofs came over the years from different models, in C. Elegans and X. Laevis Mau2,
the ortholog of Scc4, is required for axon formation (Seitan, 2006). Similar results were
the ortholog of Scc4, is required for axon formation (Seitan, 2006). Similar results were
obtained in D. Melanogaster where Nipped-B was linked to neuronal development (Pauli,
obtained in D. Melanogaster where Nipped-B was linked to neuronal development (Pauli,
2008). However the most important evidence comes from humans where NIPBL was found
2008). However the most important evidence comes from humans where NIPBL was found
frequently mutated in a developmental disorder called Cornelia de Lange syndrome (CdLS).
frequently mutated in a developmental disorder called Cornelia de Lange syndrome (CdLS).
These patients show multisystem malformations: short stature, intellectual disability,
These patients show multisystem malformations: short stature, intellectual disability,
gastroesophageal dysfunction, growth defects of the upper limbs and distinctive facial
gastroesophageal dysfunction, growth defects of the upper limbs and distinctive facial
features (Krantz, 2004; Tonkin, 2004).
features (Krantz, 2004; Tonkin, 2004).
Cell lines derived from CdLS patients and a mouse model of the syndrome, show defects in
Cell lines derived from CdLS patients and a mouse model of the syndrome, show defects in
transcription but not in cell division, since cohesion is properly established (Castronovo,
transcription but not in cell division, since cohesion is properly established (Castronovo,
2009; Revenkova, 2009). This is quite interesting, considering that also in budding yeast, the
2009; Revenkova, 2009). This is quite interesting, considering that also in budding yeast, the
amount of available cohesin can be reduced to nearly 10% of wild type level before cell
amount of available cohesin can be reduced to nearly 10% of wild type level before cell
division defects become apparent (Heidinger-Pauli, 2010). This leads to the intriguing
division defects become apparent (Heidinger-Pauli, 2010). This leads to the intriguing
concept that the canonical role of cohesin in keeping sister chromatids together requires few
concept that the canonical role of cohesin in keeping sister chromatids together requires few
active complexes, and the rest might be needed for other cellular functions.
active complexes, and the rest might be needed for other cellular functions.
Some evidence of a role for Scc2 in gene regulation can be found also in budding yeast. It has
Some evidence of a role for Scc2 in gene regulation can be found also in budding yeast. It has
been reported that Scc2 directly affects Rec8 expression during meiosis (Lin, 2011).
been reported that Scc2 directly affects Rec8 expression during meiosis (Lin, 2011).
18
18
!
Introduction
!
!
Introduction
!
Moreover, lack of Scc2 influences both general genome-wide and DNA damage response
Moreover, lack of Scc2 influences both general genome-wide and DNA damage response
specific transcription (Lindgren, 2014). A possible model for gene regulation via Scc2 in
specific transcription (Lindgren, 2014). A possible model for gene regulation via Scc2 in
yeast can be searched in the relationship between Scc2 and chromatin remodeling, since the
yeast can be searched in the relationship between Scc2 and chromatin remodeling, since the
RSC complex is needed for chromatin recruitment of the loader, and inhibition of Scc2
RSC complex is needed for chromatin recruitment of the loader, and inhibition of Scc2
results in a similar transcription profile as RSC inhibition (Lopez-Serra, 2014).
results in a similar transcription profile as RSC inhibition (Lopez-Serra, 2014).
Mechanistic insight on a role for Cohesin in gene regulation was identified in metazoans
Mechanistic insight on a role for Cohesin in gene regulation was identified in metazoans
where it was reported to co-localize with CTCF, whose presence is also necessary for cohesin
where it was reported to co-localize with CTCF, whose presence is also necessary for cohesin
positioning. Cohesin on the other hand is required for proper CTCF function as an insulator
positioning. Cohesin on the other hand is required for proper CTCF function as an insulator
affecting the expression of numerous genes (Parelho, 2008; Rubio, 2008; Stedman, 2008;
affecting the expression of numerous genes (Parelho, 2008; Rubio, 2008; Stedman, 2008;
Wendt, 2008).
Wendt, 2008).
It is still not clear how cohesin and its loader regulate gene expression, one possible
It is still not clear how cohesin and its loader regulate gene expression, one possible
explanation is that cohesin, with its ability to encircle DNA molecules, is capable of doing
explanation is that cohesin, with its ability to encircle DNA molecules, is capable of doing
this also at an intramolecular level, together with CTCF, bringing two DNA sequences in
this also at an intramolecular level, together with CTCF, bringing two DNA sequences in
close proximity, determining the formation of a loop (Sanborn, 2015) (Figure 4).
close proximity, determining the formation of a loop (Sanborn, 2015) (Figure 4).
A
B
P
E
G
C
Repression
P
G
B
Induction
Induction
E
A
E
E
Enhancer
P
Promoter
CTCF
binding site
P
G
G
P
E
G
E
Gene
C
CTCF
Repression
Cohesin
P
G
Induction
Induction
E
P
G
E
Enhancer
P
Promoter
CTCF
CTCF
binding site
Cohesin
G
Gene
Figure 4: A Model on how cohesin can affect gene expression together with CTCF. Converging CTCF
binding sequences determine the formation of a intramolecular loop stabilized by a single cohesin molecule
encircling the loop (A). An alternative version of the model in which two cohesin molecules interact together
to stabilize the intramolecular loop (B). Formation of a loop that impairs the interaction between the enhancer
and the promoter (C).
Figure 4: A Model on how cohesin can affect gene expression together with CTCF. Converging CTCF
binding sequences determine the formation of a intramolecular loop stabilized by a single cohesin molecule
encircling the loop (A). An alternative version of the model in which two cohesin molecules interact together
to stabilize the intramolecular loop (B). Formation of a loop that impairs the interaction between the enhancer
and the promoter (C).
19
19
Methodology
!
2 METHODOLOGY
Methodology
!
2 METHODOLOGY
2.1 MODEL ORGANISMS
2.1 MODEL ORGANISMS
2.1.1 Saccharomyces cerevisiae
2.1.1 Saccharomyces cerevisiae
Paper I and III are based on invesigations in S. cerevisiae. Although there are some
Paper I and III are based on invesigations in S. cerevisiae. Although there are some
differences between higher eukaryotes and S. cerevisiae, it has the advantage of a short cell
differences between higher eukaryotes and S. cerevisiae, it has the advantage of a short cell
cycle (90 minutes under optimal conditions), easy growth conditions and genetic
cycle (90 minutes under optimal conditions), easy growth conditions and genetic
manipulation procedures that allow elaborate experiments with complex genetic backgrounds
manipulation procedures that allow elaborate experiments with complex genetic backgrounds
(for example long cell cycle arrest and multiple mutations or deletions). Compared to other
(for example long cell cycle arrest and multiple mutations or deletions). Compared to other
eukaryotes S. cerevisiae has a compact genome comprising 6000 genes, with an average
eukaryotes S. cerevisiae has a compact genome comprising 6000 genes, with an average
length of 1450 bp, divided on 16 rather short chromosomes (Dujon, 1996).
length of 1450 bp, divided on 16 rather short chromosomes (Dujon, 1996).
2.1.2 Mus musculus
2.1.2 Mus musculus
In Paper IV, M. musculus, the most used vertebrate in biomedical studies is utilized. Thanks
In Paper IV, M. musculus, the most used vertebrate in biomedical studies is utilized. Thanks
to a well known genome, a life span of 1-2 years, early sexual maturity, an average of 19 days
to a well known genome, a life span of 1-2 years, early sexual maturity, an average of 19 days
of pregnancy and a litter size of 6 to 12 pups, mice are a practical mammalian model,
of pregnancy and a litter size of 6 to 12 pups, mice are a practical mammalian model,
relatively easy to modify, to breed and to grow. Especially for studies of meiosis M. musculus
relatively easy to modify, to breed and to grow. Especially for studies of meiosis M. musculus
is the favored model organism. Gametogenesis (gamete formation) takes place in
is the favored model organism. Gametogenesis (gamete formation) takes place in
seminiferous tubules of the testes (spermatogenesis), for males, and in the ovaries (oogenesis)
seminiferous tubules of the testes (spermatogenesis), for males, and in the ovaries (oogenesis)
for females. While spermatogenesis is a continuous process where new gametes are produced
for females. While spermatogenesis is a continuous process where new gametes are produced
every day, females have only a fixed number of primary oocytes generated during
every day, females have only a fixed number of primary oocytes generated during
embryogenesis, These have the potential to become mature gametes, but are initially arrested
embryogenesis, These have the potential to become mature gametes, but are initially arrested
in their development at the end of prophase I. During each menstrual cycle, due to a
in their development at the end of prophase I. During each menstrual cycle, due to a
hormonal surge, they continue meiosis and become mature ova (Hess, 2008; Pepling, 2006).
hormonal surge, they continue meiosis and become mature ova (Hess, 2008; Pepling, 2006).
The female meiosis is completely finalized only at the moment of fertilization.
The female meiosis is completely finalized only at the moment of fertilization.
2.1.3 Human cell culture
2.1.3 Human cell culture
Since research on humans for ethical reasons has multiple obvious restrictions, cell lines offer
Since research on humans for ethical reasons has multiple obvious restrictions, cell lines offer
a good compromise to study human cellular processes in vivo.
a good compromise to study human cellular processes in vivo.
The variety of available cell lines from different tissues, the possibility to administer drugs,
The variety of available cell lines from different tissues, the possibility to administer drugs,
like inhibitors for specific enzymes, and to knock down expression of genes of interest by
like inhibitors for specific enzymes, and to knock down expression of genes of interest by
siRNA, make cell cultures a powerful research tool.
siRNA, make cell cultures a powerful research tool.
21
21
Methodology
Methodology
In Paper II the commercial cell line HEK293 Flp-In™ T-REx™ (Thermo Fisher) was used.
In Paper II the commercial cell line HEK293 Flp-In™ T-REx™ (Thermo Fisher) was used.
In this system cells contain the Flp recombination target (FRT) at a specific locus in the
In this system cells contain the Flp recombination target (FRT) at a specific locus in the
genome, which allows integration of a gene of interest, thanks to the Flp recombinase
genome, which allows integration of a gene of interest, thanks to the Flp recombinase
activity, consistently at the same position in the genome. We integrated various truncations
activity, consistently at the same position in the genome. We integrated various truncations
and mutations of NIPBL, previously cloned in an expression vector, creating multiple stable
and mutations of NIPBL, previously cloned in an expression vector, creating multiple stable
cell lines. In this way experimental variation due to transfection and integration differences
cell lines. In this way experimental variation due to transfection and integration differences
could be minimized.
could be minimized.
2.2 IMMUNOFLUORESCENCE
OF
TESTICULAR
AND
OVARIAN
2.2 IMMUNOFLUORESCENCE
NUCLEAR SPREADS
OF
TESTICULAR
AND
OVARIAN
NUCLEAR SPREADS
With nuclear spreads nuclei isolated from meiotic cells are distributed on a slide for
With nuclear spreads nuclei isolated from meiotic cells are distributed on a slide for
visualization of chromosomes. We used an approach consisting of a drying-down technique
visualization of chromosomes. We used an approach consisting of a drying-down technique
that allows recovery of a sufficient number of nuclei and good preservation of chromatin
that allows recovery of a sufficient number of nuclei and good preservation of chromatin
structures (Peters, 1997). Mouse spermatocytes were obtained from young males, while
structures (Peters, 1997). Mouse spermatocytes were obtained from young males, while
prophase oocytes were obtained from mouse embryos (E16.5–E19.5). Testes and ovaries
prophase oocytes were obtained from mouse embryos (E16.5–E19.5). Testes and ovaries
were dissected and then torn to pieces in order to make single cell suspensions. Nuclei
were dissected and then torn to pieces in order to make single cell suspensions. Nuclei
extracted after incubation in a hypotonic buffer, were then fixed by spreading a drop of
extracted after incubation in a hypotonic buffer, were then fixed by spreading a drop of
suspension on slides previously dipped in paraformaldehyde.
suspension on slides previously dipped in paraformaldehyde.
Immunofluorescence is a technique where antibodies are used to recognize a specific protein.
Immunofluorescence is a technique where antibodies are used to recognize a specific protein.
The fluorescent signal derives from a secondary antibody, raised against the species of the
The fluorescent signal derives from a secondary antibody, raised against the species of the
primary antibody, carrying a fluorophore, a molecule that can emit light of a determined
primary antibody, carrying a fluorophore, a molecule that can emit light of a determined
wavelength when excited by photons coming from a specific portion of the light spectrum.
wavelength when excited by photons coming from a specific portion of the light spectrum.
Specificity of antibodies used is of utmost importance in immunofluorescence, since it is
Specificity of antibodies used is of utmost importance in immunofluorescence, since it is
impossible to tell by microscopy if the antibody binds off-target proteins. For this reason all
impossible to tell by microscopy if the antibody binds off-target proteins. For this reason all
non-commercial antibodies were also tested for specificity by western blot. Since in Paper
non-commercial antibodies were also tested for specificity by western blot. Since in Paper
IV we wished to compare genotypes, in order to reduce variation, the samples to be
IV we wished to compare genotypes, in order to reduce variation, the samples to be
compared were prepared the same day, stained with the same antibody preparation and
compared were prepared the same day, stained with the same antibody preparation and
observed using the same microscope conditions.
observed using the same microscope conditions.
2.3 MICROARRAY ANALYSIS
2.3 MICROARRAY ANALYSIS
Microarrays are powerful tools with multiple possible applications, one of which is to
Microarrays are powerful tools with multiple possible applications, one of which is to
measure transcript levels, comparing two different conditions, as in Paper III, where
measure transcript levels, comparing two different conditions, as in Paper III, where
22
22
Methodology
!
Methodology
!
differences in gene expression were observed in the presence or absence of DNA damage,
differences in gene expression were observed in the presence or absence of DNA damage,
comparing wild type cells and cells without functional Scc2. Arrays can be different in terms
comparing wild type cells and cells without functional Scc2. Arrays can be different in terms
of manufacturing method, number of samples that can be profiled (single or double channel)
of manufacturing method, number of samples that can be profiled (single or double channel)
and length of the probes (cDNA or oligonucleotides). In Paper III the GeneChip yeast
and length of the probes (cDNA or oligonucleotides). In Paper III the GeneChip yeast
genome 2.0 array from Affymetrix was used, a single channel oligonucleotide array. A
genome 2.0 array from Affymetrix was used, a single channel oligonucleotide array. A
limitation of microarrays is their strong sensitivity that can lead to variability in the raw data.
limitation of microarrays is their strong sensitivity that can lead to variability in the raw data.
The best course of action when performing a microarray-based study would be to reduce the
The best course of action when performing a microarray-based study would be to reduce the
sources of variability (batch, experimental variation, operator) to a minimum, which is often
sources of variability (batch, experimental variation, operator) to a minimum, which is often
for logistic reasons not possible. This is even more problematic when comparing multiple
for logistic reasons not possible. This is even more problematic when comparing multiple
conditions, with the possibility of failed experiments or outliers. For this reason it is
conditions, with the possibility of failed experiments or outliers. For this reason it is
important to pre-process the data obtained from the array. Pre-processing includes different
important to pre-process the data obtained from the array. Pre-processing includes different
steps: background correction, quantile normalization and summarization. Background
steps: background correction, quantile normalization and summarization. Background
correction is carried out to remove the effect of aspecific binding while quantile
correction is carried out to remove the effect of aspecific binding while quantile
normalization is the statistical process used to compare two distributions. Summarization is
normalization is the statistical process used to compare two distributions. Summarization is
on the other hand, the process that gives the expression values of a single gene derived from
on the other hand, the process that gives the expression values of a single gene derived from
the data collected from multiple probes (Wu, 2009).
the data collected from multiple probes (Wu, 2009).
2.4 CHROMATIN IMMUNOPRECIPITATION
2.4 CHROMATIN IMMUNOPRECIPITATION
In Paper I, to detect Scc2 binding at the site of a DSB, chromatin immunoprecipitation
In Paper I, to detect Scc2 binding at the site of a DSB, chromatin immunoprecipitation
(ChIP) coupled to quantitative real-time PCR was used (Figure 5). ChIP is the
(ChIP) coupled to quantitative real-time PCR was used (Figure 5). ChIP is the
immunoprecipitation of a specific protein (in our case a tagged version of Scc2) where the
immunoprecipitation of a specific protein (in our case a tagged version of Scc2) where the
whole cell extract derives from the lysis of cross-linked cells. This allows the proteins to stay
whole cell extract derives from the lysis of cross-linked cells. This allows the proteins to stay
linked to the DNA sequence they bind in vivo. In order to get good resolution in qPCR,
linked to the DNA sequence they bind in vivo. In order to get good resolution in qPCR,
chromatin is kept at an optimal size range of 300 to 500 bp, thanks to the sonication of the
chromatin is kept at an optimal size range of 300 to 500 bp, thanks to the sonication of the
lysate prior of the immunoprecipitation. After protein-DNA complexes are eluted from the
lysate prior of the immunoprecipitation. After protein-DNA complexes are eluted from the
beads of the IP, they are de-crosslinked and DNA is then further purified for removal of
beads of the IP, they are de-crosslinked and DNA is then further purified for removal of
proteins and RNA (Katou, 2006).
proteins and RNA (Katou, 2006).
Purified DNA can then be analyzed using different approaches: tiling microarrays (ChIP-on-
Purified DNA can then be analyzed using different approaches: tiling microarrays (ChIP-on-
chip), massive parallel sequencing (ChIP-seq) or ChIP coupled to quantitative PCR (ChIP-
chip), massive parallel sequencing (ChIP-seq) or ChIP coupled to quantitative PCR (ChIP-
qPCR). Out of the three, ChIP-qPCR offers fully quantitative data with the downside that it is
qPCR). Out of the three, ChIP-qPCR offers fully quantitative data with the downside that it is
possible to look only at few loci at a time. ChIP-on-chip and ChIP-seq allow a genome-wide
possible to look only at few loci at a time. ChIP-on-chip and ChIP-seq allow a genome-wide
analysis of the chromosomal association of a specific protein in a single experiment. In our
analysis of the chromosomal association of a specific protein in a single experiment. In our
23
23
Methodology
Methodology
case however ChIP-qPCR is the perfect tool to observe binding levels of a specific protein
case however ChIP-qPCR is the perfect tool to observe binding levels of a specific protein
around the site of a specific DNA DSB.
around the site of a specific DNA DSB.
Experiments$carried$out$in$a$strains$
containing$a$tagged$version$of$My$Favorite$
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Preparation$of$Whole$Cell$Extract$(WCE)$and$
chromatin$shearing$by$sonication.$
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containing$a$tagged$version$of$My$Favorite$
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$
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Reverse$crosslinks$and$purify$DNA.$
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Figure 5: Scheme of the different steps of the ChIP assay.
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formaldehyde.$
P$
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Figure 5: Scheme of the different steps of the ChIP assay.
2.5 DNA DAMAGE INDUCTION
2.5 DNA DAMAGE INDUCTION
In Paper I, II and III different approaches are used to induce DNA damage and observe the
In Paper I, II and III different approaches are used to induce DNA damage and observe the
effects and the dynamics that follow this event.
effects and the dynamics that follow this event.
In Paper I and III the homothallic switching endonuclease (HO) was used to induce DSBs in
In Paper I and III the homothallic switching endonuclease (HO) was used to induce DSBs in
an artificially inserted recognition cut-site on chromosome VI of the yeast genome. The
an artificially inserted recognition cut-site on chromosome VI of the yeast genome. The
endonuclease was regulated by a strong inducible promoter (GAL10), replacing the
endonuclease was regulated by a strong inducible promoter (GAL10), replacing the
endogenous HO promoter, in order to express the HO endonuclease by adding galactose to
endogenous HO promoter, in order to express the HO endonuclease by adding galactose to
liquid media.
liquid media.
In Paper II, two different methods to produce DNA lesions were used. The first one is laser
In Paper II, two different methods to produce DNA lesions were used. The first one is laser
microirradiation, a method with a long history in the field of DNA damage effects in cells. In
microirradiation, a method with a long history in the field of DNA damage effects in cells. In
our particular case cells are pre-treated with either BrdU or TMP to sensitize them towards
our particular case cells are pre-treated with either BrdU or TMP to sensitize them towards
laser treatment. Different types of laser, with different sources and power output can be used
laser treatment. Different types of laser, with different sources and power output can be used
24
24
!
Methodology
!
Methodology
in order to induce different types of DNA damage. We used a laser output of 20 Hz, 364 nm
in order to induce different types of DNA damage. We used a laser output of 20 Hz, 364 nm
that might generate interstrand cross-links in addition to DSBs, for this reason we used also a
that might generate interstrand cross-links in addition to DSBs, for this reason we used also a
second method to create “clean” DSB: The FokI endonuclease.
second method to create “clean” DSB: The FokI endonuclease.
The FokI reporter system is based on Lac operator repeats to which an inducible FokI
The FokI reporter system is based on Lac operator repeats to which an inducible FokI
endonuclease, fused to a Lac repressor and mCherry, is able to bind and induce DSBs.
endonuclease, fused to a Lac repressor and mCherry, is able to bind and induce DSBs.
Expression of FokI was regulated in a similar way as the HO system, but in this case a
Expression of FokI was regulated in a similar way as the HO system, but in this case a
destabilization domain and a modified estradiol receptor fused with the endonuclease in
destabilization domain and a modified estradiol receptor fused with the endonuclease in
combination with addition of small molecules (Shield1 ligand and 4-OHT) was responsible
combination with addition of small molecules (Shield1 ligand and 4-OHT) was responsible
for activation (Tang, 2013).
for activation (Tang, 2013).
These three different approaches produce different effects, laser microirradiation for example
These three different approaches produce different effects, laser microirradiation for example
induce DSB formation, but likely also other forms of DNA damage. On the other hand
induce DSB formation, but likely also other forms of DNA damage. On the other hand
endonucleases produce clean DSBs, but while the HO endonuclease with a single cut-site
endonucleases produce clean DSBs, but while the HO endonuclease with a single cut-site
induce a single DSB, the FokI endonuclease induces multiple breaks in the Lac operator
induce a single DSB, the FokI endonuclease induces multiple breaks in the Lac operator
region, which elicits a more robust response, possible to study with immunofluorescence
region, which elicits a more robust response, possible to study with immunofluorescence
microscopy.
microscopy.
25
25
Results and Discussion
!
Results and Discussion
!
3 RESULTS AND DISCUSSION
3 RESULTS AND DISCUSSION
The four papers in this thesis are centered around the cohesin loading complex in different
The four papers in this thesis are centered around the cohesin loading complex in different
models and in different aspects of genome stability.
models and in different aspects of genome stability.
3.1 PAPER I
3.1 PAPER I
Requirements for DNA Double Strand Break Accumulation of Scc2, Similarities and
Requirements for DNA Double Strand Break Accumulation of Scc2, Similarities and
Differences with Cohesin
Differences with Cohesin
Previous chapters have described a central role for cohesin in maintenance of genome
Previous chapters have described a central role for cohesin in maintenance of genome
stability, not only for proper chromosome separation but also during DNA damage. It is clear
stability, not only for proper chromosome separation but also during DNA damage. It is clear
that it is not cohesin per se that is important, but cohesion establishment. It should however
that it is not cohesin per se that is important, but cohesion establishment. It should however
not be forgotten that both during an unchallenged cell cycle and under DNA damage
not be forgotten that both during an unchallenged cell cycle and under DNA damage
conditions, cohesion requires proper cohesin loading through the heterodimer complex
conditions, cohesion requires proper cohesin loading through the heterodimer complex
Scc2
NIPBL
Scc4
/MAU2
NIPBLScc2/MAU2Scc4.
.
Previous work has reported Scc2 localization at DSB (Ström, 2007), the requirements for this
Previous work has reported Scc2 localization at DSB (Ström, 2007), the requirements for this
are however still entirely unknown. The aim of this study was therefore to understand what is
are however still entirely unknown. The aim of this study was therefore to understand what is
needed for Scc2 binding at DNA damage.
needed for Scc2 binding at DNA damage.
We first characterized Scc2 recruitment at a HO-induced DSB with a ChIP-based quantitative
We first characterized Scc2 recruitment at a HO-induced DSB with a ChIP-based quantitative
assay (ChIP-qPCR). Our data showed that Scc2 binding is enhanced up to 30 kb from the
assay (ChIP-qPCR). Our data showed that Scc2 binding is enhanced up to 30 kb from the
break site, with a strong enrichment around 5 kb, compared to unchallenged cells. We next
break site, with a strong enrichment around 5 kb, compared to unchallenged cells. We next
addressed which of the factors reported to influence cohesin DSB binding, under similar
addressed which of the factors reported to influence cohesin DSB binding, under similar
DNA damage condition, that would also affect Scc2 DSB recruitment. We started our
DNA damage condition, that would also affect Scc2 DSB recruitment. We started our
analysis with a MRE11 deletion and not surprisingly, lack of this subunit of the MRX
analysis with a MRE11 deletion and not surprisingly, lack of this subunit of the MRX
complex, one of the early factors in HR, strongly reduced the Scc2 accumulation at the DNA
complex, one of the early factors in HR, strongly reduced the Scc2 accumulation at the DNA
DSB.
DSB.
We then tested the possibility that the effect observed in mre11Δ strain, is due to other factors
We then tested the possibility that the effect observed in mre11Δ strain, is due to other factors
dependent on the MRX complex. A TEL1 deletion also affected Scc2 binding at the DSB
dependent on the MRX complex. A TEL1 deletion also affected Scc2 binding at the DSB
negatively, and we thus speculated that this kinase, which is activated early upon DNA
negatively, and we thus speculated that this kinase, which is activated early upon DNA
damage, might either directly phosphorylate Scc2/4 or affect Scc2 levels through H2A
damage, might either directly phosphorylate Scc2/4 or affect Scc2 levels through H2A
phosphorylation. However, a mass spectrometry analysis performed on the purified loading
phosphorylation. However, a mass spectrometry analysis performed on the purified loading
27
27
Results and Discussion
Results and Discussion
complex did not show any phosphorylated S/TQ sites that might relate to Tel1. In addition,
complex did not show any phosphorylated S/TQ sites that might relate to Tel1. In addition,
the only Scc4 residue (T597) specifically modified in response to DNA damage, had no
the only Scc4 residue (T597) specifically modified in response to DNA damage, had no
growth defect in the presence of the genotoxic drug MMS when mutated.
growth defect in the presence of the genotoxic drug MMS when mutated.
Since the hta1-S129A and hta2-S129A mutations, preventing phosphorylation of H2A,
Since the hta1-S129A and hta2-S129A mutations, preventing phosphorylation of H2A,
showed similar results as the tel1Δ, it was possible to assume that the role of the Tel1 kinase
showed similar results as the tel1Δ, it was possible to assume that the role of the Tel1 kinase
is to create a scaffold of phosphorylated histones for recruitment of Scc2. However, it is not
is to create a scaffold of phosphorylated histones for recruitment of Scc2. However, it is not
possible to completely rule out the need for a direct phosphorylation on either Scc2 or Scc4,
possible to completely rule out the need for a direct phosphorylation on either Scc2 or Scc4,
and it would certainly be interesting to also test different approaches to address this
and it would certainly be interesting to also test different approaches to address this
possibility.
possibility.
Since H2A phosphorylation is dependent also on Mec1, we next addressed the role of this
Since H2A phosphorylation is dependent also on Mec1, we next addressed the role of this
second kinase, activated as part of the DNA damage response, and also necessary for cohesin
second kinase, activated as part of the DNA damage response, and also necessary for cohesin
binding at break sites (Ünal, 2004). However, unlike for cohesin, a MEC1 deletion has no
binding at break sites (Ünal, 2004). However, unlike for cohesin, a MEC1 deletion has no
effect on Scc2 localization at DSB. Considering that Mec1 is recruited to ssDNA bound by
effect on Scc2 localization at DSB. Considering that Mec1 is recruited to ssDNA bound by
RPA, and formed as a result of resection of DSB ends, we then tested the dependency of
RPA, and formed as a result of resection of DSB ends, we then tested the dependency of
initial end-processing for Scc2 recruitment. Start of resection of an HO DSB is independent
initial end-processing for Scc2 recruitment. Start of resection of an HO DSB is independent
of Mre11 endonuclease activity, but depends on Sae2. In a strain with SAE2 deletion
of Mre11 endonuclease activity, but depends on Sae2. In a strain with SAE2 deletion
background the Scc2 levels around the induced HO DSB are unchanged, thus resection has
background the Scc2 levels around the induced HO DSB are unchanged, thus resection has
no effect on Scc2 recruitment.
no effect on Scc2 recruitment.
This leads to some intriguing considerations, first of all it would be interesting to test if end-
This leads to some intriguing considerations, first of all it would be interesting to test if end-
resection is required for cohesin binding, which could explain the different effects observed
resection is required for cohesin binding, which could explain the different effects observed
in mec1Δ strains between cohesin and its loader. In this respect it should be remembered that
in mec1Δ strains between cohesin and its loader. In this respect it should be remembered that
Mec1 is recruited to DSBs upon start of resection. Another open question is why Mec1
Mec1 is recruited to DSBs upon start of resection. Another open question is why Mec1
affects cohesin loading at DSBs, but not Scc2 localization. It is possible to speculate that
affects cohesin loading at DSBs, but not Scc2 localization. It is possible to speculate that
Mec1 might be necessary to actively drive the cohesin loading process in case of DNA
Mec1 might be necessary to actively drive the cohesin loading process in case of DNA
damage, either acting directly on cohesin or indirectly on the loader or even on something
damage, either acting directly on cohesin or indirectly on the loader or even on something
else yet to be defined. This could mean that Mec1 is required to drive a specific DNA damage
else yet to be defined. This could mean that Mec1 is required to drive a specific DNA damage
dependent, postreplicative, cohesin loading, if not genome-wide at least around break sites.
dependent, postreplicative, cohesin loading, if not genome-wide at least around break sites.
In the last part of this paper we focused on elucidating, in more depth, the role of the MRX
In the last part of this paper we focused on elucidating, in more depth, the role of the MRX
complex. Since resection was shown to be unnecessary for Scc2 binding and Scc2 levels
complex. Since resection was shown to be unnecessary for Scc2 binding and Scc2 levels
were reduced in the absence of Xrs2, known to be required for Tel1 recruitment (Nakada,
were reduced in the absence of Xrs2, known to be required for Tel1 recruitment (Nakada,
2003), it is possible to assume that MRX is needed only to recruit other factors of the DNA
2003), it is possible to assume that MRX is needed only to recruit other factors of the DNA
damage response cascade. However, it should be noted that Xrs2 also partially affects the
damage response cascade. However, it should be noted that Xrs2 also partially affects the
28
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!
Results and Discussion
!
Results and Discussion
MRX bridging function (Trujillo, 2003) and reduced Scc2 binding at break sites in a rad50Δ
MRX bridging function (Trujillo, 2003) and reduced Scc2 binding at break sites in a rad50Δ
background seems to point in the direction of an active role for the MRX complex. It would
background seems to point in the direction of an active role for the MRX complex. It would
be interesting to observe a direct recruitment of Scc2 by the MRX complex, similar to the
be interesting to observe a direct recruitment of Scc2 by the MRX complex, similar to the
physical interaction between cohesin and MRN that has been reported in human cells (J. S.
physical interaction between cohesin and MRN that has been reported in human cells (J. S.
Kim, 2002), and if that is the case, which subunit of the MRX complex is involved.
Kim, 2002), and if that is the case, which subunit of the MRX complex is involved.
In conclusion, we would like to suggest that the MRX complex bridging activity and likely a
In conclusion, we would like to suggest that the MRX complex bridging activity and likely a
Tel1 interaction are required for Scc2 binding at DNA breaks. Moreover it is possible to
Tel1 interaction are required for Scc2 binding at DNA breaks. Moreover it is possible to
speculate on a model where Scc2 arrives early after damage induction, before resection starts,
speculate on a model where Scc2 arrives early after damage induction, before resection starts,
while cohesin is likely to be recruited to the DSB after resection is initiated, as can be inferred
while cohesin is likely to be recruited to the DSB after resection is initiated, as can be inferred
by the two different profiles of Scc2 and cohesin at DSBs. The first one is binding on the
by the two different profiles of Scc2 and cohesin at DSBs. The first one is binding on the
DNA sequence next to the break, similarly to Mre11, while cohesin is leaving a gap around
DNA sequence next to the break, similarly to Mre11, while cohesin is leaving a gap around
the DSB similarly to the pattern of H2A phosphorylation.
the DSB similarly to the pattern of H2A phosphorylation.
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Results and Discussion
Results and Discussion
3.2 PAPER II
3.2 PAPER II
Independent Mechanisms Recruit the Cohesin Loader Protein NIPBL to Sites of DNA
Independent Mechanisms Recruit the Cohesin Loader Protein NIPBL to Sites of DNA
Damage
Damage
In this study, similarly to Paper I, we aimed to understand the requirements for DNA
In this study, similarly to Paper I, we aimed to understand the requirements for DNA
damage recruitment of the cohesin loader. However here we investigated NIPBL, the human
damage recruitment of the cohesin loader. However here we investigated NIPBL, the human
ortholog of Scc2, thus shifting from budding yeast to human cell lines. Previous work has
ortholog of Scc2, thus shifting from budding yeast to human cell lines. Previous work has
described the recruitment of a transiently transfected, Halo tagged NIPBL at I-PpoI
described the recruitment of a transiently transfected, Halo tagged NIPBL at I-PpoI
dependent DSBs in the region of 28S rDNA. In Paper II on the other hand we used confocal
dependent DSBs in the region of 28S rDNA. In Paper II on the other hand we used confocal
microscopy paired with laser microirradiation and the FokI endonuclease for DNA damage
microscopy paired with laser microirradiation and the FokI endonuclease for DNA damage
induction. Moreover a system comprised of HEK-293 cells stably transfected with inducible
induction. Moreover a system comprised of HEK-293 cells stably transfected with inducible
GFP tagged NIPBL, through a tetracycline repressor system, was generated.
GFP tagged NIPBL, through a tetracycline repressor system, was generated.
We first showed in HEK-293 cells, that both NIPBL isoforms, A (316 kDa) and B (304 kDa),
We first showed in HEK-293 cells, that both NIPBL isoforms, A (316 kDa) and B (304 kDa),
are recruited to laser damage and FokI induced breaks.
are recruited to laser damage and FokI induced breaks.
A MAU2 GFP tagged trans-gene was also recruited to laser damage, but did not form visible
A MAU2 GFP tagged trans-gene was also recruited to laser damage, but did not form visible
foci in the FokI system, likely due to the fact that MAU2 requires NIPBL to localize to the
foci in the FokI system, likely due to the fact that MAU2 requires NIPBL to localize to the
nucleus, confirmed by the fact that the ample majority of ectopic MAU2 remains
nucleus, confirmed by the fact that the ample majority of ectopic MAU2 remains
cytoplasmic. Moreover, MAU2, at least in response to DNA damage, fails to act as chromatin
cytoplasmic. Moreover, MAU2, at least in response to DNA damage, fails to act as chromatin
adaptor of the loading complex, since NIPBL proteins carrying a missense mutation (glycine
adaptor of the loading complex, since NIPBL proteins carrying a missense mutation (glycine
15 to arginine) that disrupts the NIPBL-MAU2 interaction, still localized at H2AX foci, both
15 to arginine) that disrupts the NIPBL-MAU2 interaction, still localized at H2AX foci, both
at laser damage tracks and FokI induced breaks.
at laser damage tracks and FokI induced breaks.
We next tested whether a NIPBL mutation, located inside the HP1 binding motif localized in
We next tested whether a NIPBL mutation, located inside the HP1 binding motif localized in
the N-terminal part of NIPBL, had any affect on DNA damage localization of NIPBL, a
the N-terminal part of NIPBL, had any affect on DNA damage localization of NIPBL, a
PxVxL to PxAxA modification is known to abolish the interaction between NIPBL and HP1,
PxVxL to PxAxA modification is known to abolish the interaction between NIPBL and HP1,
and in doing so preventing NIPBL recruitment to I-PpoI induced DSB. We found that
and in doing so preventing NIPBL recruitment to I-PpoI induced DSB. We found that
PxAxA
did not form foci in the FokI system, but that recruitment to laser damage still
NIPBLPxAxA did not form foci in the FokI system, but that recruitment to laser damage still
occurred, which led us to think of an additional, HP1 independent, recruitment mechanism
occurred, which led us to think of an additional, HP1 independent, recruitment mechanism
for NIPBL. To further investigate and understand this dual mechanism multiple truncations
for NIPBL. To further investigate and understand this dual mechanism multiple truncations
of NIPBL were generated (Figure 6).
of NIPBL were generated (Figure 6).
NIPBL
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Results and Discussion
!
Results and Discussion
!
Domains & Motifs
NIPBLA
Domains & Motifs
Mau2 binding domain
N
HP1 motif
NIPBL
I
NIPBLA
Mau2 binding domain
N
HP1 motif
I
NIPBL
NIPBL
NLS rich region
NIPBL
NLS rich region
NIPBLII
HEAT repeat motif
NIPBLII
HEAT repeat motif
C
C
NIPBL
NIPBL
Figure 6: Scheme of the different NIPBL truncations used in Paper II. Different color boxes represent
domains and motifs according to the legend.
Figure 6: Scheme of the different NIPBL truncations used in Paper II. Different color boxes represent
domains and motifs according to the legend.
While all truncations were recruited to laser damage, only NIPBL fragments containing the
While all truncations were recruited to laser damage, only NIPBL fragments containing the
HP1 binding motif were recruited to FokI foci. This was also confirmed by depletion of HP1γ
HP1 binding motif were recruited to FokI foci. This was also confirmed by depletion of HP1γ
where the same NIPBL truncations did not localize at DNA damage.
where the same NIPBL truncations did not localize at DNA damage.
In order to better understand the nature of these independent recruitment pathways, we
In order to better understand the nature of these independent recruitment pathways, we
evaluated the role of ATM and ATR, known to be required, in yeast, for cohesin loading at
evaluated the role of ATM and ATR, known to be required, in yeast, for cohesin loading at
DNA damage. Using specific inhibitors for ATM (KU-60019) and ATR (AZD6738) we
DNA damage. Using specific inhibitors for ATM (KU-60019) and ATR (AZD6738) we
observed that single inhibition had no effect, while double inactivation of ATM and ATR
observed that single inhibition had no effect, while double inactivation of ATM and ATR
C
N
showed reduced recruitment of NIPBL but had no effect on NIPBL . It would be interesting
showed reduced recruitment of NIPBLC but had no effect on NIPBLN. It would be interesting
to understand the role of ATM/ATR on the cohesin loader further, if they act directly on
to understand the role of ATM/ATR on the cohesin loader further, if they act directly on
NIPBL or as part of a cascade involving additional factors? Certainly there are a few S/TQ
NIPBL or as part of a cascade involving additional factors? Certainly there are a few S/TQ
sites in NIPBL found phosphorylated in mass spectrometry that could be interesting to mutate
sites in NIPBL found phosphorylated in mass spectrometry that could be interesting to mutate
in order to test their effect in DNA damage recruitment (Stokes, 2007).
in order to test their effect in DNA damage recruitment (Stokes, 2007).
Following the same thread we wanted to investigate if the ubiquitin ligase pathway starting
Following the same thread we wanted to investigate if the ubiquitin ligase pathway starting
with RNF168, and reported to affect NIPBL recruitment to I-PpoI induced DNA breaks (Oka,
with RNF168, and reported to affect NIPBL recruitment to I-PpoI induced DNA breaks (Oka,
2011), had an effect on NIPBL recruitment in our systems. We showed that reduction of free
2011), had an effect on NIPBL recruitment in our systems. We showed that reduction of free
ubiquitin, obtained by inhibiting the proteasome activity with MG132 caused reduced
ubiquitin, obtained by inhibiting the proteasome activity with MG132 caused reduced
accumulation of NIPBLN and NIPBLC at laser damage lines, which is in line with a possible
accumulation of NIPBLN and NIPBLC at laser damage lines, which is in line with a possible
involvement of the ubiquitin ligases RNF8 and RNF168. Furthermore, cells depleted of
involvement of the ubiquitin ligases RNF8 and RNF168. Furthermore, cells depleted of
N
C
RNF8 and RNF168 failed to accumulate both NIPBL and NIPBL at DNA damage. Various
RNF8 and RNF168 failed to accumulate both NIPBLN and NIPBLC at DNA damage. Various
hypotheses can be formulated to explain the effect of RNF8/RNF168 depletion and
hypotheses can be formulated to explain the effect of RNF8/RNF168 depletion and
proteasome inhibition on the recruitment of NIPBL. A first explanation could be a direct
proteasome inhibition on the recruitment of NIPBL. A first explanation could be a direct
ubiquitination of NIPBL, which can be proven by ubiquitin pull-down coupled to NIPBL
ubiquitination of NIPBL, which can be proven by ubiquitin pull-down coupled to NIPBL
detection. A possible follow up would be to discover by MS the potentially modified
detection. A possible follow up would be to discover by MS the potentially modified
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31
Results and Discussion
Results and Discussion
residues, understanding the effect of ubiquitination by mutating the cohesin loader on such
residues, understanding the effect of ubiquitination by mutating the cohesin loader on such
residues and discover the E3 ligase responsible for the modification. A second possibility is
residues and discover the E3 ligase responsible for the modification. A second possibility is
the dependency of other factors in the RNF8/RNF168 cascade such as 53BP1 or RAP80, for
the dependency of other factors in the RNF8/RNF168 cascade such as 53BP1 or RAP80, for
DNA damage recruitment of NIPBL.
DNA damage recruitment of NIPBL.
In conclusion this paper shows a dual mechanism for NIPBL recruitment, one dependent on
In conclusion this paper shows a dual mechanism for NIPBL recruitment, one dependent on
HP1 and another dependent on ATM/ATR. Moreover we have described a link between
HP1 and another dependent on ATM/ATR. Moreover we have described a link between
ubiquitination and RNF8/RNF168, with NIPBL binding at DNA damage. There are of course
ubiquitination and RNF8/RNF168, with NIPBL binding at DNA damage. There are of course
many open questions regarding the dual mechanism for NIPBL damage recruitment. Does it
many open questions regarding the dual mechanism for NIPBL damage recruitment. Does it
depend on different types of damage? Or is it a redundant mechanism to guarantee that
depend on different types of damage? Or is it a redundant mechanism to guarantee that
NIPBL is recruited at DNA damage.
NIPBL is recruited at DNA damage.
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Results and Discussion
!
Results and Discussion
!
3.3 PAPER III
3.3 PAPER III
Inactivation of the Budding Yeast Cohesin Loader Scc2 alters Gene Expression both
Inactivation of the Budding Yeast Cohesin Loader Scc2 alters Gene Expression both
Globally and in Response to a Single DNA Double Strand Break.
Globally and in Response to a Single DNA Double Strand Break.
In Paper III we aimed to understand if Scc2 influenced gene expression in budding yeast, in
In Paper III we aimed to understand if Scc2 influenced gene expression in budding yeast, in
the presence and absence of DNA damage.
the presence and absence of DNA damage.
To achieve that we made use of a microarray, testing over 5800 open reading frames (ORFs)
To achieve that we made use of a microarray, testing over 5800 open reading frames (ORFs)
comparing wild type cells with a strain containing a temperature sensitive allele for Scc2, in
comparing wild type cells with a strain containing a temperature sensitive allele for Scc2, in
the presence or absence of DNA damage, in form of a single DSB on chromosome VI (Figure
the presence or absence of DNA damage, in form of a single DSB on chromosome VI (Figure
7 experiment 1).
7 experiment 1).
The transcriptional profiles showed that when comparing Scc2-deficient cells to wild type,
The transcriptional profiles showed that when comparing Scc2-deficient cells to wild type,
both in the presence or absence of DNA damage, 754 and 567 probe sets respectively, were
both in the presence or absence of DNA damage, 754 and 567 probe sets respectively, were
significantly affected. However 399 probe sets that showed differential expression were in
significantly affected. However 399 probe sets that showed differential expression were in
common with or without DSB, which left 168 probe sets uniquely affected in response to
common with or without DSB, which left 168 probe sets uniquely affected in response to
DNA damage and 355 with lack of DNA damage, when comparing wild type and scc2
DNA damage and 355 with lack of DNA damage, when comparing wild type and scc2
deficient cells.
deficient cells.
Experiment 1
- break
+ break
WT
WT
vs
vs
Scc2ts
Scc2ts
Experiment 2
- break
WT
vs
Experiment 3
+ break
- break
WT
Scc2ts
Experiment 1
+ break
vs
Scc2ts
- break
+ break
WT
WT
vs
vs
Scc2ts
Scc2ts
Experiment 2
- break
WT
vs
Experiment 3
+ break
- break
WT
Scc2ts
+ break
vs
Scc2ts
Figure 7: Three different microarray analysis with the experimental conditions used.
Figure 7: Three different microarray analysis with the experimental conditions used.
Genes with FDR≤0.05 were considered to significantly deviate from the expected genome
Genes with FDR≤0.05 were considered to significantly deviate from the expected genome
frequency. Affected genes were then categorized based on biological process using the
frequency. Affected genes were then categorized based on biological process using the
Saccharomyces Genome Data base Gene Ontology (SGD GO) slim mapping. Our findings
Saccharomyces Genome Data base Gene Ontology (SGD GO) slim mapping. Our findings
showed that even though a majority of genes with altered expression, between scc2-4 and
showed that even though a majority of genes with altered expression, between scc2-4 and
wild type cells, were not involved in break induction, three pieces of evidence pointed to
wild type cells, were not involved in break induction, three pieces of evidence pointed to
Scc2 also affecting the transcriptional response caused by DNA damage. First, the number of
Scc2 also affecting the transcriptional response caused by DNA damage. First, the number of
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Results and Discussion
Results and Discussion
affected genes was higher in wild type than in the scc2 mutant in the presence of DNA
affected genes was higher in wild type than in the scc2 mutant in the presence of DNA
damage, thus cells lacking functional Scc2 are likely incapable of proper DNA damage
damage, thus cells lacking functional Scc2 are likely incapable of proper DNA damage
dependent transcriptional changes. Moreover when looking at the profiles and comparing
dependent transcriptional changes. Moreover when looking at the profiles and comparing
presence or absence of DNA break, from cells lacking functional Scc2, the down-regulated
presence or absence of DNA break, from cells lacking functional Scc2, the down-regulated
genes belonged to categories such as; enhanced processes for “DNA damage”, “DNA repair“
genes belonged to categories such as; enhanced processes for “DNA damage”, “DNA repair“
and “DNA recombination”. Wild type cells on the contrary, had several up-regulated genes of
and “DNA recombination”. Wild type cells on the contrary, had several up-regulated genes of
the DNA damage response but only in the presence of the DSB. In order to better understand
the DNA damage response but only in the presence of the DSB. In order to better understand
the effect of Scc2 in the DNA damage transcriptional response it was necessary to study lack
the effect of Scc2 in the DNA damage transcriptional response it was necessary to study lack
of Scc2 in isolation, comparing presence or absence of HO induction, without including wild
of Scc2 in isolation, comparing presence or absence of HO induction, without including wild
type cells in the same experiment, since it was evident from the initial experiment that the
type cells in the same experiment, since it was evident from the initial experiment that the
transcriptional profiles from wild type and Scc2 deficient cells were so different that the
transcriptional profiles from wild type and Scc2 deficient cells were so different that the
alteration in gene transcription caused by a single DSB in Scc2 deficient cells were then
alteration in gene transcription caused by a single DSB in Scc2 deficient cells were then
masked.
masked.
In order to do that it was necessary to make sure, that a single break on chromosome VI was
In order to do that it was necessary to make sure, that a single break on chromosome VI was
enough to cause a typical transcriptional change in DNA repair related genes, similarly to
enough to cause a typical transcriptional change in DNA repair related genes, similarly to
what has been previously reported for other DNA damage inducing agents. We thus initially
what has been previously reported for other DNA damage inducing agents. We thus initially
tested the effect of the single DSB on wild type cells (Figure 7 experiments 2). Our findings
tested the effect of the single DSB on wild type cells (Figure 7 experiments 2). Our findings
showed that both the number (113) and type of affected genes were in accordance with
showed that both the number (113) and type of affected genes were in accordance with
existing data.
existing data.
Since our system reflected a standard DNA damage response situation, we analyzed the effect
Since our system reflected a standard DNA damage response situation, we analyzed the effect
of DNA damage on gene expression in scc2-4 cells and found 976 altered genes (Figure 7
of DNA damage on gene expression in scc2-4 cells and found 976 altered genes (Figure 7
experiment 3). Many of the traditionally induced genes of the DNA damage response were
experiment 3). Many of the traditionally induced genes of the DNA damage response were
upregulated, similarly to what was observed in wild type cells of the previous experiment (2).
upregulated, similarly to what was observed in wild type cells of the previous experiment (2).
However a clear difference for genes of the cohesin network could be detected between wild
However a clear difference for genes of the cohesin network could be detected between wild
type and Scc2-4 cells.
type and Scc2-4 cells.
We then further analyzed the two data sets (experiments 2 and 3) using SGD GO slim
We then further analyzed the two data sets (experiments 2 and 3) using SGD GO slim
mapping. As expected in wild type cells the most enhanced processes were “cellular response
mapping. As expected in wild type cells the most enhanced processes were “cellular response
to DNA damage stimulus” and “DNA repair”. For the Scc2 mutant cells however, even
to DNA damage stimulus” and “DNA repair”. For the Scc2 mutant cells however, even
though some of the genes classically induced upon DNA damage were upregulated, none of
though some of the genes classically induced upon DNA damage were upregulated, none of
the processes enhanced in wild type cells could be observed. Instead other processes were
the processes enhanced in wild type cells could be observed. Instead other processes were
affected, like “response to chemical stimuli”, “oxidative stress” and “starvation”. Moreover
affected, like “response to chemical stimuli”, “oxidative stress” and “starvation”. Moreover
ribosome production was impaired in the scc2-4 cells. Very interestingly, a similar effect was
ribosome production was impaired in the scc2-4 cells. Very interestingly, a similar effect was
observed in a zebrafish model for CdLS and in Eco1 mutants, pointing out that the cohesin
observed in a zebrafish model for CdLS and in Eco1 mutants, pointing out that the cohesin
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!
Results and Discussion
!
Results and Discussion
network can be responsible for the ribosomal processes by affecting rRNA production (Lu,
network can be responsible for the ribosomal processes by affecting rRNA production (Lu,
2014; B. Xu, 2015).
2014; B. Xu, 2015).
In conclusion, it appears that Scc2 seems to have a role in maintaining gene regulation across
In conclusion, it appears that Scc2 seems to have a role in maintaining gene regulation across
the genome, in line with other results both for metazoans and also for yeast (Lopez-Serra,
the genome, in line with other results both for metazoans and also for yeast (Lopez-Serra,
2014). It is not yet clear however if in our case this function is independent of cohesin and
2014). It is not yet clear however if in our case this function is independent of cohesin and
might relate to the fact that Scc2 binds active promoters, or is a consequence of altered
might relate to the fact that Scc2 binds active promoters, or is a consequence of altered
cohesin binding.
cohesin binding.
Our ChIP-seq maps, where we compared Scc1 chromatin association genome wide in
Our ChIP-seq maps, where we compared Scc1 chromatin association genome wide in
unchallenged cells versus after induction of DNA damage, did not display any difference. It
unchallenged cells versus after induction of DNA damage, did not display any difference. It
should be noted however that the binding pattern reflects binding of Scc1 in pre-replication
should be noted however that the binding pattern reflects binding of Scc1 in pre-replication
loaded cohesin as well as complexes loaded in response to DNA damage. It would have been
loaded cohesin as well as complexes loaded in response to DNA damage. It would have been
more relevant to look at G2 specific break induced cohesin loading. Even though genome
more relevant to look at G2 specific break induced cohesin loading. Even though genome
wide cohesin binding did not change upon Scc2 inactivation, cohesin surrounding the break
wide cohesin binding did not change upon Scc2 inactivation, cohesin surrounding the break
site was affected. As previously reported genes next to the break had reduced expression in
site was affected. As previously reported genes next to the break had reduced expression in
wild type cells, after break induction, but only three out of six genes close to the break as
wild type cells, after break induction, but only three out of six genes close to the break as
tested by qRT-PCR, were repressed upon DNA damage in scc2-4 cells. This result might
tested by qRT-PCR, were repressed upon DNA damage in scc2-4 cells. This result might
indicate a possible effect of cohesin and its loader in silencing of gene expression around the
indicate a possible effect of cohesin and its loader in silencing of gene expression around the
break.
break.
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Results and Discussion
Results and Discussion
3.4 PAPER IV
3.4 PAPER IV
Localisation of the SMC loading complex Nipbl/Mau2 during Mammalian Meiotic
Localisation of the SMC loading complex Nipbl/Mau2 during Mammalian Meiotic
Prophase I
Prophase I
SMC complexes are master regulators of chromosome stability, not only in mitosis but also
SMC complexes are master regulators of chromosome stability, not only in mitosis but also
during meiosis. Still very little is known about the role of the cohesin loader NIPBL/MAU2
during meiosis. Still very little is known about the role of the cohesin loader NIPBL/MAU2
in meiosis.
in meiosis.
In Paper IV we investigated the NIPBL/MAU2 chromosomal localization during both male
In Paper IV we investigated the NIPBL/MAU2 chromosomal localization during both male
and female mouse meiosis. Moreover, by using a mouse model for CdLS we aimed at
and female mouse meiosis. Moreover, by using a mouse model for CdLS we aimed at
addressing the effect of lacking one copy of Nipbl.
addressing the effect of lacking one copy of Nipbl.
Staining of Nipbl and Mau2 in spermatocytes from testicular spreads showed an increasing
Staining of Nipbl and Mau2 in spermatocytes from testicular spreads showed an increasing
intensity during prophase I, and relocation from chromosomal axes in leptotene to
intensity during prophase I, and relocation from chromosomal axes in leptotene to
chromocentres in mid-pachytene. In oocytes derived from female embryos between E16.5
chromocentres in mid-pachytene. In oocytes derived from female embryos between E16.5
and E19.5 displayed a similar initial Nipbl and Mau2 distribution, with the clear difference
and E19.5 displayed a similar initial Nipbl and Mau2 distribution, with the clear difference
that the complex was retained on chromosome axes during pachytene and showed a diffuse
that the complex was retained on chromosome axes during pachytene and showed a diffuse
staining in the nucleus, unseen in male meiosis. This difference is quite interesting and it is
staining in the nucleus, unseen in male meiosis. This difference is quite interesting and it is
tempting to explain it with a mechanism that requires Nipbl/Mau2 to maintain cohesion
tempting to explain it with a mechanism that requires Nipbl/Mau2 to maintain cohesion
during dictyate, a condition where oocytes can stay for months.
during dictyate, a condition where oocytes can stay for months.
The loading complex showed no co-localization with the meiosis specific cohesin subunit
The loading complex showed no co-localization with the meiosis specific cohesin subunit
Smc1β, moreover it was interesting to note that cohesin, but not NIPBL, remained on
Smc1β, moreover it was interesting to note that cohesin, but not NIPBL, remained on
chromosome axes after pachytene.
chromosome axes after pachytene.
In spermatocytes derived from Nipbl+/- mice, a reduction of Nipbl levels in both western blot
In spermatocytes derived from Nipbl+/- mice, a reduction of Nipbl levels in both western blot
and staining intensity was observed. However Nipbl binding was only partly affected and
and staining intensity was observed. However Nipbl binding was only partly affected and
cohesin was loaded as in wild type. We could conclude that lack of one functional copy of
cohesin was loaded as in wild type. We could conclude that lack of one functional copy of
Nipbl did not affect cohesin loading as in somatic cells and certainly had no effect on
Nipbl did not affect cohesin loading as in somatic cells and certainly had no effect on
maintaining cohesin binding at chromosomal stages later than zygotene. A similar effect was
maintaining cohesin binding at chromosomal stages later than zygotene. A similar effect was
also true for other SMC complexes, neither condensin nor Smc5/6 staining changed in mutant
also true for other SMC complexes, neither condensin nor Smc5/6 staining changed in mutant
mice. This lack of difference can possibly be explained for condensin and Smc5/6, with the
mice. This lack of difference can possibly be explained for condensin and Smc5/6, with the
fact that Nipbl might have no impact on loading of other SMC complexes than cohesin in
fact that Nipbl might have no impact on loading of other SMC complexes than cohesin in
mouse meiosis. Certainly more surprising is the lack of effect on cohesin, however this can be
mouse meiosis. Certainly more surprising is the lack of effect on cohesin, however this can be
due to the fact that cohesin loading, at least for chromosome segregation, is unaffected by
due to the fact that cohesin loading, at least for chromosome segregation, is unaffected by
Nipbl gene dosage. Another possible reason is that variations are too small to be detected
Nipbl gene dosage. Another possible reason is that variations are too small to be detected
with immunofluorescence.
with immunofluorescence.
36
36
Results and Discussion
!
!
Results and Discussion
In Sycp1-/- and Sycp3-/- male and female germ cells, chromosomal axes are more
In Sycp1-/- and Sycp3-/- male and female germ cells, chromosomal axes are more
disorganized and Nipbl/Mau2 had weaker intensity on the axes and were more diffuse in the
disorganized and Nipbl/Mau2 had weaker intensity on the axes and were more diffuse in the
nuclei. However Nipbl binding could still be observed even in the absence of SC
nuclei. However Nipbl binding could still be observed even in the absence of SC
components, possibly indicating that NIPBL binds prior to the SC formation.
components, possibly indicating that NIPBL binds prior to the SC formation.
Given the role of cohesin and its loader in DSB repair we checked the localization of γH2AX
Given the role of cohesin and its loader in DSB repair we checked the localization of γH2AX
and Rad51 in relation to Nipbl. It appeared that the majority of the loader bound
and Rad51 in relation to Nipbl. It appeared that the majority of the loader bound
independently of these DNA damage and recombination markers. This held true in both wild
independently of these DNA damage and recombination markers. This held true in both wild
+/-
mutant germ cells as well as in MEFs with irradiation induced γH2AX foci.
type and Nipbl+/- mutant germ cells as well as in MEFs with irradiation induced γH2AX foci.
However an interesting difference was observed between wild type and Nipbl+/-
However an interesting difference was observed between wild type and Nipbl+/-
spermatocytes. From late zygotene γH2AX staining normally starts to disappear from the
spermatocytes. From late zygotene γH2AX staining normally starts to disappear from the
type and Nipbl
+/-
spermatocytes however, γH2AX
nucleus and is visible only at the sex body. In Nipbl+/- spermatocytes however, γH2AX
nuclear staining was still strong even at mid-pachytene. This difference, together with the fact
nuclear staining was still strong even at mid-pachytene. This difference, together with the fact
that reduction of Smc1β results in irregular γH2AX foci at late prophase, can mean that
that reduction of Smc1β results in irregular γH2AX foci at late prophase, can mean that
unlike canonical cohesin loading, localization of cohesin at meiotic DSBs is affected by
unlike canonical cohesin loading, localization of cohesin at meiotic DSBs is affected by
Nipbl dosage. However DNA repair defects were not on the same level as in mitotic cells
Nipbl dosage. However DNA repair defects were not on the same level as in mitotic cells
with altered γH2AX organization (Vrouwe, 2007). This could point to the possibility that
with altered γH2AX organization (Vrouwe, 2007). This could point to the possibility that
meiotic cells can somehow protect themselves from the reduced Nipbl levels. An additional
meiotic cells can somehow protect themselves from the reduced Nipbl levels. An additional
nucleus and is visible only at the sex body. In Nipbl
+/-
indication is that lethality in Nipbl mice, as previously reported, is a post-natal event, taking
indication is that lethality in Nipbl+/- mice, as previously reported, is a post-natal event, taking
place between conception and weaning (Kawauchi, 2009) while it seems that the majority of
place between conception and weaning (Kawauchi, 2009) while it seems that the majority of
adult male mice are fertile. Lack of offspring for certain animals might depend on physical or
adult male mice are fertile. Lack of offspring for certain animals might depend on physical or
behavioral alteration.
behavioral alteration.
37
37
Future Perspective
!
4 FUTURE PERSPECTIVE
Future Perspective
!
4 FUTURE PERSPECTIVE
4.1 HOW DOES THE LOADER WORK
4.1 HOW DOES THE LOADER WORK
Even though the importance of NIPBLScc2 is apparent, its mechanism of action is, for most
Even though the importance of NIPBLScc2 is apparent, its mechanism of action is, for most
parts, still unclear. Over the years significant details were uncovered, such as the possible
parts, still unclear. Over the years significant details were uncovered, such as the possible
entry gate of cohesin or the effect of ATP hydrolysis in modifying the cohesin ring
entry gate of cohesin or the effect of ATP hydrolysis in modifying the cohesin ring
conformation to allow DNA entry. The most important question however still remains: how
conformation to allow DNA entry. The most important question however still remains: how
cohesin is loaded onto DNA.
cohesin is loaded onto DNA.
Previous works have shown the necessity for ATP hydrolysis in cohesin loading (Hu, 2011),
Previous works have shown the necessity for ATP hydrolysis in cohesin loading (Hu, 2011),
moreover it appears that the cohesin loader stimulates the reaction carried out by the cohesin
moreover it appears that the cohesin loader stimulates the reaction carried out by the cohesin
HEAD domain (Murayama, 2014). A possible model for loading is that ATP hydrolysis
HEAD domain (Murayama, 2014). A possible model for loading is that ATP hydrolysis
induces conformational changes in the coiled-coiled domain of the SMC proteins ultimately
induces conformational changes in the coiled-coiled domain of the SMC proteins ultimately
leading to the opening of the HINGE (Gruber, 2006). However, recent work has proposed a
leading to the opening of the HINGE (Gruber, 2006). However, recent work has proposed a
different model of loading in which entry and exit gates coincide in the region of contact
different model of loading in which entry and exit gates coincide in the region of contact
between the kleisin subunit and Smc3. A conformational change induced by the loader will
between the kleisin subunit and Smc3. A conformational change induced by the loader will
turn the cohesin ring “inside out” in order for the residues responsible of sensing DNA to
turn the cohesin ring “inside out” in order for the residues responsible of sensing DNA to
contact the nucleic acid. This is possible thanks to the interaction of Scc2 with the HINGE of
contact the nucleic acid. This is possible thanks to the interaction of Scc2 with the HINGE of
Smc3 (Murayama, 2015). Not unlikely more clues are hidden in the Scc2 structure, yet to be
Smc3 (Murayama, 2015). Not unlikely more clues are hidden in the Scc2 structure, yet to be
resolved.
resolved.
Recent studies have managed to obtain Pds5 crystals (B.-G. Lee, 2016; Muir, 2016; Ouyang,
Recent studies have managed to obtain Pds5 crystals (B.-G. Lee, 2016; Muir, 2016; Ouyang,
2016), the cohesin associated protein that shares with Scc2 a repetition of HEAT domains.
2016), the cohesin associated protein that shares with Scc2 a repetition of HEAT domains.
This similarity might be of help in resolving the structure of Scc2 and/or NIPBL which can
This similarity might be of help in resolving the structure of Scc2 and/or NIPBL which can
give useful information regarding the region of contacts between cohesin and its loader, since
give useful information regarding the region of contacts between cohesin and its loader, since
the core ring structure of cohesin is well characterized. As a matter of fact the domains in the
the core ring structure of cohesin is well characterized. As a matter of fact the domains in the
cohesin subunits that bind the loader are known in S. pombe, but not vice versa. Mutation
cohesin subunits that bind the loader are known in S. pombe, but not vice versa. Mutation
analysis of residues with functional importance can tell us more of the conformational
analysis of residues with functional importance can tell us more of the conformational
changes induced by the loader and the effect on ATP hydrolysis.
changes induced by the loader and the effect on ATP hydrolysis.
Understanding the mechanism of Scc2 in S. cerevisiae can obviously be of great importance
Understanding the mechanism of Scc2 in S. cerevisiae can obviously be of great importance
also for NIPBL studies. As mentioned before, the human cohesin loader has likely additional
also for NIPBL studies. As mentioned before, the human cohesin loader has likely additional
functions, or forms of regulation, that mirror the necessity of a genome with a higher degree
functions, or forms of regulation, that mirror the necessity of a genome with a higher degree
of complexity. By understanding the conserved loading process, and the regions of the
of complexity. By understanding the conserved loading process, and the regions of the
39
39
Future Perspective
Future Perspective
protein that are involved in it, it will be possible to dissect the additional roles of NIPBL,
protein that are involved in it, it will be possible to dissect the additional roles of NIPBL,
potentially the most interesting will be those important for gene regulation.
potentially the most interesting will be those important for gene regulation.
One crucial region of the NIPBLScc2 protein is the C-terminal that has been suggested to be
One crucial region of the NIPBLScc2 protein is the C-terminal that has been suggested to be
the cohesin-binding region (Kogut, 2009). In our case tagging of NIPBL at the C-terminal
the cohesin-binding region (Kogut, 2009). In our case tagging of NIPBL at the C-terminal
greatly destabilized the protein, which seems to strengthen this possibility. Thus either the C-
greatly destabilized the protein, which seems to strengthen this possibility. Thus either the C-
terminal end is functionally important, or a highly ordered structure is destroyed by addition
terminal end is functionally important, or a highly ordered structure is destroyed by addition
of an affinity tag. A similar effect is however not seen for Scc2, despite that it is the C-
of an affinity tag. A similar effect is however not seen for Scc2, despite that it is the C-
terminal part that is more conserved. It would certainly be interesting to better understand this
terminal part that is more conserved. It would certainly be interesting to better understand this
difference and define precisely what region of the protein that is required for proper cohesin
difference and define precisely what region of the protein that is required for proper cohesin
loading.
loading.
Another interesting fact is that cohesion seems independent of the available concentration of
Another interesting fact is that cohesion seems independent of the available concentration of
NIPBL in the cell (Castronovo, 2009). At the same time NIPBL haploinsufficiency causes
NIPBL in the cell (Castronovo, 2009). At the same time NIPBL haploinsufficiency causes
multiple and serious developmental defects (Krantz, 2004; Tonkin, 2004). Even more
multiple and serious developmental defects (Krantz, 2004; Tonkin, 2004). Even more
surprisingly also duplication of NIPBL leads to disease (Novara, 2013), even though with a
surprisingly also duplication of NIPBL leads to disease (Novara, 2013), even though with a
different phenotype than CdLS. This seems to strengthen the possibility that NIPBL is
different phenotype than CdLS. This seems to strengthen the possibility that NIPBL is
required in precise amount, and possibly not acting simply as the cohesin loader.
required in precise amount, and possibly not acting simply as the cohesin loader.
4.2 THE MYSTERY PROTEIN: MAU2SCC4
4.2 THE MYSTERY PROTEIN: MAU2SCC4
Another intriguing issue concerns MAU2Scc4; to this date it is still not clear what the true
Another intriguing issue concerns MAU2Scc4; to this date it is still not clear what the true
function of this second subunit of the cohesin loader is.
function of this second subunit of the cohesin loader is.
MAU2Scc4 is an essential protein, however it is quite interesting to notice that no CdLS
MAU2Scc4 is an essential protein, however it is quite interesting to notice that no CdLS
patients with MAU2 mutations have been found so far. Is this because MAU2 has no relevant
patients with MAU2 mutations have been found so far. Is this because MAU2 has no relevant
function in the complex, or during development? Or is it possibly so that MAU2 mutations
function in the complex, or during development? Or is it possibly so that MAU2 mutations
completely impair the activity of the cohesin loader? determining prenatal death. Or do we
completely impair the activity of the cohesin loader? determining prenatal death. Or do we
need to consider the possibility that MAU2 mutations can lead to a disease with a completely
need to consider the possibility that MAU2 mutations can lead to a disease with a completely
different phenotype than CdLS?
different phenotype than CdLS?
The only CdLS mutation that can somehow relate to this situation is the NIPBL G15R
The only CdLS mutation that can somehow relate to this situation is the NIPBL G15R
mutation that disrupts the interaction between NIPBL and MAU2 (Braunholz, 2012). In
mutation that disrupts the interaction between NIPBL and MAU2 (Braunholz, 2012). In
Paper II we have shown the effect of this mutation in NIPBL recruitment in case of DNA
Paper II we have shown the effect of this mutation in NIPBL recruitment in case of DNA
damage, however it should be remembered that G15R causes CdLS with a mild phenotype
damage, however it should be remembered that G15R causes CdLS with a mild phenotype
without heart and limbs defects, and it is possible that some residual interaction, not
without heart and limbs defects, and it is possible that some residual interaction, not
detectable with yeast-two-hybrid or western blot is enough to guarantee the complex
detectable with yeast-two-hybrid or western blot is enough to guarantee the complex
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Future Perspective
!
Future Perspective
!
function. A more in depth characterization of this mutation, or additional ones affecting the
function. A more in depth characterization of this mutation, or additional ones affecting the
interaction between NIPBL and MAU2, would be very interesting to carry out in order to
interaction between NIPBL and MAU2, would be very interesting to carry out in order to
better elucidate the relationship between MAU2 and NIPBL.
better elucidate the relationship between MAU2 and NIPBL.
It is quite intriguing that an in vitro study on S. pombe Scc4 showed that it does not
It is quite intriguing that an in vitro study on S. pombe Scc4 showed that it does not
participate in any of the reported functions of the loader, neither ATP hydrolysis, nor DNA
participate in any of the reported functions of the loader, neither ATP hydrolysis, nor DNA
binding (Murayama, 2014). Another report in budding yeast did however show that Scc4 is
binding (Murayama, 2014). Another report in budding yeast did however show that Scc4 is
required for cohesin loading at centromeres, and it would be interesting to see if similar
required for cohesin loading at centromeres, and it would be interesting to see if similar
results can be obtained also in human. It is possible to speculate that the main function of
results can be obtained also in human. It is possible to speculate that the main function of
Scc4
is to maintain the structure of the loading complex, or help the folding of
MAU2Scc4 is to maintain the structure of the loading complex, or help the folding of
NIPBLScc2. To support this hypothesis it should be remembered that MAU2 has no nuclear
NIPBLScc2. To support this hypothesis it should be remembered that MAU2 has no nuclear
localization signal and that the formation of the complex should take place immediately after
localization signal and that the formation of the complex should take place immediately after
protein synthesis. The recently published structure of Scc4 can be important to address all
protein synthesis. The recently published structure of Scc4 can be important to address all
these questions. Even though the small degree of conservation between Scc4 and MAU2
these questions. Even though the small degree of conservation between Scc4 and MAU2
might make these studies difficult to transpose from yeast to human.
might make these studies difficult to transpose from yeast to human.
More data collected from Scc4 mutants can give us information regarding its interaction with
More data collected from Scc4 mutants can give us information regarding its interaction with
kinetochore subunits or the need for histone modifications that can explain the cohesin loader
kinetochore subunits or the need for histone modifications that can explain the cohesin loader
recruitment at centromeres. More effort should also be put on finding possible suppressor
recruitment at centromeres. More effort should also be put on finding possible suppressor
mutants that can rescue an Scc4 deletion or temperature sensitive allele, which can point to
mutants that can rescue an Scc4 deletion or temperature sensitive allele, which can point to
other cellular pathways required for cohesin loading.
other cellular pathways required for cohesin loading.
MAU2
4.3 REMODELLING, TRANSCRIPTION AND COHESIN LOADING
4.3 REMODELLING, TRANSCRIPTION AND COHESIN LOADING
A recent study showed a strong correlation between DNA binding of the cohesin loader and
A recent study showed a strong correlation between DNA binding of the cohesin loader and
chromatin remodeling by the RSC complex (Lopez-Serra, 2014). This finding opens to
chromatin remodeling by the RSC complex (Lopez-Serra, 2014). This finding opens to
various speculations. First of all is this mechanism conserved in metazoans? The fact that the
various speculations. First of all is this mechanism conserved in metazoans? The fact that the
Coffin-Siris Syndrome, caused by mutations in the gene encoding the human ortholog of
Coffin-Siris Syndrome, caused by mutations in the gene encoding the human ortholog of
RSC, has a similar phenotype as CdLS patients points in this direction. Finding CdLS
RSC, has a similar phenotype as CdLS patients points in this direction. Finding CdLS
patients lacking NIPBL, but carrying mutations in the chromatin remodeler genes, or Coffin-
patients lacking NIPBL, but carrying mutations in the chromatin remodeler genes, or Coffin-
Siris Syndrome patients with NIPBL mutations would strengthen this concept.
Siris Syndrome patients with NIPBL mutations would strengthen this concept.
This could also provide information on the relationship between the two complexes. Are they
This could also provide information on the relationship between the two complexes. Are they
physically interacting, and if so, through which subunits and protein regions? Again structural
physically interacting, and if so, through which subunits and protein regions? Again structural
information on Scc2 might help addressing these questions. Regardless, this link could
information on Scc2 might help addressing these questions. Regardless, this link could
strengthen the concept that cohesin is translocated to binding sites by transcription. However
strengthen the concept that cohesin is translocated to binding sites by transcription. However
41
41
Future Perspective
Future Perspective
this model still requires clarification. For example, some evidences point out the fact that
this model still requires clarification. For example, some evidences point out the fact that
cohesin sliding is strongly reduced by obstacles on DNA such as nucleosomes (Stigler,
cohesin sliding is strongly reduced by obstacles on DNA such as nucleosomes (Stigler,
2016). Moreover the RSC complex, more specifically Rsc2 and Rsc7, were found to be
2016). Moreover the RSC complex, more specifically Rsc2 and Rsc7, were found to be
important for cohesin loading at an HO-induced break site (Oum, 2011). The connection
important for cohesin loading at an HO-induced break site (Oum, 2011). The connection
between cohesin binding via the RSC complex, at DNA damage and during the unchallenged
between cohesin binding via the RSC complex, at DNA damage and during the unchallenged
cell cycle seems clear and it is very possible that the described effect depends on Scc2. It
cell cycle seems clear and it is very possible that the described effect depends on Scc2. It
would still be interesting to see which of the two existing RSC complexes that affect cohesin
would still be interesting to see which of the two existing RSC complexes that affect cohesin
and its loader. The other intriguing possibility involves transcription and DNA damage. Loss
and its loader. The other intriguing possibility involves transcription and DNA damage. Loss
of transcription can be observed in the vicinity of a DSB due to resection (Manfrini, 2015). A
of transcription can be observed in the vicinity of a DSB due to resection (Manfrini, 2015). A
model for cohesin sliding via the transcription machinery is valid also during a DNA damage
model for cohesin sliding via the transcription machinery is valid also during a DNA damage
response? Thus, the similarity between the cohesin binding profile and H2A phosphorylation
response? Thus, the similarity between the cohesin binding profile and H2A phosphorylation
around the break is very intriguing. Does the gap in localization of the two in direct vicinity
around the break is very intriguing. Does the gap in localization of the two in direct vicinity
of the cut-site depend on resection, and the consequent lack of transcription? On the other
of the cut-site depend on resection, and the consequent lack of transcription? On the other
hand why is Scc2 then located directly on the cut-site?
hand why is Scc2 then located directly on the cut-site?
4.4 FINAL REMARKS
4.4 FINAL REMARKS
Soon the cohesin field will celebrate 20 years from the discovery of the complex that holds
Soon the cohesin field will celebrate 20 years from the discovery of the complex that holds
sister chromatids together. It is a relatively recent field of research however outstanding steps
sister chromatids together. It is a relatively recent field of research however outstanding steps
forward were made to understand one of the basic and yet so important mechanisms of life.
forward were made to understand one of the basic and yet so important mechanisms of life.
Still there is more to discover: the role of cohesin in DNA damage, in gene regulation, and
Still there is more to discover: the role of cohesin in DNA damage, in gene regulation, and
the correlation with replication and topology to mention some processes where the cohesin
the correlation with replication and topology to mention some processes where the cohesin
network has been suggested to play important roles.
network has been suggested to play important roles.
This thesis describes different forms of action of the cohesin loader in different cellular
This thesis describes different forms of action of the cohesin loader in different cellular
processes. More data should be collected from mutants of conserved residues with a
processes. More data should be collected from mutants of conserved residues with a
functional importance in cohesin, NIPBL
Scc2
Scc4
and MAU2
in order to dissect the various
functional importance in cohesin, NIPBLScc2 and MAU2Scc4 in order to dissect the various
steps in the loading process or the different roles of each protein.
steps in the loading process or the different roles of each protein.
To select these mutants, large screenings utilizing protein arrays, studies of crystal structure,
To select these mutants, large screenings utilizing protein arrays, studies of crystal structure,
and deletion libraries should be carried out.
and deletion libraries should be carried out.
42
42
!
Acknowledgements
!
Acknowledgements
5 ACKNOWLEDGEMENTS
5 ACKNOWLEDGEMENTS
There are certainly many people I should thank and likely I have not enough space, so I
There are certainly many people I should thank and likely I have not enough space, so I
would like to thank all the present and past people of the department, the CHaSE members,
would like to thank all the present and past people of the department, the CHaSE members,
the DSA and all my mates from university. Thank you.
the DSA and all my mates from university. Thank you.
Lena: Thank you for taking me in as your student and for all this years. I remember during
Lena: Thank you for taking me in as your student and for all this years. I remember during
my interview you asked me why do you want to do a PhD? My reply was that at the point
my interview you asked me why do you want to do a PhD? My reply was that at the point
was the best job for me, doing research, growing independent, study and discussing on top of
was the best job for me, doing research, growing independent, study and discussing on top of
that in an international environment. After a little more than four years I would stick to the
that in an international environment. After a little more than four years I would stick to the
same response. It was a great, fun period, I learned a lot and I did tons of experience.
same response. It was a great, fun period, I learned a lot and I did tons of experience.
Piergiorgio: my first co-supervisor, thank you for your kindness and for being an inspiring
Piergiorgio: my first co-supervisor, thank you for your kindness and for being an inspiring
scientist.
scientist.
Chris: my second co-supervisor, I enjoyed our work together and our scientific discussions.
Chris: my second co-supervisor, I enjoyed our work together and our scientific discussions.
Too bad we did not figure out what NIPBL/Scc2 does. I really hope you like your new place
Too bad we did not figure out what NIPBL/Scc2 does. I really hope you like your new place
and I wish the best of luck for the future.
and I wish the best of luck for the future.
Per: my mentor, thank you for your help and kindness I really enjoy discussing with you.
Per: my mentor, thank you for your help and kindness I really enjoy discussing with you.
Davide: Il mio corrispettivo di campagna, é stato un piacere trovarti dall´altra parte del
Davide: Il mio corrispettivo di campagna, é stato un piacere trovarti dall´altra parte del
corridoio, penso sempre che non sarebbe stato lo stesso senza di te. Ti auguro tutta la fortuna
corridoio, penso sempre che non sarebbe stato lo stesso senza di te. Ti auguro tutta la fortuna
del mondo per te e la tua nuova famiglia e chissá… magari ci ritroveremo nel prossimo
del mondo per te e la tua nuova famiglia e chissá… magari ci ritroveremo nel prossimo
corridoio.
corridoio.
Ana: My little portoguese little sister. No is not a mistake is just to emphasize your littleness,
Ana: My little portoguese little sister. No is not a mistake is just to emphasize your littleness,
which is however just physical because you are a very big person, kindhearted, smart and fun.
which is however just physical because you are a very big person, kindhearted, smart and fun.
Of course you are a little bit grumpy, often in a mad mood but hej! No one is perfect! I really
Of course you are a little bit grumpy, often in a mad mood but hej! No one is perfect! I really
enjoyed the time we spent together and I wish the best for you and Bennie.
enjoyed the time we spent together and I wish the best for you and Bennie.
Kristian: I genuinely hope I will find someone like you in my next working place (well first I
Kristian: I genuinely hope I will find someone like you in my next working place (well first I
hope to find a next working place) but I fear you are one of a kind. I will probably not find
hope to find a next working place) but I fear you are one of a kind. I will probably not find
someone smart, annoying and fun to talk with. I wish you the best, good luck in Japan.
someone smart, annoying and fun to talk with. I wish you the best, good luck in Japan.
Pei Shang: we have spent little time together, so I think I still know little of you but I know
Pei Shang: we have spent little time together, so I think I still know little of you but I know
you are an hard worker and you will do well. Good luck with the rest of you PhD. Go get
you are an hard worker and you will do well. Good luck with the rest of you PhD. Go get
them witch!
them witch!
43
43
Acknowledgements
Acknowledgements
Daniela: A special thanks to you for your help, you were always very patience and very
Daniela: A special thanks to you for your help, you were always very patience and very
helpful and your smile was always a source of energy.
helpful and your smile was always a source of energy.
A great thank you to all the people that are still in the corridor Martin, Ana, Sonata, Stefina,
A great thank you to all the people that are still in the corridor Martin, Ana, Sonata, Stefina,
Liu, Camilla, Takaharu, Christer, the ones who have already left, Sidney, Kristian C,
Liu, Camilla, Takaharu, Christer, the ones who have already left, Sidney, Kristian C,
Abrahan, Andreas, Ingrid, Elin and a special thanks to Torkild and Emma.
Abrahan, Andreas, Ingrid, Elin and a special thanks to Torkild and Emma.
All the members of the pub crew, it fells like ages ago since I organized a CMB pub but I
All the members of the pub crew, it fells like ages ago since I organized a CMB pub but I
enjoyed spending time with you guys. A particular thanks to Helena, Thibaud, Shahul for
enjoyed spending time with you guys. A particular thanks to Helena, Thibaud, Shahul for
making all that friday nights.
making all that friday nights.
My family: Glauco, mamma e papá, é stato un lungo viaggio ed é cominciato molto tempo
My family: Glauco, mamma e papá, é stato un lungo viaggio ed é cominciato molto tempo
fa. Vi ringrazio per tutto tutto il vostro aiuto, supporto e affetto che mi avete dato in questi
fa. Vi ringrazio per tutto tutto il vostro aiuto, supporto e affetto che mi avete dato in questi
anni. Ovviamente un pensiero anche al piccolo Kai nuovo entrato della famiglia e ai miei zii:
anni. Ovviamente un pensiero anche al piccolo Kai nuovo entrato della famiglia e ai miei zii:
Vittoria, Aristide e Rosella.
Vittoria, Aristide e Rosella.
Giulia: Ultima ma non meno importante. Qualsiasi cosa io possa scrivere sarebbe riduttivo di
Giulia: Ultima ma non meno importante. Qualsiasi cosa io possa scrivere sarebbe riduttivo di
quello che provo. Ti ringrazio dal profondo per tutto il tuo supporto, questo é la linea di
quello che provo. Ti ringrazio dal profondo per tutto il tuo supporto, questo é la linea di
partenza per la nostra prossima avventura.
partenza per la nostra prossima avventura.
44
44
!
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Thesis for doctoral degree (Ph.D.)
2016
Thesis for doctoral degree (Ph.D.) 2016
The role of the cohesin loader in genome stability:
a journey from yeast to human
The role of the cohesin loader in genome stability: a journey from yeast to human
Fosco Giordano
Fosco Giordano