Gene regulation during development by
chromatin and the Super Elongation
Complex
Olle Dahlberg
Department of Molecular Biosciences
The Wenner-Gren Institute
Stockholm University 2014
1
Doctoral thesis 2014
Department of Molecular Biosciences
The Wenner-Gren Institute
Stockholm University
Stockholm, Sweden
©Olle Dahlberg, Stockholm 2014
ISBN 978-91-7447-932-4
Printed in Sweden by Universitetsservice US-AB, Stockholm 2014
Distributor: Stockholm University
2
”Nothing in biology makes sense except in the light of evolution”
This book is dedicated to my
wife Jessie and our F1 generation
3
4
Abstract
Developmental processes are carefully controlled at the level of transcription to ensure that the fertilized egg develops into an adult organism. The mechanisms that controls transcription of protein-coding
genes ultimately ensure that the Pol II machine synthesizes mRNA
from the correct set of genes in every cell type. Transcriptional control
involves Pol II recruitment as well as transcriptional elongation. Recent genome-wide studies shows that recruitment of Pol II is often
followed by an intermediate step where Pol II is halted in a promoterproximal paused configuration. The release of Pol II from promoterproximal pausing is thus an additional and commonly occurring
mechanism in metazoan gene regulation. The serine kinase P-TEFb is
part of the Super Elongation Complex that regulates the release of
paused Pol II into productive elongation. However, little is known
about the role of P-TEFb mediated gene expression in development.
We have investigated the function of P-TEFb in early Drosophila embryogenesis and find that P-TEFb and other Super Elongation Complex subunits are critical for activation of the most early expressed
genes. We demonstrate an unexpected function for Super Elongation
Complex in activation of genes with non-paused Pol II. Furthermore,
the Super Elongation Complex shares phenotypes with subunits of the
Mediator complex to control the activation of essential developmental
genes. This raises the possibility that the Super Elongation Complex
has an unappreciated role in the recruitment of Pol II to promoters.
The unique chromatin landscape of each cell type is comprised of
post-translational chromatin modifications such as histone methylations and acetylations. To study the function of histone modifications
during development, we depleted the histone demethylase KDM4A in
Drosophila to evaluate the role of KDM4A and histone H3 lysine 36
trimethylation (H3K36me3) in gene regulation. We find that KDM4A
has a male-specific function and regulates gene expression both by
catalytic-dependent and independent mechanisms. Furthermore, we
used histone replacement to investigate the direct role of H3K14 acetylation in a multicellular organism. We show that H3K14 acetylation
is essential for development, but is not cell lethal, suggesting that
H3K14 acetylation has a critical role in developmental gene regulation. This work expands our knowledge of the mechanisms that precisely controls gene regulation and transcription, and in addition highlights
the
complexity
of
metazoan
development.
5
6
List of Publication
I
Olle Dahlberg, Olga Shilkova and Mattias Mannervik
P-TEFb, the Super Elongation Complex and Mediator regulate
non-paused, rapidly transcribed genes during early Drosophila
embryo development
(Submitted)
II
Filip Crona, Olle Dahlberg, Lina E Lundberg, Jan Larsson
and Mattias Mannervik (2013) Gene regulation by the lysine
demethylase KDM4A in Drosophila. Dev.Biol.373(2):453463.
III
Olle Dahlberg, Roshan Vaid and Mattias Mannervik
Histone 3 lysine 14 is essential in Drosophila. (Manuscript)
Article 2 is reprinted with permission from publisher Elsevier
7
Contents
Introduction
Transcription
RNA Polymerase II
Pol II pausing
DNA pause motifs
Factors involved in pausing
P-TEFb
Super Elongation Complex
The Mediator
Cis-regulatory modules
Chromatin and epigenetics
Post translational modifications of histones
Lysine methyltransferases
Lysine demetylases
Histone acetyltransferases
Histone deacetylases
Drosophila early development
Cellularization
Future perspectives
Paper I
Paper II
Paper III
Acknowledgements
References
8
10
12
14
17
18
19
20
21
22
24
27
29
29
30
33
34
35
37
40
42
45
46
48
50
Abbreviations
Pol II
CTD
MBT
MZT
Ser2-p
SEC
CRM
H3K36me3
H3K14ac
KMT
KDM
HAT
HDAC
RNA Polymerase II
Pol II C-terminal domain
Mid-blastula transition (MBT=MZT)
Maternal to zygotic transition
Phosphorylation of CTD Serine 2
the Super Elongation Complex
Cis-regulatory module (enhancer)
Histone 3 Lysine 36 trimethylation
Histone 3 Lysine 14 acetylation
Lysine transferase
Lysine demethylase
Histone acetyltransferace
Histone deacetylace
Bp
Base pairs
9
Introduction
“They are in you and in me; they created us, body and mind; and their
preservation is the ultimate rationale for our existence. They have
come a long way, those replicators. Now they go by the name of
genes, and we are their survival machines.”
-Richard Dawkins, The selfish gene.
The vast numbers of species that exists today or ever lived show an
endless variety of beautiful forms and shapes. Marvelous species populate every corner of our planet and have done so for billions of years.
Even more amazing is that when looking at any of the species on our
planet, we find that they all are related with each other. All organisms
share a common ancestor. All living organisms use DNA to pass down
genetic information from one generation to the next. Considering that
all species share a common ancestor, our own existence is explained
by an unbroken chain of generations transferring their genetic information from the first organism that ever lived, until today. Our bodies
eventually become old and die, but our genes are immortal and will
live on. They are passed down to our children, and continue to exist.
As the genes are passed on, the transmission through generations leads
to mutations and changes in the genetic code. This is a prerequisite for
adaptation through evolution. Evolution works many times by changing old genes to perform new functions. By altering how genes are
expressed, changes in development takes place 1. One of the most
astounding facts about biology is that the same genes building an insect body are also involved in making up the human body. The conservation of gene function throughout the evolutionary tree is intriguing and gives us the opportunity to learn about general biological
mechanisms by studying any living organism.
Developmental biology is the study of an organism going from a single fertilized egg up to the point of adulthood. The mechanisms of the
development of multicellular organisms have been one of the biggest
mysteries in biology, and are still a great challenge for the developmental biologist. It has been known for many years that genes determine when and where different cell types are to be formed during em10
bryogenesis. Genes also determine how many cells each cell type will
have, where limbs will grow out and how to shape the morphology of
our bodies. Animal development is among the most fascinating events
ever to take place in nature. During embryonic development, a single
fertilized egg cell goes through multiple divisions to form an adult
organism containing different cell types and organs, ready to transfer
its genes to new generations. Some of the fundamental questions in
developmental biology are how cells know what kind of cell they will
form and what the fundamental differences are between different cell
types. What factors determines what part of the embryo that will become left and right, anterior/posterior and dorsal/ventral?
These questions have puzzled people, and the field of developmental
biology has emerged to address them.
Development is driven by a precise regulation of genes involved in
forming the function, shape and size of cells and tissue. The study of
gene regulation is of great importance to understand development.
11
Transcription
Transcription is the mechanism where the genetic information existing
in the DNA is transferred into RNA. The regulation of transcription
drives development in all organisms. A fundamental knowledge about
how transcription is regulated is essential when deciphering the mechanisms of development. Messenger RNA (mRNA) can further be used
to synthesize protein in the process of translation. Much of the noncoding part of the genome is transcribed to generate RNA that can
function as structural units such as rRNA and tRNA. In addition, noncoding RNAs such as long non-coding RNA have in more recent
years caught attention. The eukaryotic genome is transcribed by the
three RNA polymerase machines Pol I, Pol II and Pol III. RNA polymerase II (Pol II) transcribes protein-coding genes. To transcribe a
certain DNA sequence, Pol II needs access to the promoter sequence
upstream of the DNA to be transcribed. A major obstacle for getting
access to promoters is nucleosomes that occupy the promoter. Nucleosomes are an essential part of the eukaryotic chromatin and are important transcriptional regulatory components. Depletion of nucleosomes leads to both upregulation and downregulation of transcription
2
. Prior to Pol II promoter access, nucleosomes positioned around the
promoter needs to be removed to make room for the transcription machinery. If any cis-regulatory modules are to be used in the gene activation, they too need to be cleared from nucleosomes. DNA-binding
transcription factors bind nucleosome occupied region and recruits
nucleosome-remodelling factors to move the nucleosomes. This will
create an open and nucleosome-free region. This provides an opportunity for the transcription machinery to access the DNA (FIG 1). A
very basic transcriptional model includes a core promoter plus the
transcribed DNA sequence. The core promoter contains DNA elements that are recognized and bound by general transcription factors
as well as gene-specific transcription factors 3. When access to the
core promoter is granted, the Pol II machine binds in concert with a
large set of general transcription factors to form a preinitiation complex. Transcription can be studied out in vitro, and test tube transcription has been used extensively as a model for transcription. DNA templates bearing a TATA-box DNA motif require a set of general transcription factors for Pol II to localize and bind the promoter in vitro 4.
TATA-binding protein (TBP) is the first factor to bind the promoter.
TBP is a subunit of the mega-dalton TFIID complex where TBP itself
binds the TATA-box DNA motif. TFIIB and TFIIA are recruited to
stabilize the interaction between TBP and the TATA-box. The TATAbox is found ~30 bp upstream the transcription start site.
12
TFIIB binds TFIIB-recognition elements (BRE) upstream and downstream of the TATA sequence, followed by binding of Pol II together
with TFIIF. The last components added is TFIIE and the TFIIH helicase/kinase complex 3 5.
FIGURE 1. A core promoter is cleared from nucleosomes to allow for transcription. An arrow marks the transcription start site. A) A pioneering DNAbinding transcription factor (PF) is recruited to the promoter. B) The PF recruits
nucleosome remodeling factors that can alter the nucleosome occupancy to create an
open chromatin structure at the core promoter. C) The nucleosome-free region allows the general transcription machinery to bind DNA at the core promoter.
13
RNA Polymerase II
Pol II is a multi-subunit complex where the largest subunit Rpb1 contains a C-terminal domain (CTD). The CTD protrudes from the Pol II
protein complex and has multiple heptad repeats. The heptad repeats
sequence contains variations of the consensus sequence
Y1S2P3T4S5P6S7. The length of the CTD varies in different organisms.
Budding yeast contain 26-29 heptad repeats, Drosophila have 45 and
humans 52 6. The serines as well as tyrosines in the CTD are subjected
to phosphorylations. However, potentially all amino acids in the CTD
could be subjected to some kind of modification such as glycosylation, phosphorylation or ubiquitination independently of each other.
This could in principle give an almost infinite number of combinations
of modifications at one single CTD. The Pol II CTD is not essential
for transcription in vitro. However, cells with 10-12 complete heptad
repeats are conditionally viable whereas less than ten repeats is cell
lethal 7. Serine phosphorylations are among the most studied posttranslational CTD modifications. A hyperphosphorylated CTD is correlated with a transcriptional engaged Pol II (chromatin associated Pol
II). Phosphorylation of Pol II CTD Ser5 (Ser5-p) and Ser7 (Ser7-p)
takes place at the promoter while Ser2 phosphorylation (Ser2-p) is
accumulated onto the CTD during elongation and reaches high levels
at the 3´ end of the gene (FIG 2). During transcriptional initiation,
Cdk7 of the TFIIH complex carries out Ser5-p. Ser5-p is considered to
be the key event in “promoter clearance” when Pol II is released from
the pre-initiation complex at the core promoter. In addition, Ser5-p is
found on transcriptionally initiated Pol II that have “paused” 20-80
base pairs downstream the transcription start site 8. Ser2-p is best
known for the role in release of Pol II from pause into transcriptional
elongation. Ser2-p is carried out by serine kinases such as the
Cdk9/Cyclin T complex P-TEFb or Cdk12/Cyclin K.
A simplified commonly accepted model for Pol II transcription can be
outlined as follows: (1) recruitment of unphosphorylated Pol II to the
promoter followed by (2) Pol II recieves Ser5-p for its promoter clearance and (3) additionally receives Ser2-p mediated by the P-TEFb
kinase for productive elongation 9 (FIG 3).
Considering the current view on how phosphorylations controls the
release of Pol II from the preinitiation complex and from pause,
changing the levels of CTD phosphorylation would have a global impact on transcription. However, Pol II phosphorylation is very complex and most likely cannot be explained by simple models. Ser7-p
mutations have been demonstrated to give an effect on shortnoncoding RNA genes, but seem to have a less important role in the
regulation of protein-coding genes 9. Yeast mutants impaired in Ser2-p
14
are viable and show little changes in global gene expression, having
defects in cytokinesis due to changes in a small set of genes with roles
in meiosis 10. Furthermore, the more distally located CTD repeats of
higher eukaryotes are subjected to lysine 7 acetylation by the acetyltransferase P300/KAT3B. The creation of Pol II CTD Lysine mutants
showed that P300 mediated acetylation of the CTD is gene-specific
and important to activate a specific set of genes during growth-factor
response 11.
Multiple steps in mRNA biogenesis occur at the same time and place,
and this “cotranscriptionality” is key for the crosstalk between components involved in processes such as elongation and splicing 12. It has
been demonstrated that Pol II CTD is important for 3´ end processing
both in cells and in test tubes. Set2 is a Histone methyltransferase that
have been implicated in elongation and splicing. During Pol II elongation, Set2 binds the CTD via Ser2-p and Ser5-p and distributes
H3K36me3 in the gene body. H3K36me3 reaches its maximal level in
the 3´-end of the transcribed gene. Depletion of Set2 results in transcriptional defects 13.
Taken together, phosphorylation of the CTD might not be as critical
for global transcription as one might think. Instead, the function of
different amino acid residues of Pol II CTD could have evolved into
controlling different sets of genes depending on the CTD posttranslational state. One could speculate that the CTD modifications
could act as a “code” where different combinations of modifications
carefully and precisely control the different steps in mRNA synthesis
and processing.
FIGURE 2. The Pol II CTD protrudes from the largest Pol II subunit and contains
multiple heptad repeats where serine 2, 5 and 7 are subjected to phosphorylations
that alter the function of the polymerase.
15
FIGURE 3. A simple model of Pol II-mediated transcription. A) Pol II is recruited to the core promoter together with general transcription factors. This forms the
preinitiation complex. B) Pol II promoter clearance is mediated by the TFIIH complex that generates Ser5-p. At this stage, Pol II start transcription but can pause
downstream the transcription start site (indicated by black arrow). C) Release of Pol
II into transcriptional elongation by P-TEFb-mediated phosphorylation of Pol II
CTD as well as negative elongation factors.
16
Pol II pausing
Recruitment of Pol II to the gene is a major rate-limiting step that controls the transcriptional activity. Once Pol II is recruited to the promoter along with the general transcription factors (FIG 3A), transcriptional elongation can be divided into two separate stages. In the first
stage (early elongating), Pol II is released from the core promoter associated pre-initiation complex and initiates RNA synthesis (FIG 3 B).
In the next stage, Pol II progresses throughout the gene body towards
the 3´ end of the gene (productive elongation) (FIG 3 C). An intermediate step has been described as a major role in metazoan transcription. This involves a promoter proximal pausing of Pol II immediately
after the step of early elongation 14. Previous yeast studies looking at
rate-limiting factors during Pol II mediated transcription showed that
recruitment of the polymerase to the gene is a major rate-limiting step
during transcription 15. However, other early data such as studies of
the heat-shock gene hsp70 in Drosophila suggested an additional, different mechanism where Pol II is recruited to the gene but unable to
go into productive elongation before heat shock induction 16 17. In embryos, the gene Sloppy-paired 1 was one of the first developmental
genes demonstrated to have paused Pol II 18. Thus, the polymerase is
positioned downstream the transcription start site in a promoter proximal paused state, and is released into transcriptional elongation only
when the gene is activated. The pausing behavior of Pol II was for
many years seen as a less common mechanism for gene activation in
metazoan organisms whereas RNA Polymerase pausing in bacteria
has been generally accepted 14. Pol II can be recruited to an inactive
gene and stay tethered to the promoter without being released into
elongation. The half-life of gene-associated Pol II has been estimated
stably pause for approximately 5-15 min at the inactive gene 19 20.
However, a majority of all genes in early Drosophila embryos have
paused Pol II and are being active at the same time. This means that
Pol II is recruited, paused and released continuously as long as the
gene is active. With new genome-wide methods introduced to the scientific community, it has become clear that Pol II pausing is a common feature and that pausing play a major role in transcription of the
eukaryotic genome. Recent genome-wide studies show that Pol II
pausing occurs 20-80 bp downstream of the transcription start site at
thousands of genes 21 8. Estimations have been made that at least 70%
of all active genes in the Drosophila S2 cell line contain paused Pol II
22
. What makes Pol II pause? Different molecular models of pausing
17
have been proposed. One model is involving a kinetic model where
Pol II elongation is competing with pausing factors inhibiting the machine from elongation. Another model depends on the position of the
+1 nucleosome acting as a physical barrier that stops Pol II from elongating any further. A third model involves pausing factors that bind
Pol II and tether it to DNA, and thus fixating the polymerase in a
paused state 14. Taken together, Pol II pausing is accepted as a major
regulatory step in gene regulation such as in the transcription of developmental genes. However, the biological and molecular functions
of the factors involved in pausing remain unclear, and the regulation
of Pol II is an extremely complex event. There is most likely no uniform mechanism that can fully explain how pausing occurs at different
paused genes occupied with different factors and having different core
promoter sequences. We can make a model where Pol II regulation is
divided into two different types. The first is a regulation based solely
on the rate of Pol II recruitment to the gene that is immediately followed by transcriptional elongation. The second type of regulation is
controlled primary at the level of Pol II recruitment to the gene plus an
additional regulation at the level of Pol II release from promoterproximal pause. Hence, genes can have paused Pol II even though
polymerase recruitment is the rate-limiting step 23.
Pause DNA motifs
Pol II pausing strength has been linked to the DNA sequence situated
around the TSS and some DNA motifs are enriched at genes showing
more pausing. One DNA-motif that is enriched at active paused Pol II
genes is the GAGA motif. GAGA-motifs are generally located upstream the core promoter at -80 to -75 bp. The GAGA-motif can recruit the GAGA-factor (GAF or Trl). In Drosophila early embryos,
GAGA-containing genes show a high degree of pausing 24. However,
over 2000 Pol II bound promoters that lack GAF instead have the core
promoter element Motif 1 25. As with many GAGA-containing genes,
Motif 1 genes are paused and lack a TATA consensus motif in the
core promoter. Thus Motif 1, GAGA and TATA tend to be mutually
exclusive. The M1BP protein binds Motif 1 and associates with a specific set of genes in Drosophila. GAF genes and M1BP bound have
different nucleosome patterns. The strong +1 nucleosome signal in
M1BP-bound genes suggests that Pol II pausing is achieved with the
assistance of a nucleosome barrier. The low nucleosome signal at
GAF-bound genes suggests that Pol II utilizes a different pausing
18
mechanism at those genes 26. So far, no mammalian counterpart of
GAF or M1BP has been described.
Factors involved in pausing
The DRB-sensitivity inducing factor (DSIF) and negative elongation
factor (NELF) were originally biochemically purified and identified as
factors that are responsible for the effect of DRB addition. Addition of
the nucleoside analog DRB to cell cultures results in an inhibition of
Pol II elongation. However, DRB does not to inhibit Pol II itself, and
in vitro assays using purified Pol II and general transcription factors
does not respond to DRB. DSIF is composed of the subunits Spt4 and
Spt5. NELF contain the four subunits A, B or C, D and E where
NELF E contains an RNA recognition motif 14.
DSIF and NELF are identified as factors involved in pausing and the
regulation of transcription. NELF and DSIF can interact with each
other 27 and stabilize Pol II pausing 14. DSIF and NELF have been
suggested to bind Pol II and also short nascent RNA as it emerges
from the polymerase 28. NELF and DSIF act as transcriptional inhibitors in vitro, 27 and are responsible for Pol II pausing on the hsp70
gene in vivo 29. Pol II-associated DSIF and NELF are subjected to
phosphorylations that mediate the release of Pol II into elongation.
Spt5 of DSIF contains a Pol II CTD-like repetitive sequence in its Cterminal region (CTR), and release of paused Pol II into elongation is
mediated by phosphorylation of the CTR domain by the P-TEFb complex. Furthermore, Ser2-p of Pol II dissociates NELF from Pol II.
However, NELF does not seem to bind directly to the Pol II CTD
since CTD-less Pol II is still being sensitive to DSIF and NELF induced pausing 27 21. In vivo data show that both DSIF and NELF are
critical for correct development. NELF associates with Pol II at active
genes and depletion of NELF by RNAi in Drosophila S2 cells results
in a decreased Pol II occupany at many promoters 20,30. However, several studies indicate a gene-specific function rather than DSIF and
NELF would be equally important for all genes. Maternal depletion of
NELF in Drosophila embryos results in embryos with multiple lethal
phenotypes including abnormal nuclear morphology. Interestingly,
several different segmentation genes are expressed at normal levels
and NELF depletion only gives specific effects on transcription of
slp1 enhancer-promoter transgenes 31. In a similar fashion, the W049
allele of DSIF subunit Spt5 results in gene-specific effects on pair-rule
genes in Drosophila embryos with derepression of some, but not all
eve and runt stripes 32. In addition, the DSIF allele foggy isolated from
zebra fish involves a point mutation in the c-terminus of Spt5, produc19
ing an allele very similar to the DrosophilaW049 allele. The foggyallele also give specific developmental defects such as a reduction of
dopamine-containing neurons as well as an increase in serotonincontaining neurons in the hypothalamus 33. The gene specific phenotypes of the DSIF and NELF alleles suggest that some genes depends
more on NELF and DSIF than others.
P-TEFb
The core P-TEFb complex consists of two components, the kinase
Cdk9 and Cyclin T. P-TEFb can be found as a part of the Super Elongation Complex (SEC) or stored inactive in a complex consisting of a
7SK RNA and HEXIM 34. In addition, recent mass-spectrometric approaches have revealed that P-TEFb can be found in several other protein complexes 35.
P-TEFb is considered to be the main factor that ultimately releases Pol
II from pausing. This is mediated via phosphorylation of Pol II CTD
Ser2 (FIG 3 C) 36 37 38 39 as well as phosphorylation of NELF and
DSIF 40. The P-TEFb kinase activity can be blocked by the drug flavopiridol, and treatment of cells with the inhibitor block most Pol II
mediated transcription 36 and result in an increase of paused Pol II
unable to release into productive elongation 20. In contrast, the Pol II
CTD Ser2 and the kinase activity of P-TEFb is only essential for correct processing but not for the transcriptional elongation of small nuclear RNAs (snRNA) 41. These studies indicates two different roles for
P-TEFb: One as having the role of being the main Pol II pause to release switch, and in addition having a different role in cotranscriptional processes.
Although P-TEFb has been considered to work as a Pol II pause-torelease factor, in vitro assays using phosphorylated peptides show
specificity towards CTD Ser5-p. This is not considered to correlate
with Pol II pause-to-release but rather with Pol II in transcriptional
initiation 42. In mouse ES cells, knockdown of either of two existing
Cyclin T proteins give distinctly different effects on gene regulation
with different groups of genes being affected 43. This suggests that PTEFb might have specific target genes rather than function as a general transcription factor for all genes. In Drosophila, depletion of PTEFb subunit Cdk9 leads to lower levels of chromatin associated
Ser2-phosphorylated Pol II. When RNAi was used to knock down
Cdk9, CTD Ser2-p levels were reduced in an expected manner 44. Interestingly, the level of Ser5-p was reduced to the same extent. One
might speculate that the reduced Ser5-p levels could be an indirect
20
effect from a failure in TFIIH kinase activity upon Cdk9 depletion.
Another explanation could be a more direct effect where P-TEFb have
an additional role to mediate the phosphorylation of Ser5 in correlation with the in vitro findings in 42. In addition, stainings of polytene
chromosomes show that ELL of the Super Elongation Complex (discussed in next chapter) does no longer bind to chromatin in absence of
Cdk9. This indicates that Cdk9 plays a role in the recruitment or stability of the Super Elongation Complex. Depletion of Cdk9 did not
have an effect of DSIF binding, suggesting that DSIF bind independently of P-TEFb 44. Co-stainings with antibodies against P-TEFb
and hyperphosphorylated Pol II do not show a perfect correlation.
This suggests that P-TEFb do not only work in transcriptional elongation 38. In addition, the Cdk9 homolog Cdk12 have been shown to
colocalize with hyperphosphorylated (elongating) Pol II, but only
partly co-localize with P-TEFb. Interestingly, the Cdk12 distribution
over an activated hsp70 gene is spread out throughout the gene body
as if the protein is associated with Pol II during elongation rather than
associate with the promoter 45. This is in contrast to the distribution of
P-TEFb that mostly is associated with the transcription start site 46.
Cdk12 and its partner Cyclin K are in contrast to P-TEFb both essential for oogenesis. We have not been able to produce offspring maternally depleted from either Cdk12 or Cyclin K. This indicates that PTEFb and the Cdk12 complex play separate roles during oogenesis.
To study the maternal effects of Cdk12, other approaches have to be
taken. Injections of early embryos with drugs or antibodies that targets
Cdk12 could give further information about its gene-regulatory role
during early embryogenesis.
The Super Elongation Complex
The super elongation complex (SEC) is a P-TEFb-containing complex
that is involved in gene activation and can mediate rapid induction of
transcription irrespectively of Pol II pausing 47. Besides P-TEFb, the
mammalian Super elongation complex (SEC) consists of three types
of Eleven-nineteen lys-rich leukaemia (ELL) proteins (ELL1, ELL2
and ELL3) that previously were characterized as Pol II elongation
factors. Members of the AF4/FMR2 family (AFF1, AFF2, AFF3 and
AFF4) 48 are essential for the stability of the complex.
The gene MLL is often rearranged in acute childhood haematological
diseases where MLL in-frame translocations are found in a majority of
the cases. MLL is the homolog of the yeast Set1 and the Drosophila
21
protein Trithorax. Set1 was identified in yeast as a H3K4 methyltransferase and is a subunit in the large COMPASS complex (complex of
proteins associated with Set1). Among the most occurring MLL translocations in disease are fusions with nuclear-localized proteins such as
components of the SEC 49. MLL normally regulates many Hox, Pax
and Wnt target genes, and mis-activation of the MLL-associated genes
is achieved by the recruitment of the SEC complex in association with
P-TEFb.
The Drosophila protein Lilliputian (Lilli) is the homolog of AFF4 50.
Lilli controls the activation of early zygotic genes such as the cellularization gene Sry-a and the pair-rule gene fushi-tarazu (ftz) as well as
hkb 50 and sxl 51. The activation of ftz is mediated in concert with the
transcription factor Runt 51. However, Lilli does not control expression of even-skipped or tailless 50. Lilli colocalizes with elongating
hyperphosphorylated Pol II as seen on polytene chromosomes, and the
human AFF4 is recruited to heat-shock loci upon induction together
with other components of SEC 48. Our data indicates that different
subunits of the SEC are critical for early development.
The Mediator complex
The Mediator complex is considered a general transcription factor due
to its role in global regulation of transcription of protein coding-and
non-protein coding genes. Mediator is believed to regulate the formation and structure of the preinitiation complex, and most factors in
the preinitiation complex physically or functionally interact with parts
of the Mediator. The Mediator is conserved in all eukaryotic organisms and much of the subunit composition has been defined based on
mass spectrometry studies. In yeast, some Mediator subunits are required for viability and for the transcription of all genes, while other
subunits are non-essential and gene-selective. The structure of Mediator can be separated into different domains with a head, middle and
tail domain as well as a Cdk8-containing kinase domain. The Mediator has a function of transferring signals from sequence-specific transcription factors to the preinitiation complex 52 (FIG 4). The Mediator
exists in compositionally distinct variants, and the variable composition is a key factor for enabling gene and tissue specificity. Each Mediator subunit interacts with different transcription factors suggesting
a way of Mediator to act gene-specific depending on context. For example, MED1 show protein-protein interactions with nuclear receptors
and specifically regulates nuclear-receptor target genes 53. Emerging
evidence suggests that Mediator directly interacts with Pol II and thus
22
can function as a “molecular bridge” between upstream cis-regulatory
modules and the core promoter. Crystallization of the Mediator head
module demonstrates that MED6, MED8 and MED17 directly interact
with the Pol II CTD. Furthermore, the kinase activity of TFIIH is
stimulated by Mediator 54 leading to CTD Ser5-p that in turn diminishes the contact between Mediator and CTD 55. This indicates that
recruitment of the Mediator complex can mediate the release of Pol II
from the preinitiation complex. Is Mediator also involved in the recruitment of P-TEFb? Indeed, MED23 depletion decreases CTD Ser5p as well as Ser2-p at the target gene Egr1 in ES cells, whereas Pol II
occupancy stays unaffected. The decrease in Pol II phosphorylation is
explained by MED23 having a role in the recruitment of P-TEFb to
Egr1, likely due to the physical interaction with P-TEFb subunit Cdk9
56
. In addition, other Mediator subunits can release Pol II from pausing
via recruitment of the Super elongation complex. MED26 directly
recruits TFIID and later the Super Elongation Complex 57. A similar
mechanism is seen in hypoxia where the transcription factor HIF1A
enhances Pol II pause-release at hypoxia-induced genes by the recruitment of a variant of the Mediator containing CDK8, MED1 and
MED26. This is again followed by the recruitment of SEC that leads
to gene activation 58.
Taken together, the Mediator is involved in a majority of the different
stages of transcription. It interacts with the Pol II at promoters through
recruitment by gene-specific transcription factors. Next, the Mediator
directs factors that release Pol II into early elongation. Finally, Mediator recruits Pol II pause-release factors such as the SEC to drive Pol II
into productive elongation.
Although much is known about the role of Mediator, little work has
focused on the function of Mediator in early development. We maternally depleted a majority of the Mediator subunits and showed that the
head module MED20 and MED22 functionally interacts with the Super Elongation Complex during the activation of early zygotic genes.
To further characterize Mediator in development, our small Mediator
screen demonstrates the possibility to generate maternal depletion of a
number of Mediator subunits. This could in the future be a tool to further characterize the role of the different Mediator subunits in metazoan early development.
23
FIGURE 4. The multi-subunit Mediator complex can be divided into anatomical
parts: tail, middle and head module and a kinase module. Additional DNA-binding
transcription factors (TF) interact with the different subunits of Mediator to achieve
gene-specificity. The head domain containing MED20 and MED22 directly interacts
with the CTD of Pol II via MED6, MED8 and MED17.
Cis-Regulatory Modules
All multicellular organisms need to tightly regulate gene expression in
a spatial and temporal manner. The metazoan genome consists of
large intergenic sequences that do not contain protein-coding genes
but have important information about gene regulation.
During development, the organism has a need to transcribe its genes in
specific cell types and in a spatial-temporal manner to form different
cell types. The way a gene can be expressed in patterns is controlled
by cis-regulatory modules (CRM). CRM´s are often called “enhancers” due to their ability to positively affect transcription. However,
CRM´s are not only upregulating gene expression but can as well
work in the opposite direction. CRM´s consist of DNA sequences located upstream or downstream of the gene's transcription start site.
CRM´s can be positioned close to the transcription start site, in introns
or as long as several hundred kilobases from the regulated gene 59.
Historically, transcriptional enhancers were originally discovered in
viral genomes and metazoan enhancers were first demonstrated in
mouse myeloma cells to have a function in the upregulation of betaglobin genes 60. The exact mechanism for a CRM´s ability to regulate
transcription is not known, but CRM´s controls gene expression by
activating or repressing actions in a context-dependent manner. Some
studies suggests CRM´s to work by recruiting transcription factors and
form a DNA “loop” that brings the CRM in close proximity to the
24
promoter. The gene looping is mediated by factors such as the Mediator complex and the mechanism might explain why an activating protein can function on a gene from a long distance 61 (FIG 5). Recently,
genome wide data analyses have suggested that the binding of transcription factors can mark out regions that act as CRM´s. For example, ChIP-seq data show that the acetyltransferase P300 can predict
tissue-specific active CRM´s during mouse development 62. In addition, specific post-translational chromatin signatures mark out active
and inactive CRM´s. H3K4me1 in combination with H3K27ac have
been suggested as a main chromatin signature for active CRM´s in
human embryonic stem cells whereas the combination
H3K4me1/H3K27me3 mark out “poised enhancers” that will become
active in later stages 63.
Enhancers are bound and transcribed by Pol II that produces noncoding short RNA´s (eRNA). The function of eRNA transcription is
still discussed, but might have a function in keeping an open chromatin at enhancers 64.
In Drosophila, much effort has been spent trying to elucidate the function of CRM´s. A common strategy to study CRM´s has been to create
transgenic reporter constructs where the DNA sequence of the CRM is
cloned in front of a core promoter containing a transcription start site.
A DNA sequence whose transcription can be monitored (e.g. green
fluorescent protein) is placed downstream the transcription start site.
Recently, innovative large-scale assays have been designed to find all
functional CRM´s in cell culture and in vivo 66,67. However, the choice
of core promoter might impact the outcome of the assay. Core promoters differ from each other in terms of transcriptional strength and
timing 65. Using a CRM plus its gene´s endogenous core promoter will
most likely give the best result when studying spatial and temporal
activity.
25
FIGURE 5. Cis-regulatory modules can regulate transcription from long distances. A) Cell-type specific transcription factors (TF) binds the cis-regulatory
module positioned at distance from of the target gene. B) A contact between the
CRM and the promoter is established that leads to gene activation.
26
Chromatin and epigenetics
“…It is therefore very desirable that a method should be available for
obtaining many genetically identical individuals among the vertebrates. Such individuals have been produced in the case of the frog,
Xenopus laevis, by making use of the technique of nuclear transplantation.”
-J.B Gurdon, 1962
Chromatin and epigenetics are two tightly linked terms. The word
epigenetics means above genetics and was originally used by Conrad
Waddington (1905-1975) to describe a phenomenon that could not be
explained simply by genetics 68. Today, epigenetics is defined as “the
study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence”. The definition of epigenetics has changed over the years, and will most likely
continue to evolve with new discoveries. What is important to remember is the fundamental question: How can a fertilized egg cell
give rise to a whole organism 69?
A mammal contains at least two hundred different cell types, which
differ from each other in shape, size and function. Each cell type expresses defined sets of genes during its journey from a pluripotent
state to fully differentiated. During this lineage commitment, new
genes are constantly being activated at the same time as others are
silenced. In the early Drosophila embryo, some genes such as the cellularization genes are only expressed during a short period in early
development. This is followed by a complete inactivation of those
genes that is maintained during the remaining life of the fly. Anything
else could result in a complete disaster for the animal. Cell specific
gene regulatory mechanisms are maintained due to an epigenetic
memory. Epigenetic changes during linage commitment can be kept
for the whole life of an organism. For example, brain cells can continue to stay as brain cells for 100 years. However, John Gurdon showed
more than 50 years ago that it is possible to reprogram a fully differentiated genome. By inserting a somatic nucleus into a Xenopus frog
egg lacking its own nucleus, the genome of the somatic nuclei was
completely overridden and reprogrammed by the egg cell proteins 70.
The somatic chromatin was reprogrammed to recapitulate the expression of a fertilized egg cell. In this way, the artificial zygote started
dividing and developed into an adult frog. This early experiments
showed that the epigenome is indeed plastic and will change under the
right conditions.
Every cell type is maintained by cell-specific epigenetic marks that
cover its genome. Epigenetics regulate what genes that will be in a
27
silent mode, what genes that are active and what genes that will have
the ability to be turned on in later stages. In addition, epigenetics is the
reason why a daughter cell can express exactly the same set of genes
as its mother. This is due to a transfer of epigenetic marks from one
generation of cells to the next. The typical eukaryotic cell stores its
DNA in the nuclei, and in contrast to prokaryotic organisms, all
known eukaryotic organisms are storing their DNA within chromatin.
Chromatin is the term used to describe DNA wrapped around nucleosomes as well as other proteins in close proximity to DNA. Chromatin
is used to pack, store and organize the cell´s genetic material. The core
protein components of chromatin are DNA and histones. Histones are
found in octameric complexes and make up the nucleosomes. Nucleosomes are built up with two copies each of the histones H3, H4, H2A
and H2B. A sequence of 147 base pairs is wrapped around each nucleosome.
To access the information in DNA, chromatin needs to open up and
nucleosomes have to move from the sequence to be used. Evolution
has solved this problem by providing ATP-dependent nucleosomeremodeling enzymes that can slide or push nucleosomes and provide a
piece of naked DNA 71. The position of nucleosomes is established by
the combination of DNA sequences, nucleosome remodeling factors
as well as transcription factors and the general transcription machinery. Nucleosomes are depleted from active promoters and cisregulatory regions and correct nucleosome positioning is essential for
transcription 72. The position of nucleosomes around promoters is organized in a pattern of a -1 nucleosome upstream the transcription
start site and +1 nucleosome downstream. The region at the transcription start site is nucleosome-free. The +1 nucleosome has been suggested to contribute to Pol II pausing when the transcription machine
collides with the nucleosome 73. Core promoter sequence motifs correlated with Pol II pausing such as the GAGA motif (bound by GAF)
and the Motif 1 (bound by M1BP) have distinctly different nucleosome organization around the promoter. GAF-bound genes have almost devoid of an organized nucleosome positioning, whereas M1BPbound genes show highly organized nucleosomes. The GAF genes
generally have more NELF-binding than M1BP-bound genes, and
GAF genes show a higher degree of pausing 26.
28
Post-translational modifications of Histones
Much focus has been put into studying the function of the histone tails
that protrudes from the globular histone domain. The tails are subjected to a variety of post-translational modifications. Modifications of
the histone tails are correlative with the epigenetic state of genes. Several types of modifications are found on histones and the combinatorial nature of the modifications has been suggested to act as a “histone
code” 74. The epigenetic state of each gene is controlled by “writer”,
“reader” and “eraser” factors that are recruited to chromatin and modifies the histone tails. Histone modifications are well conserved from
Yeast to mammals. Histone tail substitutions and histone mutants have
been created in Yeast to elucidate the direct role of each modification.
However, the difficulties creating metazoan histone mutants have given rise to a “chicken or the egg” situation. A challenge now lies in
demonstrating whether certain modifications are the outcome of e.g.
gene activation, or if the modification itself is responsible for a certain
transcriptional state. In the following chapters, I will focus on the
modifications that are most relevant to my own work. This involves
methylations and acetylations of lysines on the Histone 3 tails (FIG 6).
Lysine methyltransferases
Histone 3 lysines are subjected to methylations that have negative or
positive effects on gene expression. Methylations are carried out by
Lysine Methyl Transferases (KMT) that distributes mono-di and trimethylation. The first KMT enzyme to be discovered that had the ability to methylate histones was the KMT1A. This class of proteins contains a SET domain that function as the catalytic unit. The discovery
of KMT1A made it possible to find other methyl transferases by homology searches. A different class of enzymes only consists of the
Dot1 protein 75. KMT´s have a high degree of substrate specificity as
well as a precise enzymatic function such as KMT1A and B that catalyze the H3K9me1 to H3K9me3 reaction, while KMT1C prefers to
catalyze H3K9me1 to H3K9me2.
The Mixed lineage leukemia (MLL) proteins contain the SET1 domain and belong to the KMT2 group that in humans have five members (MLL, MLL2, MLL3, MLL4, MLL5). The MLL proteins exists
in COMPASS complexes and can function as gene activators mediated via their function as H3K4 methyl transferases 76. MLL-mediated
H3K4me3 is often found at expressed genes, making this an active
mark. One function for H3K4me2/3 lies in the ability to directly recruit the basal transcription factor complex TFIID 77,78.
29
The recruitment of TFIID is mediated via binding of the TFIID subunit TAF3 to H3K4me2 or me3 79.
Other histone modifications are correlated with the elongating Pol II,
such as H3K36me3. During transcriptional elongation, the Pol II CTD
is associated with the KMT enzyme Set2 80 81 82. Set2 deposits
H3K36me1/me2/me3 on nucleosomes in the body of transcriptionally
active genes 83. Considering that it specifically cover exons of transcribed genes, H3K36me3 has been suggested to regulate splicing 84.
An additional mechanism have been proposed where Pol II CTD and
H3K36me3 recruits the Histone deacetylase complex Rpd3S that depletes acetylation in coding sequences. Furthermore, incorporation of
H3K36me3 inhibits insertion of H3K56ac in coding sequences. This
maintenance of hypoacetylation is essential to inhibit spurious transcription from intergenic positions 85 86 87.
Set2 is essential in development and RNAi-mediated Set2 depletion in
Drosophila results in a global decrease in H3K36me3 levels and lethality during pupal stage. In addition, Set2 genetically interacts with
the nuclear receptor Ecdysone to regulate Ecdysone target genes 82.
Lysine demethylases
Histone demethylases are enzymes that catalyse removal of histone
methylations. As with the KMT´s, there are two classes of Lysine demethylases (KDM´s) where the first class to be discovered was the
FAD-dependant enzyme KDM1A. The second class contains a JmjDdomain that uses alpha-ketoglutarate and iron for their catalytical
function. The KDM4A family contains the JmjD domain. KDM4A
catalyzes H3K9me2/3 and H3K36me2/3 demethylation. However,
KDM4A are unable to remove H3K9me1/H3K36me1 75,88 89. In Drosophila, the KDM4A family is represented by the two proteins
KDM4A and KDM4B. KDM4A mutant flies have elevated levels of
H3K36me3 and a reduced lifespan 90, although the mutation is not
lethal. This indicates that the precise level of H3K36me3 is not critical
for viability. On the other hand, overexpression of KDM4A results in
diminished H3K36me3 levels and a male-specific lethality. The sex
specific effect suggests impaired H3K36me3-mediated dosage compensation or mis-regulation of male-specific isoforms.
30
In contrast to KDM4A, the homolog KDM4B is recessive lethal.
KDM4B expression is upregulated by p53 during UV-induced DNAdamage and required for H3K9me3 demethylation 91. Recently, it was
shown that KDM4B levels are upregulated in the Kdm4a mutant, and
that at least one copy of either Kdm4a or Kdm4b is necessary for survival. This suggests a biological redundancy between KDM4A and
KDM4B 92. Interestingly, KDM4A and B homozygous double mutants showed a phenotype that resembled phenotypes resulting in the
disruption of ecdysteroid pathways. Indeed, KDM4A physically interacts with the Ecdysone receptor and removes H3K9me3 at target gene
promoters prior to transcription 92. The functional interactions between either Set2 or KDM4A with Ecdysone indicates that H3K9 demethylation works in concert with H3K36 methylation to achieve a
specific histone code that promotes expression of Ecdysone target
genes.
FIGURE 6. Modification of histone lysine residues. An empty lysine (K) can be
acetylated by histone acetyltransferases or methylated by histone methyltransferases.
The acetylation is removed by histone deacetylases and methylations are removed
by histone demethylases.
31
FIGURE 7. The function of Histone 3 modifying enzymes during transcriptional elongation. A DNA-binding transcription factor (TF) binds a motif in an upstream regulatory sequence and in turn recruits a demethylase (KDM4A) to remove
the repressive H3K9me3 marks. MLL in the COMPASS complex distributes
H3K4me3 that recruits Sgf29. This is followed by recruitment of SAGA subunits
that acetylates H3K14ac. This allow for the recruitment of general transcription
factors such as the TFIID complex. Simultaneously, HDAC´s are recruited to participate in the turnover of Histone acetylation that further promotes gene activation and
transcription. The turnover of H3K4me3 by Lid might even more increase the recruitment and binding of the general transcription machinery. During transcriptional
elongation, Set2 associates with the Pol II CTD and distributes H3K36me in the
gene body to prohibit insertion of acetylated Histones.
32
Histone acetyltransferases
One general function for DNA-binding transcription factors is to bind
specific DNA motifs and recruit co-activators that further mediates
gene activation. Histone acetyltransferases (HAT´s) are co-activators
and have mainly been correlated with gene activation and maintenance
of active transcription. Acetylation of histone tails neutralizes their
positive charge that leads to a weaker interaction between nucleosomes and DNA. This promotes nucleosome displacement and open
up the chromatin for transcription. There are four mammalian HAT
families: GCN5/PCAF, MYST, Nuclear receptor coactivator and
CBP. Additional non-histone substrates have lead to a reclassification
where HAT´s now in addition can be referred to as KAT´s (Lysine
acetyltransferases). For example, the CREB-binding protein (CBP)
can in addition to histones also acetylate a number of non-histone targets including the RNA Pol II CTD. CPB/P300 (Drosophila Nejire)
are large proteins that contain several conserved protein-binding domains. This allow for the response and participation in a number of
different pathways 93 via interaction with a variety of transcription
factors.
The large SAGA coactivator complex (Spt-Ada2b-Gcn5 acetyltransferase) is well conserved and can tweak chromatin to promote transcription. Some of the known catalytic functions of the SAGA complex involves binding to acetylated histones, H2B deubiquitination
and histone acetylation 94. The modular nature of SAGA indicates a
way of acting gene specific through different transcription factors that
interacts various modules depending on cell type, similar to CBP. One
mechanism that recruits SAGA to chromatin is through its subunit
Sgf29 that bind H3K4me2/3 nucleosomes 95. Upon SAGA recruitment, the HAT subunit Gcn5 acetylates H3K9 and H3K14 residues 95.
Finally, the combination of H3K4me3/ H3K14ac potentiates binding
of TFIID binding via the TBP-associated factor TAF1 79. With TFIID
bound to the promoter, transcription is imminent.
In Drosophila, SAGA is responsible for H3K9ac and H3K14ac. Individual loss of various SAGA components results in developmental
defects and lethality 96. Ada2b mutants die as early pupae and are extra sensitive to irradiation-induced DNA-damage, suggesting a role for
H3K9ac/H3K14ac in DNA repair 97. Similarly, both loss of the yeast
H3K14 acetyltransferase Mst2 or mutations of H3K14 is critical for
DNA damage response 98. Mutational analyses of Lysine residues have
shown that the levels of H3K4me3 decrease when H3K14 is substituted 99. One explanation to this phenomenon was recently demonstrated
in Yeast where H3K14ac directly inhibits the enzymatic activity of the
H3K4 demethylase Jhd2 (Drosophila gene lid) 100. In this way, each
33
gene can build an extremely versatile and unique mechanism of precise gene activation by the combination of writers, readers and activators.
There are additional complexes that specifically acetylate H3K9 and
H3K14. One being the MYST family protein Sas5 that is a component
of the NuA3 complex. The NuA3 complex binds H3K4me3 and mediate H3K14ac, very similar to the function of SAGA 101.
Histone deacetylases
Histone deacetylases (HDAC´s) modifies histones by the removal of
acetylations from lysines. Like the KAT enzymes, HDAC´s can act on
non-histone proteins and are partly localized to the cytoplasm. Since
HDAC proteins are found in histone-less organisms such as bacteria,
it becomes clear that HDAC´s are essential for other mechanisms than
chromatin modification as well. The HDAC family is divided into
three different classes: class I, class II and class III.
Class I contains HDAC1/2 and HDAC3. Class II contain two subclasses: HAC4/5/7/9 and HDAC6/10. Class III contains the sirtuinfamily of HDAC proteins that has been implicated in prolonging
lifespan in Drosophila. Sir2 was originally characterized as required
for transcriptional and telomeric silencing and have later been implicated in a variety of cellular processes 102. In Yeast, Sir2 deacetylate
H3K9ac and H3K14ac to promote Clr4-mediated H3K9 trimethylation 103.
Together with DNA binding factors, HDAC´s are recruited to chromatin where they deacetylate histones and thus can function as corepressors. However, the story of biology is always more than meets
the eye, and deacetylation of histones per se might not be the repressive mechanism. For example, inhibition of HDAC3´s catalytic activity or deacetylase dead HDAC3 increases global histone acetylation as
expected. Nonetheless, the protein still function as a repressor even
without its enzymatic function 104. This indicates that the repressive
function does not only come from the deacetylation mechanism but
could rather be a result of the combined action of the whole repressive
protein complex. In addition, genome-wide data show that most
HDAC´s are associated with the transcription start site of active genes
and positively correlates with transcription 105 106. This further challenge the traditional view on HDAC´s as repressors. Recent studies
have even shown that HDACs might be important for gene activation.
For example, a screen in Drosophila identified HDAC3 as an activator
of the hsp70 gene by mediating Pol II pause-release 107.
34
The combination of HAT´s/HDAC´s at active genes might function in
acetylation turnover, possibly to “reset” the chromatin after each
round of transcription. The reset function might allow the “old”
preinitiation complex to release from its position to make room for a
new Pol II-containing preinitiation complex at the core promoter.
Drosophila early development
"Two years work wasted. I have been breeding those flies for all that
time and I've got nothing out of it." -Thomas Hunt Morgan
The fruit fly, Drosophila melanogaster have a long history as a model
organism. Drosophila used in research originates in the early 1900´s.
Thomas Hunt Morgan started to use flies in his genetic research. The
knowledge about the function of different biological systems has been
constantly increasing ever since, much due many years of extensive
fly work. Fruit fly embryos are excellent when studying developmental processes. The whole developmental process after fertilization
happens outside the female. Fly embryos are easy to collect in large
quantities and it is possible to study developing cells and organs from
the outside without the need for dissection.
The study of early development is to a great degree the study of maternal factors and how they control the formation of the embryo´s
morphology and body plan. The mechanisms of embryogenesis differ
between multicellular organisms but starts with one single cell, the
fertilized egg. A fertilized egg contains information how to form the
entire organism. The information exists both in the DNA of the organism's genome, in the messenger RNA (mRNA), proteins and other
molecules in the egg cell. In the fruit fly Drosophila melanogaster, the
female fly produces her eggs in the ovaries, where she deposits RNA
and proteins into the egg. This maternal contribution is used by her
offspring during the first period of development before the embryo´s
own zygotic transcription starts. When the egg is fertilized, the female
will deposit the egg and leave it to develop. During the first hours of
development, the embryo uses the maternally provided components
for all of its cellullar functions. The time window when the embryo
starts producing mRNA from its own genome is usually termed maternal to zygotic transition (MZT) or midblastula transition (MBT) 108.
The maternal factors are rapidly degraded during the MZT to initiate
cellularization and to control nuclear divisions. The timing of maternal
35
RNA degradation is critical and degradation is mediated by elements
in the 3´ UTR of the maternal mRNA´s. The zygotic gene expression
is critical for degradation of maternal products. Experiments using Pol
II mutants show that the onset of cellularization is dependent on zygotic expression 109.
In pre-MBT embryos, a small set of genes is expressed as early as
during nuclear cycle 8. This is the first time TATA-binding protein is
seen in the nuclei. Pol II occupies only about hundred genes in preMBT embryos, compared to 4000 genes occupied by TBP and Pol II
during MBT transition. In pre-MBT embryos, many genes are activated solely by Pol II recruitment whereas Pol II pausing only exists at
ten genes 110. Several anterior-posterior patterning genes as well as the
P-TEFb target genes Sry-a, term and CG7271 are among the nonpaused genes. Non-paused genes often have multiple Zelda binding
motifs upstream the core promoter in combination with a strong TATA-box. An advantage with the non-pausing mechanism might be to
achieve multiple rounds of transcription between each short nuclear
cycle. In contrast, MBT activated genes are paused and often have the
GAGA-motifs as well as DPE, MTE and PB motifs that correlates
with paused Pol II 30 110,111. Interestingly, the patterning genes evenskipped, sloppy-paired 1 and runt has a ”dual” mode of activation,
meaning that they are activated in pre-MBT by a non-pausing mechanism and later in MBT becomes paused. What are the factors that are
involved in switching Pol II from non-pause to a paused state during
the MBT? The non-pause to pause switch might be mediated by
known factors such as NELF together with one or more of the preMBT activated genes. However, the few genes that show Pol II pausing already in pre-MBT stage suggests that pausing at these specific
genes only depend on maternal factors.
An interesting finding is that depletion of NELF in Drosophila embryos does not result in a clear effect on the endogenous expression
levels of several patterning genes including slp1. Instead, maternal
NELF depletion shows a differential requirement for NELF in activation of slp1 and eve-reporter constructs. ChIP-data show that NELF
bind the dual genes slp1 and eve and the non-paused genes Sry-a and
ftz in 2h old embryos 31. The authors suggest that the NELF regulation
is dictated from CRM´s rather than from the core promoter. Is it possible that Pol II pausing is achieved via the activity of cis-regulatory
sequences? CRM´s might not function as early as stage 8, and genes
that are expressed that early will have a dispersed expression pattern
irrespective of the core promoter sequence. Only later, the dual genes
might come under the control of their CRM´s to achieve Pol II paus36
ing, possibly mediated via looping pausing factors to the promoters to
mediate pausing.
Zygotically expressed genes that are only active during the preMBT/MBT stage such as the non-paused Sry-a, term and CG7172
might be controlled by a much more ”simple” on/off mechanism that
lacks the precise control that can be achieved from paused genes. Experiments where CRM´s from paused genes are placed in the vicinity
of a gene such as the non-paused term would be very interesting and
give valuable information about Pol II behavior.
In addition, small screens that focus on the pre-MBT genes might reveal novel proteins involved in the non-pause to pause switch. Other
alternatives could involve protein-protein interaction assays involving
the NELF subunits in pre-MBT embryos.
Cellularization
After oviposition, the fertilized egg contains one single diploid nucleus. During the first hours, the nuclei rapidly undergo 13 nuclear divisions. The nuclei divides every 8 minutes until the embryo is filled up
with ~6000 nuclei. The 6000 nuclei are a part of a syncytial blastoderm. The syncytium is made up of multiple nuclei sharing a common
cytoplasm. The shared cytoplasm promotes the diffusion of proteins
and mRNA throughout the embryo. During this early time, a single
plasma membrane surrounds the whole egg cell and the nuclei. The
nine first nuclear divisions take place in the interior of the embryo. In
nuclear cycle 9-10, the majority of nuclei reaches the embryo´s cortex
and undergoes four more nuclear divisions. In the posterior end of the
embryo, a small subset of nuclei is enclosed by cell membranes already at stage 9. This is followed by one or two cell divisions before
they arrest in G2. This group of nuclei forms the pole cells. The pole
cells are primordial germ cells that stay separated from the other cells
at the posterior end and later follow the germ band extension towards
the dorsal side. The pole cells will later become the germ cells that
give rise to the gametes. The pole cells follow a unique transcriptional
programme to maintain their germ-cell identity. While somatic nuclei
activate RNA Polymerase II (Pol II), the pole cells express factors
such as the peptide Polar granule component (pgc) to inactivate PTEFb. This has been suggested to keep the cells in a quiescent state 112
113
(FIG 8).
37
FIGURE 8. Rapid nuclear division during the first hours of embryogenesis. A)
An egg containing a diploid nucleus. B) A subset of nuclei reaches the posterior and
becomes pole cells. C) The nuclei migrate to the embryo´s cortex. Pole cells are
visible at the posterior tip of the embryo. D) Cellularization starts at nuclear cycle
13. E) Cellularization is complete. F) Gastrulation initiates.
At stage 13, a process starts that turn all into individual cells in a process termed cellularization. Cellularization is a mechanism found in
the development of most insects and the process can be seen as a form
of cytokinesis taking place on the basal side of the cells. Cellularization involves processes such as the coordination of a dynamic cytoskeleton and membrane transport and is divided in four steps. When
cycle 13 is complete, cellularization begin the first phase by an invagination of the plasma membrane between each nuclei. In the initial
steps one and two, each nuclei elongates. Step two and three involves
a continuous invagination of the plasma membrane until the leading
edge (the furrow canal) of the membrane reaches the basal end of the
elongated nuclei. During the process of cellularization, basal and apical adherens junctions appear in the cleavage furrow. In step 4, new
lateral membranes are inserted. Step four ends when an actomyosin
contractile ring consisting of myosin II and actin at the basal side of
each cell pinches off the membrane. Thus, each somatic nucleus is
turned into a monolayer of individual cells forming the cellular blastoderm embryo (FIG 9). Both maternal and zygotic factors are known
to regulate cellularization. Only a handful of factors have been shown
to specifically be involved in the process. All cellularization genes
38
show a rapid induction of expression prior to cellularization, mainly
with expression throughout the whole embryo. The zygotic cellularization genes are among the small subset of genes that are expressed in
pre-MBT embryos, in contrast to the 4000 genes that are activated
during the massive maternal to zygotic transition 110. Due to short nuclear cycles in the syncytium, early zygotically transcribed genes are
much shorter that genes expressed later in development. This allows
multiple cycles of transcription between each round of mitosis that
normally shut down transcription 114.
FIGURE 9. Different steps in the cellularization process. A i) Cellularization
starts with an invagination of the plasma membrane and an elongation of nuclei.
Actin is concentrated below the plasma membrane. A ii-A iii) Actin and myosin
follow the furrow canal forming the lateral membranes. Nuclei continue to elongate.
A iv) The actomyosin machine pinch off and thereby creates the basal membrane. A
v) A monolayer of cells is formed at the embryo´s cortex. B) Apical view of the cell
monolayer after cellularization show membranes organized in a honeycomb pattern
around each nucleus.
39
Future perspectives
Initially, Drosophila research focused of pure genetics, but has for the
last three decades much relied on forward genetic screens trying to
elucidate which genes are involved in a certain process 115. Later advantages made it possible to use reverse genetics based upon the
emerging genome sequence data 116. The existing techniques and
methods for the Drosophila system have grown in number over the
years. From relying on blunt and painstaking X-ray and chemically
induced mutagenesis that creates randomly generated mutations to
advanced molecular methods. The detection of mRNA´s with RNA in
situ hybridization is commonly used in embryos and fluorescently
labeled probes has opened up for the possibilities of the generation of
three-dimensional confocal imaging in multiple colors. Recently, the
use of locked nucleic acid probes have proven to work for the detection of miRNA´s 117 118. This might be used in future assays to label
different parts of a transcript such as the 5´ and 3´ of the genes with
two colors, or used to detect two or more transcripts that can not be
distinguished from each other with conventional RNA hybridization
methods. Using fluorescently tagged proteins, it is possible to follow
cellular processes with live imaging from the early blastoderm stage
all the way up to time point of the larvae hatching 24 h after egg laying. Future fluorescent live imaging assays might include embryos
that are carrying 10 or more cellular components tagged with different
fluorescent markers. The possibilities today let us perform more or
less any kind of targeted DNA changes in the fly genome. Recent discoveries such as the CRISPR/Cas9 system 119 120 121 122 have opened
up a new world of possibilities where the limitations only lies in the
time and cost. In addition, CRISPR/Cas9 open up for novel types of
experiments such as targeted chromatin modification or immunoprecipitation of specific DNA elements. The advantage of being able to
deplete proteins or disrupt genes in vivo and follow the phenotypes
that occur is an important a very powerful tool for the fly researcher.
One of the most frequently used molecular methods is the creation of
transgenic animals. Traditional methods for making transgenic flies
have relied on P-element based insertion of DNA sequences, which is
efficient but have the disadvantage of being random in the insertion
into the genome. P-element based insertions have been used to create
large collections of RNAi constructs, gene and enhancer traps as well
as huge fly libraries of gene disruptions. Newer methods making use
of phiC31-mediated transgenesis circumvent positional effect variations among differently inserted transgenic constructs since constructs
can be inserted at the same location with base pair faithfulness 123. In
addition, phiC31-mediated transgenesis gives the advantage of inser40
tion of larger pieces of DNA e.g. bacterial artificial chromosomes into
the genome. The transgenic flies described in the work in this book
were made both with P-element based random insertion as well as
with phiC31-mediated insertions. Future methods for creating transgenic flies might involve negative selection markers such as proapoptotic alleles or the use of drug-resistant constructs to terminate all
non-transgenic flies. Automated cloning/DNA synthesis in combination with recent large-scale injection methods 124 125 could in addition
speed up the rate of transgene generation and scientific discovery.
One of the big challenges is to overcome problems with the heterogeneous nature of the embryo. Any assay that uses whole-embryo extracts from differentiated embryos deals with the issue of having a
sample that contains multiple tissues and cell-types. This can be overcome by using methods that drive the whole embryo to differentiate
into only one tissue. However, such methods are generally painstaking
and the generation of enough material can limit the project. To overcome these problems, the establishment of cell-lines from the different
cell types is a potential solution. A second option is having robust
methods to sort out cells using flow-cytometry based methods. However, this might not be an optimal method if the cell number per embryo is low.
Antibodies with high affinity are essential for all molecular labs, and
many studies rely on the quality of antibodies. One challenge for the
future will be to efficiently produce small monoclonal antibodies such
as nanobodies or peptides that are as effective as polyclonal antibodies. High affinity peptides that can be cloned and expressed in cultures
such as bacteria will overcome the issues that generally occur when
in-house produced, unique and well-working antibodies runs out.
Clonable nanobodies can additionally be tagged with fluorescent
markers and used to track dynamic mechanisms such as phosphorylations or histone modifications in live conditions 126, or used to deplete
protein function 127. The biggest challenge (and opportunities) for future science lies in the enormous amount of data that exists today and
are constantly increasing. The human mind will not able to store and
recall all relevant information that has ever been produced. Therefore,
there will be a demand for software that automatically analyze new
data and puts it in the context of all previously generated results. One
of the next big steps in science could be the introduction of robot scientists to automate more or less all steps of the scientific discovery 128.
So far, the new genome-wide datasets are at best a complement to the
essential need to carefully and painstakingly dissect each gene by
more traditional molecular methods.
41
Perspectives on Paper I
P-TEFb was initially found to have an in vitro activity in transcriptional elongation in vitro. It overcomes the repressive effects of the
negative elongation factors DSIF and NELF. The role of P-TEFb in
early embryogenesis is poorly understood. Therefore, we investigated
the function of P-TEFb by miRNA-mediated knockdown of Cdk9 and
Cyclin T in Drosophila to deplete maternal proteins. Our aim was
initially to characterize P-TEFb´s function in Pol II pause release. We
found effects in early embryos such as a posterior lack of cells and
cellularization defects. The previously identified cellularization gene
Sry-a was downregulated in P-TEFb embryos. We further identified
two more P-TEFb target genes, terminus and CG7271. Some of the
defects could be due to a failure of Pol II recruitment to the genes Srya, terminus and CG7271. In addition, we found components of the
Super elongation complex and two Mediator subunits to phenocopy
the P-TEFb depleted embryos. We could see decreased Pol II occupancy at P-TEFb target gene 5´ ends as well as in the 3´ ends. However, we did not find and evidence for P-TEFb having a role in Pol II
pause-release at target genes. Furthermore, the levels of Ser2 phosphorylated Pol II was decreased at all tested genes, suggesting a global
role for P-TEFb in Pol II CTD Ser2-p during early development. Ser2p correlates with elongating Pol II. However, little is known about the
direct effect of Ser2-p and its role in transcription. The near to normal
expressed cellularization genes bnk, nullo and slam had decreased
Ser2-p yet showed a modest decrease in Pol II occupancy. This indicates that decreased Pol II Ser2-p is neither regulating Pol II pauserelease nor Pol II elongation efficiency at these genes. Ser2-p might
play a different function such as priming Pol II for transcriptional termination or mRNA processing. Furthermore, P-TEFb target genes in
early embryos are activated solely by Pol II recruitment indicating that
a non-paused gene cannot simply be turned into a Pol II paused gene
by depletion of P-TEFb. What is the role of Mediator in early embryonic development? In our study, depletion of either of the Mediator
head domain subunits MED20 or MED22 resulted in a P-TEFb phenotype. This suggests that the head module of Mediator regulates a specific set of genes in early embryos via MED20 and MED22. Smaller
variants of Mediator complexes comprised of a few Mediator subunits
have been suggested to regulate specific sets of genes, and it is plausible to speculate that MED20 and MED22 might
42
FIGURE 10. Activation of P-TEFb-dependent genes versus P-TEFb independent genes in early embryogenesis. A) An early zygotic P-TEFb dependent gene
such as terminus is bound by the pioneering factor Zelda together with a second
unknown DNA-binding factor (X) that might recruit Med20, Med22, and SEC/PTEFb. The specific combination of factors mediates recruitment of the preinitiation
complex. B) Activation of a P-TEFb independent gene such as slam. Zelda allow for
binding of the factor Y that recruits the preinitiation complex independently of PTEFb/SEC or Med20 and Med22.
P-TEFb regulates Pol II phosphorylation at both terminus and slam, possibly in a
mechanism separate from Pol II recruitment.
belong to a Mediator variant that activates some of the most early expressed genes in pre-MBT embryos (FIG 10).
We found a function for term in early development where overexpression as well as miRNA mediated depletion of Terminus protein lead to
cellular defects in the blastocyst stage. However, the function of terminus remains unknown. The highly similar DNA and protein sequence (~99%) between term and its homolog CG7271 suggest that a
recent gene duplication event took place in the split between the two
groups D.obscura and D.melanogaster. This has resulted in two gene
copies in melanogaster and one copy in obscura. In contrast, D. simulans only have one copy, suggesting that it have lost one copy in the
split with D. sechellia. The amino acid sequence of Terminus is containing a predicted C2H2 zinc-finger, and protein-protein interaction
data (The Drosophila Interactions Database, DroID) suggests Term to
bind proteins involved in transcription and mRNA processing. The
protein Elongin A (EloA) is among the protein-protein interaction
partners (DroID database data). EloA have been suggested to be involved in suppressing transient pausing of Pol II in vitro together with
the Super elongation complex member ELL 129. Interestingly, Term
shows a protein-protein interaction with its homolog CG6885 (FIG
11). CG6885 have a 28% amino acid homology to Term and CG7171.
CG6885 is only expressed at the same developmental stage as term
and CG7271.
43
A small but potentially interesting finding is that some maternally
contributed transcripts such as pgc and nanos showed a slightly increased RNA in situ signal. The increased levels might indicate a
function for P-TEFb or any P-TEFb target genes to control the regulation of maternal transcript levels or maternal transcript destabilization.
This study demonstrates the complexity of gene regulation and indicates a novel role for P-TEFb/SEC together with Mediator in early
development. A critical next step will be to investigate and find how
gene-specificity is achieved and what DNA-binding factors that regulates Pol II recruitment to genes in pre-MBT embryos.
FIGURE 11. Protein-protein interaction data from the DroID database. Terminus
protein-protein interaction partners. The strength of each interactions was calculated
using weighted correlation.
44
Perspectives on Paper II
KDM4A can act as a demethylase that target H3K36me3 and
H3K9me3. To assess to function of the JmjC protein KDM4A, we
made a mutant fly based on a P-element jump out strategy. The
KDM4A mutant flies were used in combination with a second
KDM4A allele to avoid background effects. The mutant flies were
viable and did not show any obvious phenotype although the levels of
H3K36me3 were higher than normal. This is consistent with recent
studies that show that KDM4A and KDM4A are biologically redundant. To investigate if overexpressed KDM4A resulted in an opposite
effect, we used an Actin Gal4 driver in combination with a UASKDM4A transgene. Indeed, we could see a decrease in H3K36me3
levels in larvae overexpressing KDM4A. This further demonstrated
that KDM4A regulates the levels of histone methylation. To find potential KDM4A targets, we prepared RNA from KDM4A mutant larvae. Microarray analyses of the mutant larvae indicated that about 100
genes had more than a 1.5 fold change in gene expression. This was
validated by measuring the levels of cDNA from a subset of the target
genes with quantitative RT-PCR. In addition, we analyzed KDM4A
target genes in the larvae overexpressing KDM4A and found an opposite effect on gene expression compared to the KDM4A mutants. This
showed that KDM4A regulate gene expression of these genes.
To investigate how the catalytic function of KDM4A influence its
function in regulating gene expression, we created transgenic flies that
expressed a catalytic dead KDM4A (H195A) cloned together with
upstream and downstream sequences. The inactive form of KDM4A
was combined with the KDM4A mutant allele and the expression of
KDM4A target genes was again investigated by RT-qPCR. Genes that
were upregulated in the KDM4A mutant showed restored expression
levels by the H195A transgene, indicating that the enzymatic function
of KDM4A is not necessary for correct gene expression of those
genes. In contrast, H195A larvae could not restore the expression levels of the down regulated target genes suggesting that the demethylase
function of KDM4A is needed for their correct expression levels.
Next, we studied the levels of methylated nucleosomes positioned at
the KDM4A target genes by performing chromatin immune precipitation. We speculated that genes with higher levels of KDM4A substrates H3K9me3 and H3K36me3 would be more sensitive to the levels of KDM4A protein. Surprisingly, all of the tested genes had a minimal amount of H3K9me3 and H3K36me3 in wild type and in
KDM4A mutants. This was not due to the genes being inactive since
the expression levels of some of the genes are high. One explanation
for this could be that KDM4A regulates expression of these genes by
45
proxy, or via a so far unappreciated mechanism that does not involve
H3K36me3 or H3K9me3.
HP1a was previously demonstrated to interact with KDM4A and to
stimulate its enzymatic function in vitro. In contrast to this synergistic
effect, we found that the expression levels of some KDM4A target
genes were restored in KDM4A/HP1 double mutant animals. This
points to HP1A and KD4A having antagonizing functions. However,
the potential of HP1a to stimulate KDM4A might not function at
genes having very low levels of H3K36me3. At genes having high
H3K36me3 levels, HP1a might function differently.
It was previously reported that male KDM4A mutant flies have a reduced lifespan suggesting that KDM4A might preferentially target
male-specific gene expression. Therefore, we overexpressed KDM4A
and investigated the effects on adult flies. We found a sex-specific
effect where male flies were sensitive to increased levels of KDM4A
to a much higher degree that females. In addition, we found that
KDM4A regulates a subset of highly expressed genes on the Xchromosome. Other studies have shown that H3K36me3 affect binding of the dosage-compensating MSL complex that might explain
some of the male lethal effects.
Together, this strengthens the evidence that KDM4A is involved in a
male specific gene regulation, possibly through the dosagecompensation mechanism. The effects could also be a result of
KDM4A working in male-specific splicing. To further characterize
the function of KDM4A, there might be a need for sex-selection of
embryos/larvae to specifically investigate gene regulation by KDM4A
in males.
Perspectives on Paper III
Studying the direct biological outcome of histone modifications have
until recently only been possible in Yeast systems. Much of the obstacles have come from scattered locations of histone genes in multicellular organisms as well as large copy numbers of each histone gene.
Lately, advantages in Drosophila genetics have opened up the possibilities for flies as a useful model to study histone modifications. In
Drosophila, the canonical histone genes are clustered within one region. Recently, flies devoid of the histone cluster were made by the
creation of a histone deficient chromosome. Furthermore, the histone
deficiency flies were rescued by the addition of twelve transgenic his46
tone copies positioned in trans 130. This histone replacement method is
carried out by the introduction of multiple vectors where each is containing a histone gene unit (HisGU). Each HisGU contains one copy
of histone 1, histone 2A, histone 2B, histone 3 and histone 4. In addition to this, histone replacement with mutant histones was previously
proven to work 131. We have used the histone replacement approach in
order to study the direct effects of several histone post-translational
modifications in a multi-cellular model system. We mutated the previously described HisGU plasmids at the different residues H3K14,
H3K18, H3K36 and H3K79 in order to generate four different histone
mutant fly strains. The transgenic flies carrying twelve copies of HisGUH3K14R were used to study the effects of an organism devoid of
H3K14 acetylation. Furthermore, we used the H3K14R flies to generate mitotic clones in imaginal wing discs. We show that H3K14ac is
essential for viability and that H3K14 replacement leads to lethality in
the embryo and larvae stage. Additionally, we conclude that
H3K14ac-mediated inhibition of H3K4 demethylation is an evolutionary conserved feature and that depletion of H3K14ac is not cell-lethal.
We will further investigate the role of H3K14 acetylation and explore
the three additional created histone mutations. One possibility to expand the usefulness of this system could involve temporally controlled
onset of the histone depletion. This could be achieved by having a
wild-type histone 3 transgene under the control of e.g. a heat-shock
promoter could be introduced in the histone replaced flies. This would
allow for expression of wild-type histone 3 until a certain stage. To
investigate the role of histone modifications in early embryos, there
might be an idea to explore the possibilities of maternally deplete
wild-type histones using miRNA-mediated knock down while at the
same time simply overexpressing miRNA resistant mutant histones
using the same maternal Gal4 driver.
47
Acknowledgements
Usually when getting someone’s thesis, the acknowledgement section
is the first and sometimes the only part in the book that people read.
So I am taking a risk by not listing all people I have ever worked with
or met during my PhD studies. Instead, I will try to mention as few
people as possible by name and just say that I am very happy for the
friends I have made during my time here. Especially I would like to
acknowledge the colleagues I have worked together with in my lab
through days, nights and weekends. I am very grateful that I had the
opportunity to get to know all of you and that I got the chance to work
with you. I am also very grateful for my family that for some mysterious reason put up with my bad mood and grumpiness during the times
of hard work in the lab.
How did I end up as a PhD student in Stockholm?
I´ve always had an interest in how things around me are constructed,
and the passion for inventing small gadgets or picking apart broken
electronics have followed me since I was young. After finishing
school, I wrote an application to study ”green biology” at Umeå University. However, I was drafted by the army and went on to do my
service as a fire fighter. After this non-military service, I spent a year
working in Stockholm and one year studying Swedish folk music in
Dalarna. Next time I sent in my application to Umeå University, I
aimed for molecular biology (white biology), even if I didn’t really
knew what it was. It turned out that the first year was dedicated to
chemistry courses. It was hard work but lots of fun and I remember
that I was among the few students that seemed to like organic chemistry. My time in Umeå was great, and I met many people that still are
some of my closest friends. Umeå have a long history of fly genetics
and even used to have a Drosophila stock center, and it was hard to
avoid being taught the basics of fly genetics as a molecular biology
student. I also had the chance to do work in research groups that used
flies, such as Dan Hultmark´s lab. During my last months as a student,
I did my exam work in Erik Johansson´s lab at the department of medical chemistry where I purified proteins. That was the time when I met
Jessie. Since Jessie lived in Växjö, far away from Umeå, I decided to
move to Växjö after finishing my studies. Living in Växjö was not
very inspiring for either of us, and when I came in contact with my
supervisor Mattias, we decided to move to Stockholm.
Instead of taking apart radios to see how they work, I got the opportunity to do similar things but on a molecular level. The time as a PhD
student has been a great experience in many ways. I have made tons of
friends, played lots of sports and had thousands of nice lunches at
Lantis. I have learned a lot about so many cool things like how one
48
can take apart and put together pieces of DNA, and how to put the
new homemade DNA back into an organism. Mattias and my other
colleagues have really helped me to develop my skills in critical and
scientific thinking. Much of the training has come from the hundreds
of journal clubs, data clubs and group meetings where we spent endless hours discussing figures and data. Mattias has always been supportive and allowed me do things in my own way, and at the same
time tried hard to turn me into a thinking, independent researcher. I
am very grateful that I got the chance of doing a PhD, and I have enjoyed every second of it.
“Somewhere, something incredible is waiting to be known”
-Carl Sagan
49
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
50
Carroll, S. B. Evo-devo and an expanding evolutionary synthesis: A genetic
theory of morphological evolution. Cell 134, 25-36, doi:Doi
10.1016/J.Cell.2008.06.030 (2008).
Iyer, V. R. Nucleosome positioning: bringing order to the eukaryotic
genome. Trends Cell Biol 22, 250-256, doi:Doi 10.1016/J.Tcb.2012.02.004
(2012).
Fuda, N. J., Ardehali, M. B. & Lis, J. T. Defining mechanisms that regulate
RNA polymerase II transcription in vivo. Nature 461, 186-192, doi:Doi
10.1038/Nature08449 (2009).
Luse, D. S. Promoter clearance by RNA polymerase II. Bba-Gene Regul
Mech 1829, 63-68, doi:Doi 10.1016/J.Bbagrm.2012.08.010 (2013).
He, Y., Fang, J., Taatjes, D. J. & Nogales, E. Structural visualization of key
steps in human transcription initiation. Nature 495, 481-+, doi:Doi
10.1038/Nature11991 (2013).
Chapman, R. D., Haidemann, M., Hintermair, C. & Eick, D. Molecular
evolution of the RNA polymerase IICTD. Trends in Genetics 24, 289-296,
doi:Doi 10.1016/J.Tig.2008.03.010 (2008).
Nonet, M., Sweetser, D. & Young, R. A. Functional Redundancy and
Structural Polymorphism in the Large Subunit of Rna Polymerase-Ii. Cell
50, 909-915, doi:Doi 10.1016/0092-8674(87)90517-4 (1987).
Adelman, K. & Lis, J. T. Promoter-proximal pausing of RNA polymerase
II: emerging roles in metazoans. Nat Rev Genet 13, 720-731, doi:Doi
10.1038/Nrg3293 (2012).
Napolitano, G., Lania, L. & Majello, B. RNA polymerase II CTD
modifications: how many tales from a single tail. J Cell Physiol 229, 538544, doi:10.1002/jcp.24483 (2014).
Saberianfar, R., Cunningham-Dunlop, S. & Karagiannis, J. Global Gene
Expression Analysis of Fission Yeast Mutants Impaired in Ser-2
Phosphorylation of the RNA Pol II Carboxy Terminal Domain. Plos One 6,
doi:ARTN e24694
DOI 10.1371/journal.pone.0024694 (2011).
Schroder, S. et al. Acetylation of RNA Polymerase II Regulates GrowthFactor-Induced Gene Transcription in Mammalian Cells. Mol Cell 52, 314324, doi:Doi 10.1016/J.Molcel.2013.10.009 (2013).
Perales, R. & Bentley, D. "Cotranscriptionality": The Transcription
Elongation Complex as a Nexus for Nuclear Transactions. Mol Cell 36,
178-191, doi:Doi 10.1016/J.Molcel.2009.09.018 (2009).
Richard, P. & Manley, J. L. Transcription termination by nuclear RNA
polymerases. Gene Dev 23, 1247-1269, doi:10.1101/gad.1792809 (2009).
Kwak, H. & Lis, J. T. Control of Transcriptional Elongation. Annu Rev
Genet 47, 483-508, doi:Doi 10.1146/Annurev-Genet-110711-155440
(2013).
Ptashne, M. & Gann, A. Transcriptional activation by recruitment. Nature
386, 569-577, doi:Doi 10.1038/386569a0 (1997).
Gilmour, D. S. & Lis, J. T. Rna Polymerase-Ii Interacts with the Promoter
Region of the Noninduced Hsp70 Gene in Drosophil-Melanogaster Cells.
Mol Cell Biol 6, 3984-3989 (1986).
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
Rasmussen, E. B. & Lis, J. T. In vivo transcriptional pausing and cap
formation on three Drosophila heat shock genes. Proc Natl Acad Sci U S A
90, 7923-7927 (1993).
Wang, X. L., Lee, C., Gilmour, D. S. & Gergen, J. P. Transcription
elongation controls cell fate specification in the Drosophila embryo. Gene
Dev 21, 1031-1036, doi:Doi 10.1101/Gad.1521207 (2007).
Buckley, M. S., Kwak, H., Zipfel, W. R. & Lis, J. T. Kinetics of promoter
Pol II on Hsp70 reveal stable pausing and key insights into its regulation.
Gene Dev 28, 14-19, doi:Doi 10.1101/Gad.231886.113 (2014).
Henriques, T. et al. Stable Pausing by RNA Polymerase II Provides an
Opportunity to Target and Integrate Regulatory Signals. Mol Cell 52, 517528, doi:Doi 10.1016/J.Molcel.2013.10.001 (2013).
Yamaguchi, Y., Shibata, H. & Handa, H. Transcription elongation factors
DSIF and NELF: Promoter-proximal pausing and beyond. Bba-Gene Regul
Mech 1829, 98-104, doi:Doi 10.1016/J.Bbagrm.2012.11.007 (2013).
Core, L. J. et al. Defining the Status of RNA Polymerase at Promoters. Cell
Rep 2, 1025-1035, doi:Doi 10.1016/J.Celrep.2012.08.034 (2012).
Saunders, A., Core, L. J., Sutcliffe, C., Lis, J. T. & Ashe, H. L. Extensive
polymerase pausing during Drosophila axis patterning enables high-level
and pliable transcription. Gene Dev 27, 1146-1158, doi:Doi
10.1101/Gad.215459.113 (2013).
Chen, K. et al. A global change in RNA polymerase II pausing during the
Drosophila midblastula transition. Elife 2, doi:ARTN e00861
DOI 10.7554/eLife.00861 (2013).
Ohler, U., Liao, G. C., Niemann, H. & Rubin, G. M. Computational
analysis of core promoters in the Drosophila genome. Genome Biol 3,
RESEARCH0087 (2002).
Li, J. & Gilmour, D. S. Distinct mechanisms of transcriptional pausing
orchestrated by GAGA factor and M1BP, a novel transcription factor.
Embo Journal 32, 1829-1841, doi:Doi 10.1038/Emboj.2013.111 (2013).
Yamaguchi, Y. et al. NELF, a multisubunit complex containing RD,
cooperates with DSIF to repress RNA polymerase II elongation. Cell 97,
41-51, doi:Doi 10.1016/S0092-8674(00)80713-8 (1999).
Missra, A. & Gilmour, D. S. Interactions between DSIF (DRB sensitivity
inducing factor), NELF (negative elongation factor), and the Drosophila
RNA polymerase II transcription elongation complex. Proc Natl Acad Sci
U S A 107, 11301-11306, doi:Doi 10.1073/Pnas.1000681107 (2010).
Wu, C. H. et al. NELF and DSIF cause promoter proximal pausing on the
hsp70 promoter in Drosophila. Gene Dev 17, 1402-1414, doi:Doi
10.1101/Gad.1091403 (2003).
Gilchrist, D. A. et al. Pausing of RNA polymerase II disrupts DNAspecified nucleosome organization to enable precise gene regulation. Cell
143, 540-551, doi:10.1016/j.cell.2010.10.004 (2010).
Wang, X. L., Hang, S. Y., Prazak, L. & Gergen, J. P. NELF Potentiates
Gene Transcription in the Drosophila Embryo. Plos One 5, doi:ARTN
e11498
DOI 10.1371/journal.pone.0011498 (2010).
51
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
52
Jennings, B. H. et al. Locus-specific requirements for Spt5 in
transcriptional activation and repression in Drosophila. Current Biology 14,
1680-1684, doi:Doi 10.1016/J.Cub.2004.08.066 (2004).
Guo, S. et al. A regulator of transcriptional elongation controls vertebrate
neuronal development. Nature 408, 366-369 (2000).
Li, Q. T. et al. Analysis of the large inactive P-TEFb complex indicates that
it contains one 7SK molecule, a dimer of HEXIM1 or HEXIM2, and two PTEFb molecules containing Cdk9 phosphorylated at threonine 186. Journal
of
Biological
Chemistry
280,
28819-28826,
doi:Doi
10.1074/Jbc.M502712200 (2005).
Ramakrishnan, R. et al. Identification of novel CDK9 and Cyclin T1associated protein complexes (CCAPs) whose siRNA depletion enhances
HIV-1 Tat function. Retrovirology 9, doi:Artn 90
Doi 10.1186/1742-4690-9-90 (2012).
Chao, S. H. & Price, D. H. Flavopiridol inactivates P-TEFb and blocks
most RNA polymerase II transcription in vivo. Journal of Biological
Chemistry 276, 31793-31799, doi:Doi 10.1074/Jbc.M102306200 (2001).
Ni, Z. Y. et al. P-TEFb is critical for the maturation of RNA polymerase II
into productive elongation in vivo. Mol Cell Biol 28, 1161-1170, doi:Doi
10.1128/Mcb.01859-07 (2008).
Lis, J. T., Mason, P., Peng, J., Price, D. H. & Werner, J. P-TEFb kinase
recruitment and function at heat shock loci. Gene Dev 14, 792-803 (2000).
Price, D. H. P-TEFb, a cyclin-dependent kinase controlling elongation by
RNA polymerase II. Mol Cell Biol 20, 2629-2634, doi:Doi
10.1128/Mcb.20.8.2629-2634.2000 (2000).
Peterlin, B. M. & Price, D. H. Controlling the elongation phase of
transcription with P-TEFb. Mol Cell 23, 297-305, doi:Doi
10.1016/J.Molcel.2006.06.014 (2006).
Medlin, J. et al. P-TEFb is not an essential elongation factor for the
intronless human U2 snRNA and histone H2b genes. Embo Journal 24,
4154-4165, doi:Doi 10.1038/Sj.Emboj.7600876 (2005).
Czudnochowski, N., Bosken, C. A. & Geyer, M. Serine-7 but not serine-5
phosphorylation primes RNA polymerase II CTD for P-TEFb recognition.
Nat Commun 3, 842, doi:10.1038/ncomms1846 (2012).
Kohoutek, J. et al. Cyclin T2 Is Essential for Mouse Embryogenesis. Mol
Cell Biol 29, 3280-3285, doi:Doi 10.1128/Mcb.00172-09 (2009).
Eissenberg, J. C., Shilatifard, A., Dorokhov, N. & Michener, D. E. Cdk9 is
an essential kinase in Drosophila that is required for heat shock gene
expression, histone methylation and elongation factor recruitment.
Molecular Genetics and Genomics 277, 101-114, doi:Doi 10.1007/S00438006-0164-2 (2007).
Bartkowiak, B. et al. CDK12 is a transcription elongation-associated CTD
kinase, the metazoan ortholog of yeast Ctk1. Gene Dev 24, 2303-2316,
doi:Doi 10.1101/Gad.1968210 (2010).
Boehm, A. K., Saunders, A., Werner, J. & Lis, J. T. Transcription factor
and polymerase recruitment, modification, and movement on dhsp70 in
47
48
49
50
51
52
53
54
55
56
57
58
59
60
vivo in the minutes following heat shock. Mol Cell Biol 23, 7628-7637,
doi:Doi 10.1128/Mcb.23.21.7628-7637.2003 (2003).
Luo, Z., Lin, C. & Shilatifard, A. The super elongation complex (SEC)
family in transcriptional control. Nat Rev Mol Cell Biol 13, 543-547,
doi:10.1038/nrm3417 (2012).
Lin, C. et al. AFF4, a component of the ELL/P-TEFb elongation complex
and a shared subunit of MLL chimeras, can link transcription elongation to
leukemia. Mol Cell 37, 429-437, doi:10.1016/j.molcel.2010.01.026 (2010).
Mohan, M., Lin, C., Guest, E. & Shilatifard, A. Licensed to elongate: a
molecular mechanism for MLL-based leukaemogenesis. Nat Rev Cancer
10, 721-728, doi:10.1038/nrc2915 (2010).
Tang, A. H., Neufeld, T. P., Rubin, G. M. & Muller, H. A. J.
Transcriptional regulation of cytoskeletal functions and segmentation by a
novel maternal pair-rule gene, lilliputian. Development 128, 801-813
(2001).
VanderZwan-Butler, C. J., Prazak, L. M. & Gergen, J. P. The HMG-box
protein Lilliputian is required for Runt-dependent activation of the pair-rule
gene
fushi-tarazu.
Dev
Biol
301,
350-360,
doi:Doi
10.1016/J.Ydbio.2006.10.027 (2007).
Yin, J. W. & Wang, G. The Mediator complex: a master coordinator of
transcription and cell lineage development. Development 141, 977-987,
doi:10.1242/dev.098392 (2014).
Poss, Z. C., Ebmeier, C. C. & Taatjes, D. J. The Mediator complex and
transcription regulation. Crit Rev Biochem Mol 48, 575-608, doi:Doi
10.3109/10409238.2013.840259 (2013).
Kim, Y. J., Bjorklund, S., Li, Y., Sayre, M. H. & Kornberg, R. D. A
Multiprotein Mediator of Transcriptional Activation and Its Interaction
with the C-Terminal Repeat Domain of Rna-Polymerase-Ii. Cell 77, 599608, doi:Doi 10.1016/0092-8674(94)90221-6 (1994).
Robinson, P. J. J., Bushnell, D. A., Trnka, M. J., Burlingame, A. L. &
Kornberg, R. D. Structure of the Mediator Head module bound to the
carboxy-terminal domain of RNA polymerase II. Proc Natl Acad Sci U S A
109, 17931-17935, doi:Doi 10.1073/Pnas.1215241109 (2012).
Wang, W. et al. Mediator MED23 regulates basal transcription in vivo via
an interaction with P-TEFb. Transcription 4, 39-51, doi:10.4161/trns.22874
(2013).
Takahashi, H. et al. Human Mediator Subunit MED26 Functions as a
Docking Site for Transcription Elongation Factors. Cell 146, 92-104,
doi:Doi 10.1016/J.Cell.2011.06.005 (2011).
Galbraith, M. D. et al. HIF1A Employs CDK8-Mediator to Stimulate
RNAPII Elongation in Response to Hypoxia. Cell 153, 1327-1339, doi:Doi
10.1016/J.Cell.2013.04.048 (2013).
Shlyueva, D., Stampfel, G. & Stark, A. Transcriptional enhancers: from
properties to genome-wide predictions. Nature reviews. Genetics 15, 272286, doi:10.1038/nrg3682 (2014).
Banerji, J., Olson, L. & Schaffner, W. A Lymphocyte-Specific Cellular
Enhancer Is Located Downstream of the Joining Region in
53
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
54
Immunoglobulin Heavy-Chain Genes. Cell 33, 729-740, doi:Doi
10.1016/0092-8674(83)90015-6 (1983).
Carlsten, J. O. P., Zhu, X. F. & Gustafsson, C. M. The multitalented
Mediator complex. Trends Biochem Sci 38, 531-537, doi:Doi
10.1016/J.Tibs.2013.08.007 (2013).
Visel, A. et al. ChIP-seq accurately predicts tissue-specific activity of
enhancers. Nature 457, 854-858, doi:Doi 10.1038/Nature07730 (2009).
Rada-Iglesias, A. et al. A unique chromatin signature uncovers early
developmental enhancers in humans. Nature 470, 279-+, doi:Doi
10.1038/Nature09692 (2011).
Kim, T. K. et al. Widespread transcription at neuronal activity-regulated
enhancers. Nature 465, 182-U165, doi:Doi 10.1038/Nature09033 (2010).
Lagha, M. et al. Paused Pol II Coordinates Tissue Morphogenesis in the
Drosophila
Embryo.
Cell
153,
976-987,
doi:Doi
10.1016/J.Cell.2013.04.045 (2013).
Arnold, C. D. et al. Genome-Wide Quantitative Enhancer Activity Maps
Identified by STARR-seq. Science 339, 1074-1077, doi:Doi
10.1126/Science.1232542 (2013).
Pfeiffer, B. D. et al. Tools for neuroanatomy and neurogenetics in
Drosophila. Proc Natl Acad Sci U S A 105, 9715-9720, doi:Doi
10.1073/Pnas.0803697105 (2008).
Goldberg, A. D., Allis, C. D. & Bernstein, E. Epigenetics: A landscape
takes shape. Cell 128, 635-638, doi:Doi 10.1016/J.Cell.2007.02.006 (2007).
Felsenfeld, G. A brief history of epigenetics. Cold Spring Harb Perspect
Biol 6, doi:10.1101/cshperspect.a018200 (2014).
Gurdon, J. B. Multiple Genetically Identical Frogs. J Hered 53, 5-& (1962).
Mueller-Planitz, F., Klinker, H. & Becker, P. B. Nucleosome sliding
mechanisms: new twists in a looped history. Nat Struct Mol Biol 20, 10261032, doi:Doi 10.1038/Nsmb.2648 (2013).
Struhl, K. & Segal, E. Determinants of nucleosome positioning. Nat Struct
Mol Biol 20, 267-273, doi:Doi 10.1038/Nsmb.2506 (2013).
Mavrich, T. N. et al. Nucleosome organization in the Drosophila genome.
Nature 453, 358-U327, doi:Doi 10.1038/Nature06929 (2008).
Jenuwein, T. & Allis, C. D. Translating the histone code. Science 293,
1074-1080, doi:Doi 10.1126/Science.1063127 (2001).
Black, J. C., Van Rechem, C. & Whetstine, J. R. Histone lysine methylation
dynamics: establishment, regulation, and biological impact. Mol Cell 48,
491-507, doi:10.1016/j.molcel.2012.11.006 (2012).
Shilatifard, A. The COMPASS Family of Histone H3K4 Methylases:
Mechanisms of Regulation in Development and Disease Pathogenesis.
Annual Review of Biochemistry, Vol 81 81, 65-95, doi:Doi
10.1146/Annurev-Biochem-051710-134100 (2012).
Smith, E., Lin, C. & Shilatifard, A. The super elongation complex (SEC)
and MLL in development and disease. Gene Dev 25, 661-672,
doi:10.1101/gad.2015411 (2011).
Jakovcevski, M. & Akbarian, S. Epigenetic mechanisms in neurological
disease. Nat Med 18, 1194-1204, doi:Doi 10.1038/Nm.2828 (2012).
79
80
81
82
83
84
85
86
87
88
89
90
91
Vermeulen, M. et al. Selective anchoring of TFIID to nucleosomes by
trimethylation of histone H3 lysine 4. Cell 131, 58-69, doi:Doi
10.1016/J.Cell.2007.08.016 (2007).
Li, J. X., Moazed, D. & Gygi, S. P. Association of the histone
methyltransferase Set2 with RNA polymerase II plays a role in
transcription elongation. Journal of Biological Chemistry 277, 4938349388, doi:Doi 10.1074/Jbc.M209294200 (2002).
Li, B., Howe, L., Anderson, S., Yates, J. R. & Workman, J. L. The Set2
histone methyltransferase functions through the phosphorylated carboxylterminal domain of RNA polymerase II. Journal of Biological Chemistry
278, 8897-8903, doi:Doi 10.1074/Jbc.M212134200 (2003).
Stabell, M., Larsson, J., Aalen, R. B. & Lambertsson, A. Drosophila dSet2
functions in H3-K36 methylation and is required for development. Biochem
Biophys Res Commun 359, 784-789, doi:Doi 10.1016/J.Bbrc.2007.05.189
(2007).
Kizer, K. O. et al. A novel domain in Set2 mediates RNA polymerase II
interaction and couples histone H3 K36 methylation with transcript
elongation. Mol Cell Biol 25, 3305-3316, doi:10.1128/MCB.25.8.33053316.2005 (2005).
Luco, R. F., Allo, M., Schor, I. E., Kornblihtt, A. R. & Misteli, T.
Epigenetics in Alternative Pre-mRNA Splicing. Cell 144, 16-26, doi:Doi
10.1016/J.Cell.2010.11.056 (2011).
Li, B. et al. Histone H3 Lysine 36 Dimethylation (H3K36me2) Is Sufficient
to Recruit the Rpd3s Histone Deacetylase Complex and to Repress
Spurious Transcription. Journal of Biological Chemistry 284, 7970-7976,
doi:Doi 10.1074/Jbc.M808220200 (2009).
Carrozza, M. J. et al. Histone H3 methylation by Set2 directs deacetylation
of coding regions by Rpd3S to suppress spurious intragenic transcription.
Cell 123, 581-592, doi:Doi 10.1016/J.Cell.2005.10.023 (2005).
Venkatesh, S. et al. Set2 methylation of histone H3 lysine 36 suppresses
histone exchange on transcribed genes. Nature 489, 452-U145, doi:Doi
10.1038/Nature11326 (2012).
Klose, R. J. et al. The transcriptional repressor JHDM3A demethylates
trimethyl histone H3 lysine 9 and lysine 36. Nature 442, 312-316,
doi:10.1038/nature04853 (2006).
Whetstine, J. R. et al. Reversal of histone lysine trimethylation by the
JMJD2 family of histone demethylases. Cell 125, 467-481,
doi:10.1016/j.cell.2006.03.028 (2006).
Lorbeck, M. T. et al. The histone demethylase Dmel\Kdm4A controls
genes required for life span and male-specific sex determination in
Drosophila. Gene 450, 8-17, doi:10.1016/j.gene.2009.09.007 (2010).
Palomera-Sanchez, Z., Bucio-Mendez, A., Valadez-Graham, V., Reynaud,
E. & Zurita, M. Drosophila p53 Is Required to Increase the Levels of the
dKDM4B Demethylase after UV-induced DNA Damage to Demethylate
Histone H3 Lysine 9. Journal of Biological Chemistry 285, 31370-31379,
doi:Doi 10.1074/Jbc.M110.128462 (2010).
55
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
56
Tsurumi, A., Dutta, P., Yan, S. J., Sheng, R. & Li, W. X. Drosophila Kdm4
demethylases in histone H3 lysine 9 demethylation and ecdysteroid
signaling. Sci Rep-Uk 3, doi:Artn 2894
Doi 10.1038/Srep02894 (2013).
Bedford, D. C., Kasper, L. H., Fukuyama, T. & Brindle, P. K. Target gene
context influences the transcriptional requirement for the KAT3 family of
CBP and p300 histone acetyltransferases. Epigenetics-Us 5, 9-15 (2010).
Samara, N. L. & Wolberger, C. A new chapter in the transcription SAGA.
Curr Opin Struc Biol 21, 767-774, doi:10.1016/j.sbi.2011.09.004 (2011).
Bian, C. B. et al. Sgf29 binds histone H3K4me2/3 and is required for
SAGA complex recruitment and histone H3 acetylation. Embo Journal 30,
2829-2842, doi:Doi 10.1038/Emboj.2011.193 (2011).
Weake, V. M. et al. Post-transcription initiation function of the ubiquitous
SAGA complex in tissue-specific gene activation. Gene Dev 25, 14991509, doi:Doi 10.1101/Gad.2046211 (2011).
Qi, D., Larsson, J. & Mannervik, M. Drosophila Ada2b is required for
viability and normal histone H3 acetylation. Mol Cell Biol 24, 8080-8089,
doi:Doi 10.1128/Mcb.24.18.8080-8089.2004 (2004).
Wang, Y. et al. Histone H3 Lysine 14 Acetylation Is Required for
Activation of a DNA Damage Checkpoint in Fission Yeast. Journal of
Biological Chemistry 287, 4386-4393, doi:Doi 10.1074/Jbc.M111.329417
(2012).
Nakanishi, S. et al. A comprehensive library of histone mutants identifies
nucleosomal residues required for H3K4 methylation. Nat Struct Mol Biol
15, 881-888, doi:Doi 10.1038/Nsmb.1454 (2008).
Maltby, V. E. et al. Histone H3K4 demethylation is negatively regulated by
histone H3 acetylation in Saccharomyces cerevisiae. Proc Natl Acad Sci U
S A 109, 18505-18510, doi:Doi 10.1073/Pnas.1202070109 (2012).
Taverna, S. D. et al. Yng1 PHD finger binding to H3 trimethylated at K4
promotes NuA3 HAT activity at K14 of H3 and transcription at a subset of
targeted
ORFs.
Mol
Cell
24,
785-796,
doi:Doi
10.1016/J.Molcel.2006.10.026 (2006).
Park, S., Mori, R. & Shimokawa, I. Do sirtuins promote mammalian
longevity?: A Critical review on its relevance to the longevity effect
induced by calorie restriction. Mol Cells 35, 474-480, doi:Doi
10.1007/S10059-013-0130-X (2013).
Alper, B. J. et al. Sir2 is required for Clr4 to initiate centromeric
heterochromatin assembly in fission yeast. Embo Journal 32, 2321-2335,
doi:Doi 10.1038/Emboj.2013.143 (2013).
Sun, Z. et al. Deacetylase-Independent Function of HDAC3 in
Transcription and Metabolism Requires Nuclear Receptor Corepressor. Mol
Cell 52, 769-782, doi:Doi 10.1016/J.Molcel.2013.10.022 (2013).
Wang, Z. B. et al. Genome-wide Mapping of HATs and HDACs Reveals
Distinct Functions in Active and Inactive Genes. Cell 138, 1019-1031,
doi:Doi 10.1016/J.Cell.2009.06.049 (2009).
Negre, N. et al. A cis-regulatory map of the Drosophila genome. Nature
471, 527-531, doi:10.1038/nature09990 (2011).
107
108
109
110
111
112
113
114
115
116
117
118
119
120
Achary, B. G., Campbell, K. M., Co, I. S. & Gilmour, D. S. RNAi screen in
Drosophila larvae identifies histone deacetylase 3 as a positive regulator of
the hsp70 heat shock gene expression during heat shock. Biochim Biophys
Acta, doi:10.1016/j.bbagrm.2014.02.018 (2014).
Tadros, W. & Lipshitz, H. D. The maternal-to-zygotic transition: a play in
two acts. Development 136, 3033-3042, doi:Doi 10.1242/Dev.033183
(2009).
Sung, H. W., Spangenberg, S., Vogt, N. & Grosshans, J. Number of
Nuclear Divisions in the Drosophila Blastoderm Controlled by Onset of
Zygotic Transcription. Current Biology 23, 133-138, doi:Doi
10.1016/J.Cub.2012.12.013 (2013).
Chen, K. et al. A global change in RNA polymerase II pausing during the
Drosophila midblastula transition. Elife 2, e00861, doi:10.7554/eLife.00861
(2013).
Hendrix, D. A., Hong, J. W., Zeitlinger, J., Rokhsar, D. S. & Levine, M. S.
Promoter elements associated with RNA Pol II stalling in the Drosophila
embryo. Proc Natl Acad Sci U S A 105, 7762-7767,
doi:10.1073/pnas.0802406105 (2008).
Hanyu-Nakamura, K., Sonobe-Nojima, H., Tanigawa, A., Lasko, P. &
Nakamura, A. Drosophila Pgc protein inhibits P-TEFb recruitment to
chromatin in primordial germ cells. Nature 451, 730-U737, doi:Doi
10.1038/Nature06498 (2008).
Lesch, B. J. & Page, D. C. Genetics of germ cell development. Nat Rev
Genet 13, 781-794, doi:Doi 10.1038/Nrg3294 (2012).
Heyn, P. et al. The Earliest Transcribed Zygotic Genes Are Short, Newly
Evolved, and Different across Species. Cell Rep 6, 285-292, doi:Doi
10.1016/J.Celrep.2013.12.030 (2014).
Venken, K. J. & Bellen, H. J. Chemical mutagens, transposons, and
transgenes to interrogate gene function in Drosophila melanogaster.
Methods, doi:10.1016/j.ymeth.2014.02.025 (2014).
St Johnston, D. Using mutants, knockdowns, and transgenesis to investigate
gene function in Drosophila. Wiley Interdiscip Rev Dev Biol 2, 587-613,
doi:10.1002/wdev.101 (2013).
Kloosterman, W. P., Wienholds, E., de Bruijn, E., Kauppinen, S. &
Plasterk, R. H. A. In situ detection of miRNAs in animal embryos using
LNA-modified oligonucteotide probes. Nat Methods 3, 27-29, doi:Doi
10.1038/Nmeth843 (2006).
Lagendijk, A. K., Moulton, J. D. & Bakkers, J. Revealing details: whole
mount microRNA in situ hybridization protocol for zebrafish embryos and
adult tissues. Biol Open 1, 566-569, doi:10.1242/bio.2012810 (2012).
Bassett, A. & Liu, J. L. CRISPR/Cas9 mediated genome engineering in
Drosophila. Methods, doi:10.1016/j.ymeth.2014.02.019 (2014).
Gratz, S. J. et al. Highly Specific and Efficient CRISPR/Cas9-Catalyzed
Homology-Directed
Repair
in
Drosophila.
Genetics,
doi:10.1534/genetics.113.160713 (2014).
57
121
122
123
124
125
126
127
128
129
130
131
58
Bassett, A. R., Tibbit, C., Ponting, C. P. & Liu, J. L. Mutagenesis and
homologous recombination in Drosophila cell lines using CRISPR/Cas9.
Biol Open 3, 42-49, doi:10.1242/bio.20137120 (2014).
Gratz, S. J. et al. Genome Engineering of Drosophila with the CRISPR
RNA-Guided Cas9 Nuclease. Genetics 194, 1029-+, doi:Doi
10.1534/Genetics.113.152710 (2013).
Bateman, J. R., Lee, A. M. & Wu, C. T. Site-specific transformation of
Drosophila via phiC31 integrase-mediated cassette exchange. Genetics 173,
769-777, doi:10.1534/genetics.106.056945 (2006).
Bischof, J. et al. A versatile platform for creating a comprehensive UASORFeome library in Drosophila. Development 140, 2434-2442, doi:Doi
10.1242/Dev.088757 (2013).
Delubac, D. et al. Microfluidic system with integrated microinjector for
automated Drosophila embryo injection. Lab Chip 12, 4911-4919, doi:Doi
10.1039/C2lc40104e (2012).
Rothbauer, U. et al. Targeting and tracing antigens in live cells with
fluorescent
nanobodies.
Nat
Methods
3,
887-889,
doi:Doi
10.1038/Nmeth953 (2006).
Caussinus, E., Kanca, O. & Affolter, M. Fluorescent fusion protein
knockout mediated by anti-GFP nanobody. Nat Struct Mol Biol 19, 117U142, doi:Doi 10.1038/Nsmb.2180 (2012).
Sparkes, A. et al. Towards Robot Scientists for autonomous scientific
discovery. Autom Exp 2, 1, doi:10.1186/1759-4499-2-1 (2010).
Shilatifard, A., Conaway, R. C. & Conaway, J. W. The RNA polymerase II
elongation complex. Annu Rev Biochem 72, 693-715, doi:Doi
10.1146/Annurev.Biochem.72.121801.161551 (2003).
Gunesdogan, U., Jackle, H. & Herzig, A. A genetic system to assess in vivo
the functions of histones and histone modifications in higher eukaryotes.
EMBO Rep 11, 772-776, doi:10.1038/embor.2010.124 (2010).
Pengelly, A. R., Copur, O., Jackle, H., Herzig, A. & Muller, J. A histone
mutant reproduces the phenotype caused by loss of histone-modifying
factor Polycomb. Science 339, 698-699, doi:10.1126/science.1231382
(2013).