Linköping University Medical Dissertations No. 1086
Histone H1
Subtypes and phosphorylation in cell life
and death
Anna Gréen
Division of Cell Biology
Department of Clinical and Experimental Medicine
Faculty of Health Sciences
Linköping University
SE-581 85 Linköping, SWEDEN
Linköping 2009
Cover:
A picture of Bråviken in Kolmården
© Anna Gréen, 2008
All rights reserved.
ISBN: 978-91-7393-757-3
ISSN: 0345-0082
Published articles have been reprinted with permission from the publishers:
Paper I © 2005 Wiley-Blackwell, FEBS journal.
Paper II © 2008 American Chemical Society, Biochemistry.
Printed in Sweden by LTAB Linköpings Tryckeri AB, Sweden, 2008.08-0813.
To my family
Henrik, Linnea & Julia
Mamma & Pappa
Lars & Rebecka
-
I am among those who think
that science has great beauty. A
scientist in his laboratory is not
only a technician: he is also a
child placed before natural
phenomena which impress him
like a fairy tale
Marie Curie, 1933
TABLE OF CONTENTS
ABSTRACT ................................................................................................... 10
POPULÄRVETENSKAPLIG SAMMANFATTNING............................. 12
PAPERS IN THE PRESENT THESIS ....................................................... 15
ABBREVIATIONS ....................................................................................... 16
INTRODUCTION......................................................................................... 17
THE ORGANISATION OF DNA INTO CHROMATIN ........................................... 17
DNA and the chromosomes ....................................................................................... 17
The nucleosome ......................................................................................................... 18
Chromosome structure ............................................................................................... 20
REGULATION OF THE CHROMATIN STRUCTURE-REGULATION OF GENES........ 21
THE STRUCTURAL ROLE AND PHYSIOLOGICAL FUNCTION OF HISTONE H1 .... 22
Chromatin structure.................................................................................................... 22
Gene regulation .......................................................................................................... 22
Inhibition of replication.............................................................................................. 23
Apoptosis.................................................................................................................... 23
THE STRUCTURE OF HISTONE H1.................................................................. 25
HISTONE H1 GENES, REGULATION AND EXPRESSION .................................... 29
HISTONE H1 SUBTYPES................................................................................. 30
THE SIGNIFICANCE OF MULTIPLE H1 SUBTYPES ............................................ 34
Sequence conservation ............................................................................................... 34
Gene regulation .......................................................................................................... 34
Knock-out experiments .............................................................................................. 34
Affinity for DNA and chromatin................................................................................ 35
Distribution in chromatin ........................................................................................... 35
POSTTRANSLATIONAL MODIFICATIONS OF H1 HISTONES .............................. 36
PHOSPHORYLATION OF HISTONE H1 ............................................................. 38
Significance of H1 phosphorylation........................................................................... 38
Histone H1 phosphorylating and dephosphorylating enzymes .................................. 38
H1 phosphorylation in gene regulation ...................................................................... 40
Histone H1 phosphorylation and chromatin condensation ........................................ 40
H1 phosphorylation in the cell cycle.......................................................................... 41
Histone H1 phosphorylation in apoptosis .................................................................. 43
Histone H1 phosphorylation pattern in malignant transformation............................. 44
AIMS OF THE THESIS............................................................................... 45
MATERIAL AND METHODS.................................................................... 47
HISTONE H1 EXTRACTION ............................................................................ 48
SEPARATION OF H1 HISTONES AND THEIR PHOSPHORYLATED VARIANTS ...... 50
Reversed Phase-High Performance Liquid Chromatography (RP-HPLC) of H1
histones ........................................................................................................................... 50
Hydrophilic Interaction Liquid Chromatography (HILIC) ........................................ 51
High Performance Capillary Electrophoresis (HPCE)............................................... 51
GENETIC ANALYSIS ...................................................................................... 52
DNA-samples............................................................................................................. 52
PCR amplification of H1 genes or gene fragments.................................................... 53
DNA sequencing ........................................................................................................ 54
Detection of sequence variations ............................................................................... 54
Restriction Fragment Length Polymorphism (RFLP) analysis ......................................... 54
Denaturing HPLC (Wave) ................................................................................................ 55
FLOW CYTOMETRY ....................................................................................... 57
Cell cycle distribution ................................................................................................ 58
Cell tracing ................................................................................................................. 60
Apoptosis detection.................................................................................................... 60
Cell Sorting using FACS Aria Special order system ................................................. 63
CELL CULTURE ............................................................................................. 64
Jurkat .......................................................................................................................... 64
T cell activation.......................................................................................................... 64
Ag1523....................................................................................................................... 65
IMMUNOCYTOCHEMISTRY ............................................................................ 65
RESULTS AND DISCUSSION ................................................................... 67
THE EXISTENCE OF H1 MICROSEQUENCE VARIANTS ..................................... 67
HISTONE H1 AND H1 PHOSPHORYLATION IN APOPTOSIS ............................... 68
HISTONE H1 SUBTYPE DISTRIBUTION DURING CELL DIVISION ....................... 70
HISTONE H1 PHOSPHORYLATION IN THE CELL CYCLE ................................... 72
CONCLUSIONS............................................................................................ 75
FUTURE ASPECTS ..................................................................................... 77
ACKNOWLEDGEMENTS.......................................................................... 79
REFERENCES .............................................................................................. 84
PAPERS ......................................................................................................... 99
ABSTRACT
The genetic information of a human diploid cell is contained within
approximately 2 metres of linear DNA. The DNA molecules are compacted
and organized in various ways to fit inside the cell nucleus. Various kinds of
histones are involved in this compaction. One of these histones, histone H1 is
the topic of the present thesis. In addition to its structural role, H1 histones
have been implicated in various processes, for example gene regulation and
inhibition of chromatin replication.
H1 histones, also termed linker histones, are relatively conserved proteins,
and the various subtypes seem to have different and important functions even
though redundancy between the subtypes has been demonstrated. Despite the
sequence conservation of H1 subtypes, two sequence variations were detected
within the H1.2 and H1.4 subtypes using hydrophilic interaction liquid
chromatographic separation of H1 proteins from K562 and Raji cell lines in
Paper I in the present thesis. The variations were confirmed by genetic
analysis, and the H1.2 sequence variation was also found in genomic DNA of
normal blood donors, in an allele frequency of 6.8%. The H1.4 sequence
variation was concluded to be Raji specific. The significance of H1
microsequence variants is unclear, since the physiological function of H1
histones remains to be established.
H1 histones can be phosphorylated at multiple sites. Changes in H1
phosphorylation has been detected in apoptosis, the cell cycle, gene
regulation, mitotic chromatin condensation and malignant transformation.
Contradictory data have been obtained on H1 phosphorylation in apoptosis,
and many results indicate that H1 dephosphorylation occurs during apoptosis.
We and others hypothesized that cell cycle effects by the apoptosis inducers
may have affected previous studies. In Paper II, the H1 phosphorylation
pattern was investigated in early apoptosis in Jurkat cells, taking cell cycle
effects into account. In receptor-mediated apoptosis, apoptosis occurs with a
mainly preserved phosphorylation pattern, while Camptothecin induced
apoptosis results in rapid dephosphorylation of H1 subtypes, demonstrating
that H1 dephosphorylation is not a general event in apoptosis, but may occur
upon apoptosis induction via the mitochondrial pathway. The
dephosphorylation may also be a result of early cell cycle effects or signalling.
Therefore, the H1 phosphorylation pattern in the cell cycle of normal
activated T cells was investigated in Paper IV in this thesis. Some studies,
which have been made using cancer cell lines from various species and cell
synchronization, have indicated a sequential addition of phosphate groups
across the cell cycle. Normal T cells and cell sorting by flow cytometry were
used to circumvent side-effects from cell synchronization. The data
demonstrate that a pattern with phosphorylated serines is established in late
G1/early S phase, with some additional phosphorylation occurring during S,
and further up-phosphorylation seems to occur during mitosis. Malignant
transformation may lead to an altered G1 H1 phosphorylation pattern, as was
demonstrated using sorted Jurkat T lymphoblastoid cells.
During mitosis, certain H1 subtypes may be relocated to the cytoplasm. In
Paper III, the location of histones H1.2, H1.3 and H1.5 during mitosis was
investigated. Histone H1.3 was detected in cell nuclei in all mitotic stages,
while H1.2 was detected in the nucleus during prophase and telophase, and
primarily in the cytoplasm during metaphase and early anaphase. H1.5 was
located mostly to chromatin during prophase and telophase, and to both
chromatin and cytoplasm during metaphase and anaphase. Phosphorylated
H1 was located in chromatin in prophase, and in both chromatin and
cytoplasm during metaphase, anaphase and telophase, indicating that the
mechanism for a possible H1 subtype relocation to the cytoplasm is
phosphorylation.
In conclusion, data obtained during this thesis work suggest that H1
histones and their phosphorylation may participate in the regulation of events
in the cell cycle, such as S-phase progression and mitosis, possibly through
altered interactions with chromatin, and/or by partial or complete removal of
subtypes or phosphorylated variants from chromatin.
POPULÄRVETENSKAPLIG SAMMANFATTNING
I varje cell i människokroppen finns ungefär 2 meter DNA. DNA kodar
för proteiner som utför alla nödvändiga funktioner i cellen. För att den långa
DNA molekylen ska få plats i cellens kärna, och för att undvika brott och
trassel av DNA, så kompakteras och organiseras DNA molekylerna till en
struktur som kallas kromatin. Den första nivån av kompaktering medför att en
grupp av proteiner, histoner, organiserar DNA. Det innebär att DNA lindas
ungefär 2 varv runt en ”oktamer” av core histoner, vilket sker längs hela DNA
molekylen, som nu liknar ett pärlhalsband, eller en rad av små trådrullar med
tråd emellan. På ut/ingången av DNA sitter ytterligare ett histon protein
bundet, histon H1. Histon H1 och dess funktion i cellen är ämnet för denna
avhandling.
Trots att histon H1 finns i stor mängd i våra celler så är proteinets funktion
oklar. Förutom att histon H1 har en roll i att kompaktera och organisera
DNA, så tros den också vara inblandad i reglering av gener, celldelning och
celldöd (apoptos). Histon H1 är egentligen en familj av proteiner, ibland med
skilda funktioner. I denna avhandling har jag studerat de H1 subtyper som
finns i de flesta celltyper, histon H1.2, H1.3, H1.4 och H1.5. Dessutom kan
H1 proteinerna modifieras med fosforyleringar. Också funktionen av dessa
fosforyleringar är oklar, men tros involvera reglering av genuttryck, celldelning
och celldöd och kan därför också vara involverad i uppkomst av cancer.
H1 histoner är relativt konserverade proteiner, d v s deras
aminosyrasekvens är relativt lika i olika arter. I denna avhandling upptäcktes
små variationer i vissa H1 typer då vi använt olika tekniker för att separera H1
histoner från olika cellinjer. Dessa variationer detekterades i histonerna H1.2
och H1.4. Med hjälp av olika genetiska analyser har jag kunnat bekräfta de
korresponderande variationerna i DNA från cellinjerna, samt funnit att en av
variationerna också finns hos normala blodgivare, medan den andra endast
hittats hos en cellinje. Eftersom funktionen av H1 histonerna är oklar, så är
betydelsen av sekvensvariationerna svårbedömd.
Vidare har det i denna avhandling också undersökts om det finns en
koppling mellan H1 histonernas fosforyleringar och programmerad celldöd,
vilket har indikerats i andra studier. Dessa studier har inte undersökt om en
eventuell bieffekt av påverkan av cellcykeln (processen där en cell fördubblar
sitt DNA och slutligen delar sig i två dotterceller) kan vara förklaringen till
den snabba defosforyleringen man observerat. I vår studie visas att tidig
apoptos, inducerad via Fas receptorn i Jurkatceller (T lymfoblastoida celler)
sker med bibehållen fosforylering, medan apoptos inducerad via DNA skador
leder till defosforylering utan uppmätt påverkan på cellcykeln. Av detta har
dragits slutsatsen att defosforylering inte är en generell mekanism i tidig
apoptos, men kan vara ett resultat av apoptos som inducerats via cellens
mitokondrier. Eftersom H1s fosforyleringsmönster i cellcykeln hos humana
celler är oklar, kan vi inte säkert veta om den mätta defosforyleringen är ett
resultat av cellcykeleffekter som ej kan mätas.
Jag har därför fortsatt att undersöka histon H1s fosforylering i cellcykeln
hos normala, aktiverade T celler från blodgivare. Vi har sedan sorterat cellerna
i de olika cellcykelfaserna G1, S och G2/M, och analyserat H1 histonerna och
deras fosforyleringar i dessa faser. I vissa tidigare studier av olika cellinjer från
hamster, mus, råtta och humana cancerceller har man observerat en gradvis
stigande fosforylering genom cellcykeln, men då använt kemiska ämnen och
tekniker som stoppar cellcykeln i olika faser. Eftersom detta kan ha olika
bieffekter så har vi istället använt oss av cellsortering. I vår studie har vi funnit
att ett visst fosforyleringsmönster etableras i sen G1-fas eller i tidig S-fas, sen
sker endast viss vidare fosforylering i S-fas, medan maximal fosforylering
antagligen sker under mitosfasen. Möjligen kan det första
fosforyleringsmönstret vara kopplat till processen där DNA kopieras
(replikation). Vi har också undersökt den cellcykelspecifika fosforyleringen i
cellcykeln hos Jurkat celler. I dessa celler har vi funnit att det första
fosforyleringsmönstret uppkommer tidigare i cellcykeln. Detta kan bero på att
dessa celler delar sig snabbare eller att det är en del av den mekanism som
leder till att cancerceller delar sig ohämmat.
Vi har också undersökt de olika H1 subtypernas lokalisation under själva
celldelningens (mitosens) olika faser med immunocytokemi, och funnit att
H1.3 finns i cellkärnan under alla faser. H1.2 detekterades i kärnan under
profas och telofas, medan det mesta av H1.2 är lokaliserat i cellens cytoplasma
under metafasen och tidig anafas. Distributionen av H1.5 liknar den av H1.2,
förutom att H1.5 är lokaliserat till både cellkärnan och cytoplasman under
metafas och anafas. Jag arbetar nu för att verifiera dessa resultat med andra
metoder. Omlokalisering av olika H1 subtyper kan vara viktigt för
”omprogrammering” av cellens arvsmassa under celldelningen.
PAPERS IN THE PRESENT THESIS
This thesis is based on the following Papers, which will be referred to by their Roman
numerals:
I.
Characterization of sequence variations in human histone H1.2 and
H1.4 subtypes
Bettina Sarg, Anna Gréen, Peter Söderkvist, Wilfried Helliger, Ingemar
Rundquist and Herbert H. Lindner. FEBS Journal 272: 3673–3683, 2005.
II.
Histone H1 Dephosphorylation Is Not a General Feature in Early
Apoptosis
Anna Gréen, Bettina Sarg, Elisavet Koutzamani, Ulrika Genheden, Herbert
H. Lindner and Ingemar Rundquist. Biochemistry 47: 7539–7547, 2008
III.
Translocation of Histone H1 Subtypes Between Chromatin and
Cytoplasm During Mitosis in Normal Human Fibroblasts.
Anna Gréen, Anita Lönn, Kajsa Holmgren Peterson, Karin Öllinger, and
Ingemar Rundquist. Submitted.
IV.
Histone H1 interphase phosphorylation pattern becomes largely
established during G1/S transition in proliferating cells.
Anna Gréen, Bettina Sarg, Henrik Gréen, Anita Lönn, Herbert Lindner
and Ingemar Rundquist
ABBREVIATIONS
The most important abbreviations used in
this thesis are listed below:
Cdk
CFSE
DHPLC
FLICA
GFP
HILIC
HPCE
IL-2
MS
PBL
PCA
PCR
PFA
PHA
PI
RFLP
RP-HPLC
SNP
TCA
TFA
TFE
cyclin dependent kinase
5(6)-carboxyfluorescein diacetate N-succinimidyl ester
denaturing high performance liquid chromatography
flourescent inhibitor of caspases
green florescent protein
hydrophilic interaction liquid chromatography
high performance capillary electrophoresis
interleukin 2
mass spectrometry
pheripheral blood lymphocytes
percloric acid
polymerase chain reaction
paraformaldehyde
phytohemagglutinin
propidium iodide
restriction fragment length polymorphism
reversed phase-high performance liquid chromatography
single nucleotide polymorphism
trichloro acetic acid
trifluoroacetic acid
trifluoroethanol
INTRODUCTION
The organisation of DNA into chromatin
DNA and the chromosomes
Life is dependent on the retrieving, storage, translation and transmission of
genetic information in cells. For a long time, the source of this genetic
information was unknown. Through pioneering work of Friedrich Miescher,
who discovered a substance he named nuclein, and work by, among others
Albrecht Kossel, Walther Flemming and Oskar Hertwig chromosomes were
suggested to be the carriers of genetic information (van Holde, 1989). When
the structure of DNA (deoxyribonucleic acid) was elucidated by Watson and
Crick in 1953, DNA was concluded to be the carrier of genetic information,
which encodes the amino acid sequence of proteins. DNA is organised as a
double helix, made from two complementary DNA strands. The strands are
composed of four different nucleotides, A, T, C and G. The order of the
nucleotides determines the sequence of all proteins. In a diploid human cell,
there are 6x109 base pairs of DNA, all together approximately 2 m of DNA
double helix. These 2 m of DNA double helix are compacted to fit in the cell
nucleus. This compaction is accomplished through organisation of DNA by
various proteins into a structure called chromatin. The word chromatin was
postulated by Flemming in 1879 for the stainable material in cell nuclei
INTRODUCTION | 17
(Paweletz, 2001). In addition to be compact, chromatin structure needs to be
flexible to be accessible for processes like replication, transcription and DNA
repair. The compaction of DNA into chromatin involves two classes of
proteins, the histones and the non-histone chromosomal proteins. Histones
were discovered and named by Albrecht Kossel in 1884 (Olins and Olins,
2003). Histones are responsible for the first level of DNA condensation into
chromatin, the organisation of DNA in nucleosomes. The basic repeating unit
in chromatin was proposed by Kornberg in 1974 to consist of 2 copies of
each core histone (H2A, H2B, H3 and H4) and 200 bp of DNA, which forms
throughout the DNA giving an appearance like ‘beads on a string’ (Kornberg,
1974), which has a diameter of 11 nm (figure 1). The same hypothesis had
also been put forward by Olins and Olins (Olins and Olins, 2003). An H1
histone is associated with the nucleosome (Thomas, 1999). Chromatin is
further compacted into chromosomes, which are dispersed in the cell nucleus
during interphase when the cell is not dividing, and become highly condensed
during mitosis (cell division) (figure 1).
The nucleosome
The basic subunit of chromatin was originally defined by digestion of
chromatin with the enzyme micrococcal nuclease, which results in the release
of mononucleosomes (figure 2). Further treatment with micrococcal nuclease
trims linker DNA (the DNA between the nucleosome core particles) and
results in a particle with approximately 166 bp of DNA, an octamer of core
histones and a H1 histone (Thomas, 1999). Even further digestion results
produces nucleosome core particles, with 146 bp of DNA and the corehistone octamer (Ramakrishnan, 1997b; Thomas, 1999). The core histones are
small, highly conserved proteins (102-135 aa), which have a common
structural motif, the histone fold, which consists of three α-helices connected
by two loops (Arents et al., 1991; Arents and Moudrianakis, 1995). There are
four different core histones, H2A, H2B, H3 and H4. In the nucleosome core
particle, H2A and H2B dimerises, and H3 and H4 dimerises (Arents et al.,
1991). Then two H3-H4 dimers form a tetramer, to which two H2A-H2B
dimers bind, to create the octamer (Arents et al., 1991). The core histones
contain many lysine and arginine residues which have a positive charge, which
attracts the negatively charged DNA
18 | INTRODUCTION
INTRODUCTION | 19
Figure 2. A schematic drawing of the nucleosome
Figure 1. The compaction of DNA into chromatin and higher order chromatin structure (adapted from
Richard Wheeeler at en.wikipedia)
backbone. In addition, many hydrogen bonds and hydrophobic interactions
take place between DNA and the core histones. The structure of the
nucleosome core particle was determined by X-ray crystallography and 146 bp
of DNA was found to be wrapped 1.65 turns around the histone octamer
(Luger et al., 1997). The positioning of the nucleosomes on DNA is
determined by specific DNA sequences (Nucleosome positioning sequences)
(Ramakrishnan, 1997b; Schnitzler, 2008) and other proteins that bind strongly
to DNA. The positions of nucleosomes are modulated by chromatin
remodelling enzymes (Schnitzler, 2008), sometimes together with histone
chaperones.
To complete the nucleosome (figure 2), a linker histone (H1 or H5) is
bound to the nucleosome core particle, and thereby protects another 20 bp of
linker DNA, which organise the entry and exit of DNA on the nucleosome
(Sivolob and Prunell, 2003). Nucleosomes are connected with linker DNA,
and the average length varies between cell types and species (Ramakrishnan,
1997a). The location of linker histones on the nucleosome has been debated
(Thomas, 1999; Travers, 1999) but is believed to be of vital importance since
linker histones are involved in the packaging of nucleosomal arrays into the 30
nm fibre (figure 1). The main issue involves symmetric or asymmetric binding
of the globular domain of linker histones to the nucleosome dyad. The
structure of the 30 nm fibre is not completely resolved yet, and different
models for the packaging have been proposed (Ramakrishnan, 1997a;
Robinson and Rhodes, 2006; Staynov, 2008). Even though chromatin
compaction can be achieved in the absence of linker histones, many studies
suggest that linker histones are either needed for the formation of the 30 nm
fibre (Robinson and Rhodes, 2006), or stabilizes the fibre once formed
(Carruthers et al., 1998; Ramakrishnan, 1997a).
Chromosome structure
The process where the 30 nm fibre is further condensed is not completely
understood, but probably involves looping of the fibre (figure 1) along a
chromosomal axis (Belmont, 2006). In the mitotic chromosomes, even further
packaging is performed probably involving condensins (Belmont, 2006;
Watrin and Legagneux, 2003).
20 | INTRODUCTION
Regulation of the chromatin structure-regulation of genes
In different parts of the genome, different types of chromatin higher order
structures occur, which is a part of gene expression control. Some of these
structures are inherited, which is called epigenetic inheritance. The term
epigenetics originally was defined as ‘the study of heritable phenotype changes
that do not involve alterations in DNA sequence’, but the term epigenetic has
developed into meaning ‘the information carried by the genome (e.g., on
chromatin) that is not coded by DNA’ (Kouzarides, 2007). There are two
types of chromatin, heterochromatin which is condensed and euchromatin,
which is less condensed during interphase compared to heterochromatin,
which was recognized by Heitz in 1928 (Heitz, 1928). Heterochromatin is
present in condensed regions of the cell nucleus, like centromeres and
telomeres. Gene expression is highly suppressed in, but not absent from
heterochromatin (Dimitri et al., 2005). Euchromatin generally corresponds to
regions with active genes. Different chromatin regions are separated by
specific DNA sequences that prevent spreading of heterochromatin into
euchromatic regions and vice versa.
The epigenetic regulation, involving the chromatin structure is executed via
mechanisms like DNA methylation (Jaenisch and Bird, 2003) and post
translational modifications of core histones. A ‘histone code’ has been
proposed (Jenuwein and Allis, 2001) where the different combinations of core
histone modifications are believed to provide signals for gene regulation via
chromatin structure. These signals may also be involved in DNA replication
and repair (Kouzarides, 2007).
Gene regulation can also be executed via ATP-dependent remodelling of
chromatin, where the structure, content and location of nucleosomes are
altered (Saha et al., 2006).
Histone H1, the topic of this thesis, has been implicated in the regulation
of chromatin structure in various ways, and a linker histone code has been
suggested (Godde and Ura, 2008).
INTRODUCTION | 21
The structural role and physiological function of histone H1
The determination of the physiological function of H1 histones has been,
and still is a challenging task. H1 histones have been implicated in a number
of processes, but the exact role of H1 histones remains to be determined. The
difficulty in H1 research is partly due to the presence of multiple subtypes and
viable H1 gene knockouts (Fan et al., 2001; Shen et al., 1995; Sirotkin et al.,
1995). Gene targeting strategies in mice have shown that the H1 subtypes are,
to a certain extent, functionally redundant but also that H1 histones are
essential in mouse development (Fan et al., 2003).
Chromatin structure
Histone H1 is believed to be involved in the compaction of chromatin
through its C-terminal tail (Allan et al., 1986). Many studies show the
involvement of H1 histones in chromatin condensation, and removal of H1
histones from chromatin results in decondensation of chromatin (Robinson
and Rhodes, 2006; Shen et al., 1995). It has also been suggested that histone
H1 is essential for stabilization of the condensed structures, rather than
promoting condensation (Carruthers et al., 1998; Ramakrishnan, 1997a).
Knockout-experiments in mice show that reduced H1 levels results in
decreased nucleosome repeat length, and local rather than global
decondensation of chromatin (Fan et al., 2005).
Histone H1 has also been implicated in the structure and segregation of
mitotic chromosomes, which was shown in experiments on Xenopus laevis egg
extracts (Maresca et al., 2005), while in other experiments chromosome
condensation was obtained without histone H1 (Ohsumi et al., 1993).
Gene regulation
The function of H1 histones in gene regulation has been debated. Since
active chromatin is relatively depleted of histone H1 compared to inactive
chromatin (Woodcock et al., 2006), histone H1 was initially assigned the role
of a general gene repressor (Weintraub, 1984), and a number of studies are in
support of this view (Brown, 2003). Other studies favour the hypothesis that
H1 exert gene-specific regulation (Crane-Robinson, 1999; Thomas, 1999;
Wolffe et al., 1997). Histone H1 may function by inhibiting chromatin
22 | INTRODUCTION
remodelling (Horn et al., 2002), competing with other chromatin-binding
proteins (Brown, 2003), or by affecting positioning and stabilization of
nucleosomes (Brown, 2003; Crane-Robinson, 1999; Zlatanova et al., 2000).
H1 phosphorylation is also believed to influence gene regulation.
Knockout experiments in Tetrahymena thermophila has demonstrated that in
this organism with a single atypical H1, histone H1 does not seem to affect
global transcription, but regulates specific genes (Shen and Gorovsky, 1996).
Knock-out experiments in mice have demonstrated that only a few genes are
affected by simultaneous H1c, H1d and H1e depletion in ES cells, leading to
embryonic lethality, and that many of the affected genes are normally
regulated by DNA methylation, suggesting that histone H1 is involved in
epigenetic control (Fan et al., 2005).
Inhibition of replication
Histone H1 has been implicated in regulation of DNA replication in
Physarum polycephalum (Thiriet and Hayes, 2008), and was also found to inhibit
DNA replication using Xenopus Egg extracts (Lu et al., 1998; Lu et al., 1999).
Apoptosis
Histone H1 has been suggested to be involved in apoptosis, but the role of
H1 in apoptosis remains to be determined. The H1.2 subtype has a specific
role in apoptosis signalling (Konishi et al., 2003). Histone H1 has been
implicated in apoptosis in a number of ways:
• Histone H1 interacts with and activates the major apoptotic nuclease,
DFF40 (CAD in mice) in vitro (Liu et al., 1999).
• Histone H1 may change location upon caspase activation, possibly to the
periphery of the nucleus (Ohsawa et al., 2008), and is found in the cytoplasm
after DNA fragmentation in some systems (Gabler et al., 2004; Wu et al.,
2002).
• Posttranslational modifications of H1 histones may be altered upon
apoptosis induction, for example poly(ADP-ribosyl)ation at the time for
DNA fragmentation (Th'ng, 2001; Yoon et al., 1996) and phosphorylation
(Th'ng, 2001).
INTRODUCTION | 23
Apoptosis
The term apoptosis, a greek word for ‘falling off’ like leaves from a tree,
was first used in a paper by Kerr, Wyllie and Currie in 1972 (Kerr et al., 1972).
Apoptosis is described as a certain mode of programmed cell death (Elmore,
2007). Apoptosis occur during, for example, development, ageing, immune
system function, regulation of tissue homeostasis, and protection against
cytotoxic agents and DNA damage, for reviews see for example (Elmore,
2007; Khosravi-Far and Esposti, 2004; Krammer, 2000; Testa, 2004; Twomey
and McCarthy, 2005). Defects in the apoptotic system may lead to, for
example, cancer, autoimmune disease and neurodegenerative disease.
Apoptosis may be executed via two main pathways, the extrinsic or intrinsic
(mitochondrial) pathway (Elmore, 2007). The extrinsic pathway is activated by
engagement of death-receptors, like FAS (CD95) and TNF, while the intrinsic
pathway is activated upon extra- or intracellular stress, like DNA damage. The
extrinsic pathway is executed by formation of the death-inducing signalling
complex (DISC) and autocatalysis of procaspase-8 after death-receptor
activation. Depending of the cell type there are two different pathways
downstream of DISC formation and caspase-8 activation. In type II cells, to
which Jurkat belongs, the low levels of active caspase-8 and DISC formed
leads to amplification of the apoptotic signal by mitochondria through
cleavage of Bid (Krammer, 2000; Scaffidi et al., 1998) which engage
mitochondria mainly like in the intrinsic pathway (Khosravi-Far and Esposti,
2004). Apoptotic activation of mitochondria leads to permeabilisation of the
outer mitochondrial membrane and loss of mitochondrial potential through a
complex process involving pro-apoptotic and anti-apoptotic Bcl-2 family
proteins (Elmore, 2007). In this process cytochrome c and other proapoptotic
factors are released. Cytochrome C forms a complex (the apoptosome) with
apoptotic protease activating factor 1 (Apaf-1), procaspase-9 and other
factors, which activates caspase 3. Caspase 3, other caspases and proapoptotic factors activated by caspase-3 gives DNA fragmentation, membrane
blebbing, nuclear and cellular fragmentation and other apoptotic features
(Hengartner, 2000).
24 | INTRODUCTION
The structure of Histone H1
Histone H1, also called linker histones due to their association with linker
DNA, is not a single protein, but a protein family with at least 11 members,
H1.1-H1.5, H1.X, H1º, testis specific H1t, H1t2 and HILS1 and oocyte
specific H1oo (Ausio, 2006; Godde and Ura, 2008). Different nomenclatures
have been used for naming the H1 subtypes, the nomenclature used here was
introduced by Albig and Doenecke for human H1 histones. The most
commonly used nomenclatures are listed in a recent review (Izzo et al., 2008).
Most H1 histones have a tripartite structure, with a central globular domain
and basic N- and C-terminal tails (Parseghian and Hamkalo, 2001)(figure 3).
Since the subtypes H1.X, H1º, H1t, H1t2 and HILS1 and H1oo are only
found in certain cell types, none of which was studied in this thesis, and
exhibit many differences compared with the H1.1-H1.5 histones (figures 4
and 5) these will be described only briefly under the section on Histone H1
subtypes, and will not be included here.
Figure 3. The three-partite structure of H1 histones
The hydrophobic globular domains of Histones H1.1 to H1.5 share high
sequence homology (figure 5), while the N- and C-terminal tails are less
conserved. The differences between the subtypes primarily reside in the Cterminal tail (figure 5). The N-terminal includes approximately 40 residues, the
globular domain approximately 80 residues and the C-terminal tail the
remaining residues (approximately 95-105 aa) (Hartman et al., 1977). In an
aqueous solution, the hydrophobic globular domain is folded, while the
hydrophilic N-and C-terminal tails remain unstructured due to the many
lysines and arginines in the tails.
The structure of the globular domain contains a three-helix bundle, with a
β-hairpin (also called ‘wing’) in the C-terminus (Ramakrishnan, 1997b). The
globular domain is believed to bind at or near the nucleosome dyad, and may
function in sealing the two turns of DNA on the nucleosome (Woodcock et
INTRODUCTION | 25
al., 2006). Using green fluorescent protein (GFP) fused to H1º and
fluorescence recovery after photo bleaching (FRAP), the globular domain was
predicted to have two DNA-binding sites, one near the nucleosome dyad and
one in linker DNA (Brown et al., 2006), in agreement with many in vitro
studies (Catez et al., 2006). The globular domains of the various H1 subtypes
may differ in their binding within the nucleosome (Brown et al., 2006).
Very little is known about the N-terminal of H1 histones. In
trifluoroethanol (TFE) solutions, which stabilizes secondary structures, two αhelices are formed close to the globular domain (Vila et al., 2002), which may
be involved in positioning the globular domain on the nucleosome (Allan et
al., 1986; Vila et al., 2002), possibly in conjunction with the C-terminal tail
(Allan et al., 1986).
The C-terminal tail is assumed to be of vital importance in DNA
condensation (Allan et al., 1986). The DNA-condensing property of the Cterminal tail probably resides in a 34 aa stretch (residue 145-178 in rat H1d)
(Bharath et al., 2002). The α-helical content in the C-terminal tail increases in
TFE solutions, and is believed to adopt a structure of a ‘kinked’ α-helix upon
binding to DNA (Clark et al., 1988). Using H1º and DNA, the α-helical
content in the C-terminal domain was found to decrease upon partial
phosphorylation, and full phosphorylation resulted in further decreased αhelical content and increase in β-structure (Roque et al., 2008).
Histone H1 binding to chromatin is believed to be highly dynamic, as
measured by GFP-H1 molecules in FRAP experiments (Catez et al., 2006).
Both the globular domain and the C-terminal tail was identified as important
factors for interactions between H1 histones and chromatin, and
phosphorylations are likely to affect this interaction (Catez et al., 2006).
26 | INTRODUCTION
INTRODUCTION | 27
Figure 4. Multiple alignment by Clustal X of the amino acid sequences of the
known human H1 histones
N-terminal domain
Globular domain
Globular domain
C-terminal domain
C-terminal domain
Figure 5. Multiple alignment by Clustal X of the amino acid sequences of the human H1.1
to H1.5 histones
28 | INTRODUCTION
Histone H1 genes, regulation and expression
Gene expression of the various human histone H1 variants varies between
the subtypes. Subtype expression is either oocyte specific (H1oo), testis
specific (H1t, H1t2 and HILS1), replication-dependent (H1.1-H1.5) or
replication independent (H1º and H1.X) (Godde and Ura, 2008). The genes of
the somatic subtypes H1.1, H1.2, H1.3, H1.4 and H1t are located in the major
human histone gene cluster at chromosome 6 while H1.5 is located in a
second histone cluster about 2MB away (Albig and Doenecke, 1997;
Doenecke et al., 1997; Volz et al., 1997). The other subtypes are located on
other chromosomes.
In addition to TATA-boxes, histone H1 genes exhibit some special features
in their regulatory sequences, for example H1 boxes, CCAATCA boxes and
CH1UE boxes (Doenecke et al., 1994; Parseghian and Hamkalo, 2001). The
presence of different combinations of these and other boxes together with
differential location in the genome may contribute to differential regulation of
H1 genes. The mRNA synthesis of Histones H1.1-H1.5 are coupled to Sphase, but H1.2 and H1.4 has also been suggested to be both S-phase
dependent and independent (Parseghian and Hamkalo, 2001). Replicationdependent histones do not contain a poly-A tail, instead they end by a highly
conserved stem-loop, and their pre-mRNAs are processed by a specialized 3’
end processing machinery. The processing of replication-dependent histone
mRNA is believed to involve for example U7 small nuclear RNP (U7
snRNP), the 100 kDa zinc finger protein (ZPF100), endonucleases CPSF 73
and 100, and the stem-loop binding protein (SLBP) (Dominski and Marzluff,
2007). Both ZPF100 and U7 snRNP are located in Cajal bodies, and a fraction
of these are located close to histone gene loci in vertebrates (Dominski and
Marzluff, 2007) and are involved in processing of histone pre-mRNA (Gall,
2001). After pre-mRNA processing, SLBP remains bound to the histone
stem-loop structure, and stimulates histone translation in the cytoplasm
(Dominski and Marzluff, 2007). A high SLBP level in S-phase is followed by
rapid decline during G2/M, and is paralleled by histone mRNA degradation,
possibly giving SLBP a role in post-transcriptional regulation of histone
expression (Dominski and Marzluff, 2007).
INTRODUCTION | 29
After translation in the cytoplasm, histone H1 is probably imported into
the cell nucleus through the nuclear pore complexes. The import is believed
to be governed by binding of H1 to a complex with importin β and importin
7, and RanGTP is required to complete the import through the nuclear pore
complex (Jakel et al., 1999). Once in the nucleus, histone H1 is associated with
chromatin, while some H1 may be present in the nucleus bound to a protein
called NASP (Alekseev et al., 2003). Histone H1 can also be transported to
nuclei by cytoplasmic factors (Kurz et al., 1997), and in absence of these by
tNASP (Alekseev et al., 2005). NASP is believed to be a histone chaperone,
which assists in the inclusion of H1 histones onto nucleosome arrays (Finn et
al., 2008).
The regulation of histone H1 subtype expression and accumulation are
poorly understood processes, since the regulation seems to be executed at
many different levels. Histone H1 expression is connected with the
proliferative capacity of cells, and the regulatory sequences in H1 genes allow
the genes to be sub-divided into embryonic, replication dependent or
differentiation-specific H1 histones (Khochbin, 2001). The somatic subtypes
H1.1-H1.5 seems to be of two different types, one where expression
continues after cell proliferation is decreased (H1.2 and H1.4) and one where
cellular quiescence results in decline of subtypes (H1.1, H1.3 and H1.5)
(Parseghian and Hamkalo, 2001). Using mouse lymphoid cells, H1a and H1b
(corresponding to human H1.1 and H1.5) was found to be expressed in large
amounts only in dividing cells, while H1c, H1d and H1e (H1.2, H1.4 and
H1.3) was expressed in both dividing and non-dividing cells and H1.3
accumulated in non-dividing cells (Lennox and Cohen, 1983).
Histone H1 subtypes
For amino acid sequence alignments of all known human H1 subtypes see
figure 4, and for H1.1-H1.5 see figure 5.
When discussing the different subtypes and their expression it is important
to keep in mind that H1 histones may also be regulated by post-transcriptional
mechanisms, since the level of mRNA expression not always coordinate with
30 | INTRODUCTION
the levels of protein expression. This has been demonstrated for the H1º
subtype (Cuisset et al., 1999).
H1oo was detected in oogenesis and embryogenesis of mice, from the
secondary follicle oocyte to the two-cell stage embryo (Tanaka et al., 2003a).
Somatic H1 histone was not detectable in fully grown oocytes, 1-cell or 2-cell
mouse embryos, but was detected in early in the 4-cell stage (Clarke et al.,
1998), in another study, somatic H1 was detected at the late two-cell stage
(Stein and Schultz, 2000). H1oo mRNA contains polyadenylation unlike
replication-dependent H1 histones, and is longer (304 aa) than other H1
subtypes (Tanaka et al., 2003a). The corresponding human protein is called
osH1 and is a 347 aa protein, with 42.3% homology with mouse H1oo and
polyadenylated mRNA (Tanaka et al., 2003b).
At least three testis-specific variants exist in mammals, H1t which is present
solely in testis (Seyedin and Kistler, 1980), Hils1 which is a spermatid specific
protein (Iguchi et al., 2003; Yan et al., 2003), and H1T2 which is selectively
expressed in male haploid cells during spermiogenesis (Martianov et al., 2005).
All three proteins are distinctly different from histones H1.1-H1.5.
Histone H1.X is approximately 30% similar to the somatic H1 histones,
and have polyadenylated mRNA (Takata et al., 2007; Yamamoto and
Horikoshi, 1996). Histone H1.X is believed to be expressed in many tissues
(Yamamoto and Horikoshi, 1996), and to be accumulated in nucleoli during
G1, and distributed evenly in cell nuclei during S and G2 phases (Stoldt et al.,
2007). H1.X may be necessary for mitotic progression in HeLa cells (Takata et
al., 2007).
Histone H1º accumulates during terminal differentiation and in nondividing cells, which has been demonstrated in many studies using cell- and
tissue systems (Doenecke et al., 1994; Sekeri-Pataryas and Sourlingas, 2007).
Histone H1º is called a replacement variant since proliferating cells and tissues
contain mainly histones H1.1-H1.5, while after cell proliferation ceases and
cells differentiate, histone H1º accumulates and H1.1-H1.5 levels decline
(Clarke et al., 1998). The H1º mRNA is polyadenylated, and the expression of
INTRODUCTION | 31
H1º is not restricted to S-phase (Doenecke et al., 1994). Histone H1º is also
detected throughout oocyte growth, 2-and 4 cell stages, in reduced amounts at
the 8-cell stage and again in higher amounts at morula and blastocyst stages
(Clarke et al., 1998).
The presence of histone H1.1 is probably restricted to a few cell types.
Histone H1.1 mouse mRNA was only detected in thymus, testis and spleen
(Franke et al., 1998), and the corresponding H1a protein has also been
detected in mouse liver, kidney and lung before 4 weeks of age (Lennox and
Cohen, 1983), and in mouse testis, neurons and lymphocytic cells (Rasheed et
al., 1989). H1.1 could not be detected in human lymphocytes (Happel et al.,
2008).
The expression of H1.2 was shown to be both replication dependent and
independent (with a polyadenylated transcript), and this subtype is present in
most cell types (Parseghian and Hamkalo, 2001), and mRNA measurements
demonstrate that H1.2 levels were relative constant in various human cell lines
(Meergans et al., 1997). H1.2 seems to be uniformly distributed in nuclei from
human cells, possibly being responsible for the foundation of a basal level of
chromatin condensation (Parseghian and Luhrs, 2006). Histone H1.2 has been
reported to have a number of specialized functions in mammalian cells.
Histone H1.2 has been shown to be an important signalling molecule in
apoptosis promoted by induction of double strand breaks, where H1.2 is
involved in mitochondrial cytochrome c release (Konishi et al., 2003). H1.2 is
also a specific inhibitor of furin, a proprotein convertase (Han et al., 2006).
H1.2 may also be specifically involved in p53-mediated transcription, acting as
a part of a repressive complex (Kim et al., 2008). Recent data indicate that
H1.2 may be needed for cell cycle progression in certain cell lines (Sancho et
al., 2008).
Histone H1.3 expression is coupled to S-phase, and is probably decreased
upon quiescence and differentiation, but has a slow turnover (Parseghian and
Hamkalo, 2001) and is found in non-dividing somatic mouse cells (Lennox
and Cohen, 1983). H1.3 may be associated primarily with inactive chromatin,
32 | INTRODUCTION
and actively transcribed chromatin was selectively depleted of H1.3
(Parseghian and Hamkalo, 2001).
Histone H1.4 is present in most cell types and tissues, and the H1.4 content
is unaltered or higher after quiescence or differentiation (Parseghian and
Hamkalo, 2001). Possibly, H1.4 is mainly associated with inactive and
condensed chromatin (Parseghian and Hamkalo, 2001).
Histone H1.5 expression is reduced in differentiated and quiescent cells
(Parseghian and Hamkalo, 2001), and H1b (corresponding to human H1.5)
was expressed in high amounts only in dividing mouse cells (Lennox and
Cohen, 1983). H1.5 may be associated with less condensed chromatin, and
H1.5 is present in actively transcribed chromatin (Parseghian and Hamkalo,
2001). Histone H1.5 may also have specialized functions, for example in gene
regulation (Kaludov et al., 1997). Also, the mouse homologue H1b interacts
with Msx1, thereby repressing myogenic differentiation (Lee et al., 2004).
INTRODUCTION | 33
The significance of multiple H1 subtypes
Sequence conservation
The somatic H1 subtypes, H1.1-H1.5 exhibit 60-85% sequence similarity.
The H1 subtypes show higher interspecies conservation than intraspecies
conservation (Eirin-Lopez et al., 2004), i.e. the similarity between human H1.5
and mouse H1b (H1.5) is higher than the similarity between human H1.2 and
human H1.5. The hydrophobic globular domain is conserved, while the Nand C-terminal tails display less conservation (figures 4 and 5). Evolutionary
data suggest that H1 subtypes have evolved with functional differentiation
(Ponte et al., 1998). The evolution of H1 subtypes may have taken place
according to a birth-and-death model, with strong purifying selection for the
subtypes due to their functional roles (Eirin-Lopez et al., 2004).
Gene regulation
H1 histone subtypes may play differential roles in gene regulation, and the
regulation may be either positive or negative (Alami et al., 2003). Experiments
where H1º was over expressed resulted in cell cycle effects, and over
expression of H1c (corresponding to human H1.2) resulted in negligible
effects on some genes, while increased expression of other genes was detected
(Brown et al., 1996). In these experiments, the nucleosomal spacing was
increased (Gunjan et al., 1999).
Knock-out experiments
H1º was early associated with the process of differentiation, but once a H1º
knockout mouse was produced no detectable abnormalities were displayed
(Sirotkin et al., 1995). This was also true after H1t elimination, and
spermatogenesis proceeded normally in these mice (Drabent et al., 2000).
Also, mice devoid of either: H1c, H1d or H1e or H1 º in combination with
H1c, H1d or H1e are viable and exhibit no anatomic or histological
abnormalities (Fan et al., 2001). In all of these studies, the remaining subtypes
seem to compensate for the loss of subtypes and the level of their expression
is increased to maintain the normal linker-to-core ratio. When H1c, H1d and
H1e mouse triple-knockouts were produced, mouse embryos died with a
34 | INTRODUCTION
number of defects, and with a 50% reduction in H1 content, showing that H1
histones are essential in mammals (Fan et al., 2003). This experiment showed
that the amount of H1 is vital in embryonic development (Fan et al., 2003),
and that nucleosomal spacing and local chromatin compaction is reduced in
H1c, H1d and H1e depleted ES cells (Fan et al., 2005). H1T2 was shown to
be essential in spermiogenesis (Martianov et al., 2005), and H1.X is essential in
mitotic progression in HeLa cells (Takata et al., 2007). Inducible knockouts of
H1 subtypes produced in a human breast cancer cell line, indicated specific
roles for H1.4 in cell survival and for H1.2 in cell cycle progression (Sancho et
al., 2008).
Affinity for DNA and chromatin
H1 histones have been shown to differ in their affinity for chromatin or
DNA in a number of experimental systems. Using rat H1 histones, H1e
(H1.4), H1d (H1.3) and H1º was shown to bind with high affinity to
chromatin, while H1b (H1.5) and H1c (H1.2) had intermediate affinity and
H1a (H1.1) had the lowest affinity (Orrego et al., 2007). These results are in
partial agreement with GFP-experiments where H1.1 and H1.2 was more
weakly bound than H1º and H1.3, and than the subtypes with highest affinity,
H1.4 and H1.5 (Th'ng et al., 2005).
Distribution in chromatin
Several studies indicate that the H1 subtypes are localized differently in the
cell nucleus, between active and inactive chromatin (Orrego et al., 2007;
Parseghian and Hamkalo, 2001; Parseghian and Luhrs, 2006; Th'ng et al.,
2005). GFP-studies indicate that H1.1, H1.2 and H1.3 are more frequent in
euchromatic regions, while H1.4 and H1.5 are common in heterochromatic
regions (Th'ng et al., 2005). Immunohistological investigations indicate
uniform distribution of H1.2, association of H1.3 and H1.4 with inactive
chromatin, and H1.5 with active chromatin (Parseghian and Hamkalo, 2001).
INTRODUCTION | 35
Posttranslational modifications of H1 histones
A number of post-translational modifications have been detected on H1
histones, including ubiquitination, poly(ADP-ribosyl)ation, methylation,
acetylation and phosphorylation (Ausio et al., 2001; Daujat et al., 2005;
Wisniewski et al., 2007). Since we have focussed on H1 phosphorylation, this
modification will be described in more detail. H1 phosphorylation is
connected to the cell cycle and mitosis, and therefore a brief summary of
these events will be presented before the description of H1 phosphorylation.
The cell cycle and cell division
The cell cycle is the essential process where all living things duplicate.
During the cell cycle, a cell duplicates its content and divides in two in a set
order of events. Cell division is required for establishment of all organisms,
like the human body. It is also necessary for maintenance of the organism, for
example to grow and replace damaged cells in the different tissues. Failure in
the cell cycle control system often results in disproportionate cell divisions
which may lead to cancer. The cell cycle includes four different phases, G1, S,
G2 and M (figure 6). The cell can also leave the cell cycle in the G1 phase and
go into a resting state called G0 (figure 6). At the restriction point, G0 and G1
cells are committed to DNA synthesis.
The G1and G2 phases are gap phases with cell growth and control of DNA
integrity, while DNA synthesis takes place in S-phase to duplicate the all
DNA in the genome of the cell, and the nucleus and the cytoplasm divide in
M (mitosis) phase. Generally, a human cell spends about 10-12 h in S-phase,
one hour in M-phase, while the time spent in the gap-phases are more
variable. Progression in the cell cycle is controlled by an intricate control
system (Murray, 2004), where the cyclins and cyclin-dependent kinases (Cdk:s)
are the major regulatory proteins. The different cyclins bind to certain Cdk:s
to form an active cyclin-Cdk complex which acts by phosphorylation of other
cell regulatory proteins. The cyclins were discovered in 1983 (Evans et al.,
1983). There are three major checkpoints in the cell cycle, the restriction
point, the G2/M checkpoint and the metaphase-to-anaphase transition. After
mitogenic stimulation in G1, active cyclin D/Cdk4/Cdk6 complexes
36 | INTRODUCTION
phosphorylate Rb, (Blomen and Boonstra, 2007). Later in G1, activation of
cyclin E/Cdk2 promotes DNA replication (Murray, 2004) and further
phosphorylates Rb leading to its inactivation which release inhibition of E2F
transcription factors which are needed for entry into S-phase (Blomen and
Boonstra, 2007). The cell cycle is negatively regulated by the cyclin-dependent
kinase inhibitors (CKIs), which inhibit the cyclin/Cdk complexes. These can
be divided into two families, the INK4 and Cip/Kip family (Blomen and
Boonstra, 2007) (figure 6). Cyclin A/Cdk2 is a factor in controlling DNA
replication, while the Cyclin A/Cdk1 complex is active in G2 phase
progression (Kaldis and Aleem, 2005). During G2, cyclin A is degraded, and
cyclin B is expressed and binds Cdk1, the cyclin B/Cdk1 regulates, among
other things, events in the G2/M transition and in mitotic progression while
inactivation of the complex ensures exit from mitosis (Malumbres and
Barbacid, 2005).
The general overview of the cell cycle regulation (figure 6) has been drawn
based on available data, but much is yet to be discovered in the field of cell
cycle regulation. New models for cell cycle regulation have been suggested
(Kaldis and Aleem, 2005).
Figure 6. Basic overview of the eukaryote cell cycle
INTRODUCTION | 37
Interphase includes G1, S and G2 stages in the cell cycle. At mitosis, nuclear
and cellular division occurs. In prophase (figure 7), chromatin begins to
condense and the mitotic spindle is formed, and in prometaphase the nuclear
membrane breaks down, and the chromosomes are attached to the mitotic
spindle at the centromeres (kinetochores), and the chromosomes are lined up
in the middle of the cell during metaphase (figure 7). Parting of the sister
chromatids takes place in anaphase and telophase (figure 7). In telophase, new
nuclear membranes are formed. Then cell division is completed through
cytokinesis, which results in two (identical) daughter cells.
Interphase
Prophase
Metaphase
Anaphase
Telophase
Figure 7. Mitosis of a normal human fibroblast, cell nuclei stained by DNA dye DAPI,
visualizing the different steps in mitosis.
Phosphorylation of histone H1
Significance of H1 phosphorylation
Histone H1 phosphorylation has been implicated in several important
processes, like gene regulation, apoptosis, chromatin condensation and cell
cycle progression. In all of these fields, the data on H1 phosphorylation has
often been contradictory and the precise functional role of H1
phosphorylation remains to be determined. Phosphorylation of H1 histones
occurs primarily in S/TPK(A)K motifs (figure 8).
Histone H1 phosphorylating and dephosphorylating enzymes
Histone H1 phosphorylation was recognized in the 1960. Despite this, the
enzymes responsible for the phosphorylation are still not completely known.
And, despite the fact that a number of histone H1 phosphorylating proteins
have been found to be H1 kinases in vitro, these properties may be different in
vivo. Also, there may be additional H1 kinases which have not yet been
recognized, or there may be H1 kinases with phosphorylating ability in vivo,
38 | INTRODUCTION
but not in vitro, like Cdk4 and Cdk6 (Matsushime et al., 1994; Meyerson and
Harlow, 1994).
H1 phosphorylation was found to be performed by “Mammalian growthassociated kinase”, later identified as a homologue to yeast cdc2+/CDC28
(Langan et al., 1989). This enzyme is known as p34cdc2, active in the G2/M
transition. The p34cdc2 kinase is believed to require the motif Ser/Thr-Pro-X-Z
(where X is a polar amino acid and Z generally is a basic amino acid) for
phosphorylation (Moreno and Nurse, 1990). In contradiction, H1 from
Chinese hamster cells (CHO), contained a mitosis-specific site without this
motif, SETAPAAPAAAPPAEK, with phosphorylations on both Ser and Thr
(Gurley et al., 1995). This site was later demonstrated to be phosphorylated by
p34cdc2 kinase when bound to various cyclins (Swank et al., 1997).
Cdk2 has also been recognized as a histone H1 kinase (Bhattacharjee et al.,
2001). Cdk2 activation and H1 phosphorylation take place in mid/late G1 and
S phases of the cell cycle (Herrera et al., 1996). Cdk2 is active in the G1/S
transition, and Cyclin E/Cdk2 and cyclin A/Cdk2 (late G1 and G1/S kinases)
phosphorylates H1b (mouse homologue to human H1.5) in vitro (Contreras et
al., 2003).
Using Chinese hamster cells (CHO), which contain mainly one H1 subtype,
there seemed to be no specificity of four different H1 kinases (p34cdc2/cyclin
A, p33cdk2/cyclin A, p34cdc2/cyclin B and p34cdc2/cyclin ?) for different
phosphorylation sites in H1 (Swank et al., 1997). The timing of
phosphorylation of the different sites during the cell cycle was hypothesized
to be explained by differential accessibility of sites (Swank et al., 1997).
In conclusion, histone H1 is not phosphorylated in G0. Probably, no
phosphorylation takes place in early G1, since histone H1 is a poor substrate
for the early G1 kinases, Cdk4 and Cdk6, at least in vitro (Matsushime et al.,
1994; Meyerson and Harlow, 1994). During late G1, histone H1 is probably
phosphorylated by Cyclin E/Cdk2, and by cyclin A/Cdk2 in the G1/S
transition. Then, in G2/M histone H1 is phosphorylated by Cdk1.
After hyperphosphorylation at mitosis, histone H1 is rapidly
dephosphorylated. This dephosphorylation is performed by one or many
phosphatases. The exact mechanism and the identity of the phosphatases
INTRODUCTION | 39
involved in the dephosphorylation process remains to be determined. The
likely candidates for H1 dephosphorylation are protein phosphatase 1(PP1)
(Paulson et al., 1996) or protein phosphatase 2A (PP2A). Dephosphorylation
of Histone H1b (homologue to H1.5) has been suggested to be
dephosphorylated by PP1 (Chadee et al., 2002). However, in vivo there may be
additional phosphatases involved in H1 dephosphorylation. Also, different
processes, like gene regulation, chromatin condensation or the cell cycle may
involve different phosphatases for H1 dephosphorylation.
H1 phosphorylation in gene regulation
Histone H1 phosphorylation has been connected to gene regulation in
some systems. In Tetrahymena, H1 phosphorylation has been connected to
both activation and repression of certain genes (Dou et al., 1999), probably by
altering the overall charge of a specific, small H1 domain (Dou and Gorovsky,
2000). Also, unphosphorylated H1 has been detected in a region of the CDC2
promotor in Tetrahymena, when CDC2 expression is reduced, but not at active
CDC2 expression (Song and Gorovsky, 2007). FRAP (fluorescence recovery
after photo bleaching) experiments in Tetrahymena with GFP-H1 mutants
demonstrated that H1 phosphorylation may result in increased rate of H1
dissociation from chromatin (Dou et al., 2002). Glucocorticoid stimulation of
the mouse mammary tumour virus (MMTV) promoter has been used as
another model system for examining the effect of histone H1 phosphorylation
on gene regulation, linking H1 phosphorylation with transcriptional
competency (Lee and Archer, 1998). This is supposed to be accomplished
through phosphorylation of histone H1 by Cdk2, subsequent dissociation of
the phosphorylated H1 from the promoter, followed by chromatin
remodelling of the promoter (Stavreva and McNally, 2006). In mouse
fibroblasts, H1b (homologue to human H1.5) phosphorylation was
substantially reduced by inhibitors of transcription (Chadee et al., 1997), and
phosphorylated H1b has been suggested to be bound to transcriptionally
active chromatin (Chadee et al., 1995).
Histone H1 phosphorylation and chromatin condensation
Histone H1 phosphorylation was initially proposed to initiate chromosome
condensation, i.e. act as a mitotic ‘trigger’ (Bradbury et al., 1974). In support
40 | INTRODUCTION
of this theory, experiments on the mouse tumour cell line FM3A with the
protein kinase inhibitor staurosporine showed that staurosporine induced H1
dephosphorylation and chromosome decondensation in metaphase arrested
cells (Th'ng et al., 1994). In contrast to this, data from some other organisms
suggested that H1 dephosphorylation was connected to condensation of
chromatin (Roth and Allis, 1992), leading to a paradox in this field. Also,
using mouse mammary tumour FT210 cells without p34cdc2 kinase activity, the
presence of condensed chromosomes without hyperphosphorylation of
histone H1 was demonstrated, implying that H1 hyperphosphorylation is not
an absolute condition for chromatin condensation (Guo et al., 1995). Possibly,
this paradox can in part be explained by the fact that dephosphorylated H1,
partially phosphorylated H1 and maximally phosphorylated H1 may be
different in structure and character, in regard to DNA affinity and DNA
compaction (Roque et al., 2008).
In human lung fibroblasts, mid/late G1 and S-phase H1 phosphorylation,
presumably by Cdk2, was connected to chromatin decondensation (Herrera et
al., 1996). In Rb-deficient mouse fibroblasts, elevated Cdk2 activities were
detected along with increased H1 phosphorylation and chromatin
decondensation in G0, G1 and S-phases (Herrera et al., 1996). In S-phase,
cdc45 recruitment leads to local decondensation of chromatin and the
presence of phosphorylated H1 (Alexandrow and Hamlin, 2005). Presumably,
cdc45 recruits Cdk2 to replication foci, with concurrent H1 phosphorylation
and chromatin decondensation (Alexandrow and Hamlin, 2005). Using GFPlabelled H1b mutants and FRAP, Cdk2 was suggested to phosphorylate H1b
and thereby affect its nuclear mobility (Contreras et al., 2003). A possible
mechanism for histone H1 phosphorylation in chromatin decondensation
during interphase has been suggested (Hale et al., 2006). Histone H1 interacts
with heterochromatin protein 1α (HP1α) in heterochromatin, and upon H1
phosphorylation by Cdk2 this interaction is disrupted, leading to
decondensation of the condensed heterochromatin (Hale et al., 2006)
H1 phosphorylation in the cell cycle
Histone H1 phosphorylation during the cell cycle has been detected in a
number of organisms. Data from these experiments are in many cases
contradictory, both within and between species.
INTRODUCTION | 41
Using Chinese hamster cells, Gurley and co-workers concluded that the
H1 histone in these cells was phosphorylated sequentially in the cell cycle
(Gurley et al., 1975). Early G1 cells were unphosphorylated, initial serine
phosphorylation took place later in the G1-phase, followed by serine
phosphorylation of another motif in the beginning of S-phase and serine
phosphorylation was continued through the S-phase with up to three
phosphates; in M-phase further phosphorylation was detected on both serines
and threonines, with up to six phosphates, followed by rapid
dephosphorylation upon G1 re-entry (Gurley et al., 1995; Gurley et al., 1975;
Hohmann et al., 1976). In further experiments, the kinase activity of four
different H1 kinases, active at different time points in the cell cycle was shown
to have no selectivity for the different phosphorylation sites in the CHO cells
in vitro (Swank et al., 1997), leading to the conclusion that the cell cycle
dependent phosphorylation was a result from differential accessibility of H1
phosphorylation sites during the cell cycle rather than from specialised
functions of different H1 kinases.
The cell cycle dependent phosphorylation of H1 histones has also been
investigated using NIH 3T3 mouse fibroblasts and rat C-6 glioma cells; and
the phosphorylation pattern was concluded to be different for different H1
subtypes (Talasz et al., 1996). In NIH 3T3 cells, H1 phosphorylation began
during late G1, with mainly unphosphorylated and few monophosphorylated
H1 subtypes (Talasz et al., 1996). H1b (homologue to human H1.5) was
mostly unphosphorylated, but with some mono- and di-phosphorylations;
during S-phase the phosphorylation increased, giving 0-1 phosphates in H1a
and H1c (homologous to H1.1 and H1.2), 0-2 phosphates in H1d (H1.4) and
0-3 phosphates in H1b and H1e (homologous to H1.5 and H1.3) in late S.
Purified metaphase cells displayed tetra-phosphorylated H1a, H1c and H1e
(H1.1, H1.2 and H1.3) and penta-phosphorylated H1b and H1d (H1.5 and
H1.4) (Talasz et al., 1996).
In early work on human HeLa cells, it was suggested that the two H1
subtypes detected at that time in HeLa cells had different phosphorylation
patterns in the cell cycle (Ajiro et al., 1981).
42 | INTRODUCTION
Recent experiments on human CEM cells have confirmed that
phosphorylation of human histones H1.2, H1.3, H1.4 and H1.5 occur site
specifically during the cell cycle, during interphase phosphorylation was
detected only on serines in SPK(A)K motifs, while H1.5 threonine
phosphorylation took place in mitosis (Sarg et al., 2006). Additional H1.2,
H1.3 and H1.4 threonine phosphorylation was also presumed to occur in
mitosis (Sarg et al., 2006).
In figure 8, detected and presumptive phosphorylation sites in human
histones H1.2-H1.5 are described, with data taken from (Sarg et al., 2006), but
additional sites may occur (Wisniewski et al., 2007).
Figure 8: Phosphorylation of H1.2, H1.3, H1.4 and H1.5 histones
Histone H1 phosphorylation in apoptosis
Some researchers have reported on increased phosphorylation of H1
histones upon apoptosis induction while many others have connected H1
INTRODUCTION | 43
dephosphorylation with the apoptotic process, or the absence of
phosphorylated H1 in apoptotic cells (Th'ng, 2001).
Histone H1 phosphorylation pattern in malignant transformation
In mouse fibroblasts transformed with ras, increased levels of H1
phosphorylation and less condensed chromatin was detected, and G1/S
arrested ras transformed cells showed more extensive H1 phosphorylation
than arrested untransformed cells (Chadee et al., 1995). This was later
concluded to be a result of increased Cdk2 activity, where ras transformation
resulted in an initial increase in p21cip1 levels, inhibiting Cdk2 activity, followed
by a decrease in p21cip1 levels and an increase in Cdk2 activity and H1b
phosphorylation (Chadee et al., 2002).
44 | INTRODUCTION
AIMS OF THE THESIS
The overall aim was to elucidate the functional role of H1 histones,
especially in apoptosis and the cell cycle, and the significance of histone H1
phosphorylation in apoptosis and the cell cycle.
Specific aims
-to determine if there where sequence variations within the histone H1
subtypes, and if so determine the allele frequency of these variations in a
normal population
-to determine if and how histone H1 phosphorylation is connected to
apoptosis, taking cell cycle effects into account
-to determine the histone H1 subtype distribution and the distribution of
phosphorylated H1 in human diploid cells during cell division
-to determine the histone H1 subtype and phosphorylation pattern during
the cell cycle in normal cells
-to investigate if malignant transformation leads to changes in the cell cycle
phosphorylation pattern
AIMS OF THE THESIS |45
46 | AIMS OF THE THESIS
MATERIAL AND METHODS
In Paper I in this thesis, the presence of sequence variants within certain
H1 subtypes was determined. Histone H1 was extracted by perchloric acid
from various cell lines and the H1 subtype composition was analysed by
hydrophilic interaction liquid chromatography (HILIC). The identity of the
detected sequence variations was determined by reversed phase-high
performance liquid chromatography (RP-HPLC), HILIC, peptide sequencing
by Edman degradation and mass spectrometric analyses of peptide digests
from the HILIC analysis. The presence of the sequence variants was
confirmed in the corresponding H1 genes in genomic DNA from K562 and
Raji cell lines by direct cycle sequencing. Genomic DNA from normal blood
donors was screened for the sequence variants using restriction fragment
length polymorphism (RFLP) or denaturing HPLC (DHPLC).
In Paper II, the histone H1 subtype and phosphorylation patterns in
apoptotic Jurkat cells were determined. Apoptosis was measured by flow
cytometry, using Annexin V and a fluorescent inhibitor of caspases as cellular
markers. Flow cytometry was also used for cell cycle analysis of cell samples,
where the DNA dyes Hoechst 33342 and propidium iodide (PI) were used for
staining of cell nuclei. Histone H1 samples were prepared by perchloric acid
extraction and the histone H1 subtype composition and phosphorylation
pattern was determined using high performance capillary electrophoresis
MATERIAL AND METHODS | 47
(HPCE). Identities of peaks in the electroferogram were determined using
RP-HPLC and HILIC separations, with mass spectrometric identification of
subtypes.
In Paper III, the location of the H1.2, H1.3 and H1.5 subtypes during
mitosis in normal human cultured fibroblasts was determined using
immunocytochemistry and confocal microscopy.
In Paper IV, the phosphorylation pattern of histone H1 subtypes in the G1,
S and G2/M phases was determined in activated T cells and Jurkat cells. The
T cell activation was assessed by flow cytometry, using the cell tracer 5(6)Carboxyfluorescein diacetate N-succinimidyl ester (CFSE), cell cycle
distribution by PI staining, CD3+ phenotyping and Annexin V as an apoptosis
marker. Activated T cells and Jurkat cells were sorted into G1, S and G2/M
populations on the basis of DNA content by flow sorting of Hoechst 33342
stained cells. The G1, S and G2/M cell populations were extracted by
perchloric acid to obtain histone H1 samples. The H1 samples were analysed
for subtype composition and phosphorylation pattern with HPCE.
In this section a brief summary of methods used in Papers I-IV, selection
of methods and the principal aspects the methods used will be presented. The
details of all used methods are described in the individual Papers.
Histone H1 extraction
Different methods have been used to extract histones from cells, most of
them based on the ability of histones to be soluble in diluted acids, like
hydrochloric acid or sulphuric acid (Shechter et al., 2007). Also, high salt
concentrations can be used to selectively extract different histones (Bolund
and Johns, 1973), with histone H1 being extracted between 0.3-0.7 M NaCl
(Loborg and Rundquist, 1997). Another, widely used method for obtaining
H1 histones, is extraction by perchloric acid (PCA). By the use of perchloric
acid, selective extraction of mostly basic proteins is accomplished. The major
component of the extracts is H1 histones, but also HMG proteins are
extracted as well as a number of other proteins (Zougman and Wisniewski,
2006). Since relatively pure H1 histones are obtained using this method, with a
48 | MATERIAL AND METHODS
relatively high recovery, this method was used to extract H1 histones from the
various cell lines used in this thesis.
All cells were harvested by centrifugation and subsequently washed in
buffer. Since histones are highly susceptible to proteases, protease inhibitors
were added in all steps until the proteins were precipitated. All samples were
kept on ice at all times to minimize the activity of proteases and phosphatases.
Since H1 histones are proteins containing many positive charges, they easily
adhere to both glass and plastic material when they are free in solution.
Therefore, all tubes and tips were pre-treated with silicone (Sigmacote)
through filling tubes and tips with Sigmacote, removing the solution and
letting them dry completely before use. Sigmacote consists of a chlorinated
organopolysiloxane in heptane, which reacts with silanol-groups to give a very
thin hydrophobic and neutral film, which repels water and prevents
adsorption of many basic proteins.
After washing, the cells were permeabilized by Triton, and cell nuclei were
recovered by centrifugation. To include a possible cytoplasmic pool of H1
histones, the permeabilisation step was omitted and H1 histones were
extracted from whole cells by perchloric acid instead. In control experiments,
we could not detect any differences between H1 samples prepared from cell
nuclei from Jurkat cells and whole Jurkat cells. Therefore, in papers II and IV,
we have extracted H1 histones from whole cells. After extraction, proteins
were precipitated with trichloroacetic acid (TCA), and protamine sulphate was
added to co-precipitate H1 proteins. The proteins were collected by rapid
centrifugation and washed in acetone. After this step, remaining proteins were
dissolved in water with β-mercaptoethanol, and then lyophilized by freezedrying using a Speed-Vac.
Depending on the method for analysis of H1 histones, different amounts
of cells were subjected to H1 extraction. For HPCE analyses a minimum of
about 10 million cells were required and for RP-HPLC and subsequent HPCE
analyses, about 25 million cells were required for analysis.
MATERIAL AND METHODS | 49
The recovery of H1 histones from cellular extracts are variable, but seem
not to be subtype specific with the exception of H1.X extraction. H1.X is
detected in PCA extracts (Zougman and Wisniewski, 2006), but is known to
be less soluble in PCA than other H1 subtypes (Happel et al., 2005a).
Therefore, the low amount of H1.X detected in Jurkat and T cells using RPHPLC is not representative and the actual amount of H1.X in these cells is
unknown.
Separation of H1 histones and their phosphorylated variants
Since the material used in this thesis is of human origin, the main objective
is the separation of human H1 histones and their phosphorylated variants.
The separation of histone H1 subtypes and their modified forms has been a
challenging task, due to the structural similarity of the subtypes. Many
methods have been used to achieve separation, for example gelelectrophoresis with different gel types, for example acetic acid urea gels, and
a number of two-dimensional systems have also been used (Lindner, 2008;
Mizzen, 2004). Separation of H1 histones by RP-HPLC with additions of
various solutes (for example TFA) in the mobile phase, lead to the separation
of some H1 subtypes, and the modified variants will not be separated using
this method (Lindner, 2008). HILIC provides certain resolution of H1
subtypes and phosphorylations (Lindner, 2008; Mizzen, 2004). HPCE can be
used for the near complete separation of H1 histones and their
phosphorylated variants. For further separation, RP-HPLC can be used in a
first dimension, and further resolution can be obtained by analysing the
relevant fractions in a second dimension, for example by HPCE or HILIC.
Reversed Phase-High Performance Liquid Chromatography (RPHPLC) of H1 histones
The basis of separation is the ability of the H1 subtypes to interact
differently with, and be retained by, the stationary hydrophobic phase of the
column, and the elution with increasing amounts of acetonitrile in the mobile
phase. The interaction with the stationary phase is probably executed
primarily through the hydrophobic globular domain of H1 histones. The H1
histones can be separated into a number of peaks using RP-HPLC, possibly in
50 | MATERIAL AND METHODS
a species dependent manner (Mizzen, 2004). Using RP-HPLC, human H1
histones can be separated into two peaks, one containing H1.5 and its
phosphorylated variants, and one the remaining subtypes and their
phosphorylated variants (Sarg et al., 2006). Identification of the different
peaks in the chromatogram was done by subsequent HILIC separation and
amino acid sequencing and/or MS.
Hydrophilic Interaction Liquid Chromatography (HILIC)
This technique is based on normal phase chromatography, where the H1
subtypes and their phosphorylated variants are differently retained in the polar
stationary phase with the use of a non-polar mobile phase. Elution is
accomplished through increasing salt gradients or increased polarity of the
mobile phase (Lindner, 2008). The retention of H1 histones and their
phosphorylated variants with the polar stationary phase is probably due to the
polar tails of H1 histones, and the differences in polarity due the addition of
negative charges upon phosphorylation, where a serine or threonine are
modified by a negative phosphate group (Lindner, 2008). Using HILIC most
unphosphorylated H1 subtypes variants can be resolved. H1 samples
separated by HILIC can be resolved in four peaks, where peak 1 is histone
H1.2, peak 2 is H1.3 and H1.5, peak 3 H1.4 and peak 4 phosphorylated H1.5
(Sarg et al., 2005). Even some site specific phosphorylations can be separated
using this technique (Sarg et al., 2006). Identification of peaks was determined
by amino acid sequencing and MS.
High Performance Capillary Electrophoresis (HPCE)
HPCE separation is based on the differential ability of molecules to migrate
in an electric field in a fused silica capillary. Various coating agents can be
used to prevent secondary interactions of H1 histones with the silica,
hydroxypropylmethylcellulose (HPMC) being very effective on H1 separation
as well as phosphorylated subtypes (Lindner, 2008). Since the separation order
of H1 histones differs between different species, further development was
needed to separate human H1 histones (Lindner, 2008). A system with TEA
phosphate/perchlorate/HPMC allowed the separation of H1 histones and
their phosphorylations from a number of species (Lindner et al., 1993), and
also allowed the near complete separation of human histones and their
phosphorylated variants (Lindner, 2008).
MATERIAL AND METHODS | 51
By this method, complete separation of un-phosphorylated H1.3, H1.4 and
H1.5, and monophosphorylated H1.4 and H1.5 was obtained (Green et al.,
2008). If HPCE was performed on fractions containing H1.5 separated by
RP-HPLC, H1.5 and all of its phosphorylated variants could be resolved (Sarg
et al., 2006). The identification of the various peaks from HPCE cannot be
done by immediate, on line, mass spectrometry because of the high charge of
the HPCE. Identification of the peaks from HPCE was done as described in
Paper II and its supplementary material (Green et al., 2008). Identification was
done using mixing experiments, which do not allow the complete
identification of the content of the peaks. Therefore, the peaks may contain
additional proteins that co-migrate with the identified H1 protein in the peak,
but in low amounts. One example is H1.X, where its migration in HPCE has
not been determined. We estimate that the low amounts of H1.X present in
RP-HPLC separations will not make any major contribution to any H1 peak
in the HPCE separation.
Genetic analysis
DNA-samples
Genomic DNA samples from the various cell lines were extracted using the
Dneasy™ tissue kit (Qiagen). To obtain good quality non-fragmented DNA,
the cells were seeded to be in log growth phase and continuously monitored
by microscopic examination. All cell samples exhibited low numbers of
damaged cells at DNA extraction.
DNA from 103 normal individuals was obtained from a larger DNA bank,
where approximately 800 samples were included after informed consent. All
donors were selected from a population register in south-east Sweden and
were 22-77 years of age. The design had been approved by the Linköping
University Hospital Ethical Committee. DNA was extracted from blood
samples taken from all donors using the QIAamp® DNA Blood Maxi kit
(Qiagen).
52 | MATERIAL AND METHODS
PCR amplification of H1 genes or gene fragments
The polymerase chain reaction (PCR) is a method for amplifying a certain
region on a DNA template. The region of amplification is determined by a set
of specific primers that anneal to a complementary sequence in the DNA.
PCR was used to amplify specific regions in two histone H1 genes in Paper I.
Histone H1 genes are relatively conserved in the N-and C-terminal and
highly conserved in the globular domain (figures 4 and 5). Therefore, specific
PCR amplification may be difficult in certain regions of the corresponding
genes. Also, Histone H1 genes lacks introns, which may further complicate
specific amplification, since introns provide regions of low sequence
conservation where specific primers can be placed. To accomplish specific
amplification, multiple alignments of H1 genes or gene fragments were
executed using the program Clustal W or Clustal X. The primers were placed
in regions of low similarity. Primer design was made using the program
Primer 0.5 or 3. This program selects primers on the basis of primer melting
temperature, primer length, GC content and primer-dimer formation, among
other things (Rozen and Skaletsky, 2000). Also, the primers were checked for
complimentary sequences in the human genome using BLAST. Primers with
high similarities to other sequences than the gene it was designed for were
excluded.
The presence of pseudo-genes may be an obstacle in PCR amplifications
from genomic DNA. H1 histones have no known pseudogenes except in
Xenopus (Eirin-Lopez et al., 2004), and no truncated sequences were detected
in the DNA sequencing.
All PCR reactions were optimized for annealing temperature and MgCl2
concentration. The optimal annealing temperature in combination with
optimal magnesium concentrations for specific PCR products were
determined using a gradient PCR, where annealing temperatures between
approximately 52 and 62 degrees were applied, and magnesium concentrations
between 1 and 3 mM were tested. Also, the number of cycles, and the
duration of the melting, annealing and extension steps were sometimes
optimised. In all PCR reactions, a non-template control was included to
MATERIAL AND METHODS | 53
exclude the possibility of contaminating DNA. All PCR products were
analysed through separation on agarose gels, with a 1000 bp ladder.
DNA sequencing
PCR products were sequenced using direct cycle sequencing based on the
dideoxy chain termination method (Sanger et al., 1977). This method is based
on a PCR-reaction using 33P labelled bases and chain-terminating
dideoxynucleotides, which are incorporated in the amplified DNA. The
labelled fragments were subsequently separated on polyacrylamide gels, and
after drying gels these where exposed to X-ray film. From these films, the
DNA sequence could be manually deduced. The H1.2 Ala17Val substitution
was found to correspond to a C→T substitution in codon 18, and the H1.4
Lys173Arg to a heterozygote A→G substitution in codon 174.
Detection of sequence variations
In paper I, we determined the sequence variations by DNA sequencing of
the corresponding gene fragments, and the assays for screening normal
individuals and cell lines were subsequently designed for detection of the
genetic variants corresponding to H1.2 Ala17Val and H1.4 Lys173Arg. RFLPanalysis is an easy and convenient method, which is based on differential
cleavage patterns of PCR products of the normal and variant DNA by a
restriction enzyme. This method is reliant on that the recognition sequence of
a restriction enzyme is present in the normal or variant sequence exactly
where the sequence variation is situated. Using the program Nebcutter
(Vincze et al., 2003), BsuRI was selected for differential cleavage of a H1.2
PCR product with the C→T substitution in codon 18, enabling RFLP
analysis. For the A→G substitution in codon 174 no appropriate enzyme
could be found. Therefore, denaturing HPLC was tested for a PCR product
from the H1.4 gene. This method requires a positive control for optimal
detection.
Restriction Fragment Length Polymorphism (RFLP) analysis
An RFLP analysis was designed for detection of the Ala17Val
polymorphism in histone H1.2. A 183 bp fragment was amplified. This
fragment was subjected to cleavage by BsuRI (HaeIII), which cleave the wild
type PCR product in three fragments with the lengths 21, 53 and 109 bp. PCR
54 | MATERIAL AND METHODS
products with the polymorphism were cut in two fragments, 53 and 130 bp.
When the digestion products (figure 9) were separated on agarose gels
according to fragment size, different patterns of separation was obtained
between wild type and variant PCR products. Inefficient cleavage reactions,
may result in incomplete digestion of the PCR product and the generation of
a false positive result for the H1.2 Ala17Val variant. To avoid this, all detected
polymorphisms were confirmed by DNA sequencing.
Figure 9. Cleavage patterns of H1.2 PCR products from wild type donors (top, GCC in
codon 18), heterozygote donors (middle, GCC and GTC in codon 18) and homozygote
donors (bottom, GTC in codon 18)
Denaturing HPLC (Wave)
A PCR product consists of two complementary strands. If the PCR
product is heated to separate the two strands and then cooled gradually,
heteroduplexes can be formed if a heterozygous sequence variation is present,
as well as homoduplexes of both wild type and variant alleles (figure 10). If a
homozygote, which carries the sequence variation to be detected in both
alleles, heteroduplexes are formed, as well as homoduplexes, if the PCR
product is mixed with wild type PCR product and gradually cooled prior to
analysis. The mix of heteroduplexes and homoduplexes are then separated by
denaturing HPLC. The HPLC column consist of a non-polar stationary phase,
where triethylammonium ions are absorbed, creating a surface for positive
interactions with the negatively charged DNA (Xiao and Oefner, 2001). Due
to differences in retention in the column and in partial denaturation between
heteroduplexes and homoduplexes in the column oven, they elute at different
times upon increasing acetonitrile concentrations (figure 11) (Xiao and
Oefner, 2001). Detection is performed by UV absorption. The optimal
temperature for analysis by denaturing HPLC of the H1.4 fragment was
predicted from the melting profile obtained by using the WAVEMAKER™
software. Also, optimal melting temperature of the fragment was confirmed
MATERIAL AND METHODS | 55
by separating the H1.4 homo- and heteroduplexes using a temperature
gradient in the DHPLC. DHPLC is considered to be a highly sensitive and
specific method with nearly 100% detection of sequence variations within
DNA fragments (Xiao and Oefner, 2001; Yu et al., 2006). The oven
temperature may vary to some extent, which may result in false negative
results. We consider this possibility to be very small in the detection of the
genomic variant H1.4 Lys173Arg since detection of the sequence variation
was stable at least ±1ºC from the temperature of analysis, which exceeded the
variations in the carefully calibrated oven temperature.
Figure 10. Homoduplex and heteroduplex formation of PCR products from wild type and
variant alleles
Figure 11. DHPLC separation of PCR products by a acetonitrile (ACN) gradient
56 | MATERIAL AND METHODS
Flow cytometry
The term cytometry can be explained by ‘measure cells’. Hydrodynamic
focussing allows the flowing of single cells into the flow chamber, where cells
travel in a fluid steam through a beam of light, usually produced by one or
multiple lasers (figure 12). The cells scatter the incident light and emit
fluorescence, which can be passed through multiple filters, and focussed on
photomultiplicators (figure 13), after which the emitted light is converted to a
digital signal that is saved in a computer. Multiple parameters can be measured
in this manner. The flow cytometers used in this thesis are controlled by
CellQuest Pro (BD LSR II) or DIVA (FACS Aria Special Order System)
softwares.
Figure 12. Sample loading and excitation
in a flow cytometer. Graphics obtained
from BD Biosciences, San Jose, CA
Figure 13.Optics in a flow cytometer.
Graphics obtained from BD Biosciences,
San Jose, CA
To visualize a cell population, a plot of the detected Forward Scatter (FSC)
versus Side Scatter (SSC) can be obtained in CellQuest Pro or DIVA, where
the FSC is proportional to the size or area of the cell, and the SSC to the
granularity of the cell. From this plot, conclusions about the homogeneity of
the cell population and the presence of cell debris can be drawn.
MATERIAL AND METHODS | 57
Various components of cells can be labelled by fluorescent compounds,
either directly, or via fluorescent primary or secondary antibodies. Fluorescent
compounds absorb light at specific wavelengths, creating an excited electron.
Upon return to its normal energetic level, a light photon is emitted. The
wavelengths of the emitted light are higher than the absorbed wavelengths.
Therefore, each fluorochrome has a specific absorption and emission
spectrum. Upon designing flow cytometric measurements with
fluorochromes, selection of fluorochromes must be done with consideration
of cytometric filter settings and lasers available (excitation source). Multiple
fluorochromes can also be detected simultaneously if the emitted light is
diverted to different PMT detectors by optical filters. If multiple colours are
used, colour compensation must be set between the different fluorochromes
if they have overlapping spectra. Data can be visualized in various ways, for
example in histograms and scatter plots.
Cell cycle distribution
In this thesis, the DNA content of various cells and cell lines were
measured to obtain the cell cycle distribution of the cell populations. Many
different DNA binding fluorochromes are available.
We have chosen to use propidium iodide staining in a procedure developed
by Vindelov et al. (Vindelov et al., 1983). This is a widely used method, for
example in standardized clinical investigations of various cancers. In this
method, the cell membranes are lysed using NP-40, proteins are removed by
trypsin, RNA is broken down by ribonuclease A, the nuclear membrane is
stabilised by spermine tetrachloride to obtain a “nuclear preparation” and
staining of the DNA in the cell nuclei is performed with propidium iodide.
Flow cytometric measurement results in a DNA histogram (figure 14) where
the measured DNA content in G1 cells have small variations, i.e. a low
coefficient of variation (CV). Propidium iodide is an intercalating DNA dye,
which stains both DNA and RNA (RNase is used to remove RNA). Based on
our own observations and others, we do not expect fragmented DNA to be
selectively lost during the staining procedure.
58 | MATERIAL AND METHODS
Figure 14. DNA histogram obtained from PI stained T cells using CellQuest Pro.
We have also used the AT-specific and cell membrane permeable stain
Hoechst 33342. This dye specifically binds electrostatically to minor grooves
in DNA. If titrated carefully, and if the detection is optimised in the flow
cytometer, the G1 peak in Hoechst 33342 histograms may have a CV like
propidium iodide, or even lower as can be seen in the measurements made at
the FACSAria Special Order system with the DIVA software (figure 18).
Viable staining of cells with Hoechst 33342 may have several disadvantages.
For example, this stain can induce apoptosis, since it is a cytotoxic substance.
This can often be observed in the FSC/SSC plot. My/our own observations
and measurements show that the effects of the dye itself on exponentially
growing viable cells are small under the conditions used, but staining of
apoptotic cells often results in accelerated, and more pronounced induction of
apoptosis. These effects can be limited by using a low concentration of the
dye, and short incubation times. Also, cell storage on ice and centrifugation at
low temperatures minimizes this problem.
Cell cycle distributions can be analysed using different programs. We have
used ModFitTM for determining the cell cycle distributions of cell populations
stained either with PI or Hoechst 33342. The models produced by ModFit are
processed by a Marquardt nonlinear least-squares analysis.
MATERIAL AND METHODS | 59
Cell tracing
In paper IV, we have used 5(6)-Carboxyfluorescein diacetate Nsuccinimidyl ester (CFSE) to determine if our activated PBLs divide. CFSE
irreversibly reacts with both intracellular and cell-surface proteins upon
staining. Some initial loss of CFSE fluorescence is detected within the first 24
hours (Wallace et al., 2008). Upon cell division, the CFSE labelling is equally
distributed between the two daughter cells, which thereby exhibit half the
fluorescence as the parent cell. Multiple cell divisions can be observed in this
manner, and the presence of a non-dividing population can be determined
(figure 15). We have observed a slightly affected cell growth by CFSE
labelling, possibly leading to a minor underestimation of the fraction of
dividing cells in unlabelled cultures.
Figure 15. CFSE tracing of T cell activation by flow cytometry at 1day (left), 3 days (middle)
and 5 days (right) after activation. Histograms of CFSE fluorescence were obtained by
CellQuest Pro.
Apoptosis detection
Apoptosis can be detected in many different ways, and at different stages in
the apoptotic progress. Also, some methods provide qualitative data (presence
of a certain apoptotic feature) and other quantitative data (fraction of
apoptotic cells in a cell population). To investigate the H1 phosphorylation
pattern in apoptotic cells, and taking cell cycle effects into account, we needed
a quantitative assay, and an assay with simultaneous cell cycle analysis. Flow
cytometric measurements of various apoptotic features provide quantitative
data. Since H1 histones are known to leave chromatin at DNA fragmentation,
we have aimed at measuring apoptosis at an early stage. Therefore we
measured Annexin V and a fluorescent inhibitor of caspases (FLICA) using
flow cytometry. Also, we have detected the extent of DNA fragmentation
using DNA laddering (results not shown). No apparent fragmentation took
60 | MATERIAL AND METHODS
place during the first 4 hours of incubation, while some fragmentation was
evident after 6 hours. Even so, the early apoptotic cultures will contain some
late stage cells.
Annexin is a widely used apoptosis marker, and often considered to
measure early apoptosis. During apoptosis, internal phosphatidylserines are
relocated across the cell membrane, to the outside of the membrane. Early
apoptotic cells retain an intact cell membrane, while late apoptotic cells are
permeable. The Annexin assay detects phosphatidylserines, and is used in
conjunction with a fluorescent counter stain which is excluded from intact
cells. We have used Annexin conjugated with Phycoerythrin, and 7-AAD as a
counter stain (figure 16). Compensation was set between the two colours
using single stained samples. Some data indicate that the DNA may already be
fragmented upon phosphatidylserine externalisation (Bacso et al., 2000), but
no loss (or selective loss) of H1 histones from nuclei could be detected during
the first 6 hours after apoptosis induction, as no differences could be detected
between H1 histones from nuclei and whole cells during this time.
Figure 16. Annexin and 7-AAD staining of apoptotic Jurkat cells. Lower left quadrant:
viable cells, upper left quadrant: apoptotic cells, upper right quadrant late apoptotic cells
and/or damaged cells, lower right quadrant: severely damaged cells.
MATERIAL AND METHODS | 61
We have also used a fluorescent inhibitor of caspases (FLICA). In this
assay active caspases are detected. FLICA binding have been shown to be an
earlier step in apoptotic progression than phosphatidylserine exposure
(Pozarowski et al., 2003). The exact mechanism of FLICA binding to the
active caspases is unclear (Pozarowski et al., 2003), but results in retention of
the reagent in apoptotic cells. Since the reagent includes carboxyfluorescein
(FAM) reporter, the FAM fluorescence detected by the flow cytometer is
related to the amount of active caspase in the cell. Propidium iodide is
included as a counter stain in this assay to determine membrane integrity. To
determine the cell cycle phase of the apoptotic cells, the cells are also stained
with Hoechst 33342. Compensation was set between the FAM and PI
fluorescence. FLICE data sometimes implicated several FLICE positive
populations, possibly reflecting the activation of different caspases (figure 17,
lower right rectangle).
Figure 17. FLICA and PI fluorescence scatter gram of apoptotic Jurkat cells obtained by
CellQuest Pro. Lower left rectangle: cells devoid of active caspases, upper rectangle: late
apoptotic and damaged cells, lower right rectangle: cells with active caspases
62 | MATERIAL AND METHODS
Cell Sorting using FACS Aria Special order system
After analysis in the flow cuvette using a 355 nm laser for excitation, the
stream is accelerated through a nozzle (we used a 70 µm nozzle), after which
the stream is broken into droplets for sorting. When a particle (cell) that meets
the criteria set for sorting in the sort layout (in our case a G1, S or G2/M cell,
defined by sorting gates in the DNA histogram, see figure 18), the stream is
supplied with an electrical charge in the moment where the droplet
(containing the selected cell for sorting) just leaves from the stream. The
charged droplet passes between two highly charged deflection plates, and is
deflected far-left, left, right or far-right depending on charge. The magnitude
and type of the supplied charge, determines the location for the sorting of the
droplet. The sorting procedure can be performed in different ways, and we
have chosen to sort on yield, rather than purity. Since at least 10 million cells
are needed for H1 extraction, this is necessary to avoid extended sorting times
and to limit the amount of cells needed for sorting.
Figure 18. DNA histogram including sorting gates obtained from Hoechst 33342 stained T
cells using FACS DIVA
MATERIAL AND METHODS | 63
Cell culture
Jurkat
Jurkat cells clone (E6.1) were originally derived from a 14 year old boy
suffering from acute T cell leukaemia. Jurkat cells are often used for apoptosis
studies due to their ability to go readily into apoptosis on apoptotic induction
by various agents. Careful cell culturing has to be performed since dense
cultures, with reduced amounts of nourishment often contain apoptotic cells.
Therefore, the Jurkat cells were sub-cultivated at least 3 times a week and the
cell concentration was kept between 0.1-1x106. The cells were counted and
trypan blue exclusion was used upon sub-culturing, to detect apoptotic
cultures if present. All cell cultures were also examined by microscopy daily or
every 2-3 days. Cells were used for approximately one month. Since Jurkat is a
T lymphoblastoid cell line, it was also used as a malignant counterpart to
normal activated T cells in Paper IV.
T cell activation
Activated T cells were chosen for studying the histone H1 phosphorylation
pattern in the cell cycle because they 1) are normal human cells, 2) are
available in large numbers as is needed for H1 protein extraction, 3) are
actively dividing cells and 4) grow in suspension and are therefore easy to sort
by flow cytometry.
Peripheral blood lymphocytes (PBLs), which were purified from buffy
coats, consist of a mixture of primarily T and B cells, but also NK cells, as
well as contaminating platelets and red blood cells. Upon stimulation with
Phytohemagglutinin (PHA), predominantly T cells are specifically activated to
begin cell division (mitogenic stimulation). The activation is polyclonal,
involving a large proportion of the target cells and stimulates only mature T
cells (Di Sabato et al., 1987). Upon PHA stimulation, signals are transmitted
via the T cell receptor, which induces IL-2 production. This IL-2 production
stimulates the proliferation of CD4+ and CD8+ T cells, which is enhanced by
the addition of IL-2 (Proleukin) in the culture media, resulting in rapidly
dividing T cells. The presence of B cells, and other non-dividing T cell subsets
as well as contaminants in the PBLs by erythrocytes and platelets gradually
64 | MATERIAL AND METHODS
disappeared during the first sub-cultivations probably through apoptosis and
dilution upon T cell expansion. Upon sorting, the culture consisted of almost
solely T cells, as demonstrated by cellular CD3+ expression measured by flow
cytometry.
Ag1523
Normal human foreskin fibroblasts (Ag1523) were used in the
immunocytochemical experiments in Paper III. These fibroblasts were grown
in 25 cm2 flasks for continuous cell culture, and seeded in 12-well plates
containing cover glasses for immunocytochemistry. After two days, most cells
have attached to the cover glasses.
Immunocytochemistry
Immunocytochemistry is used for determining the presence and location of
a protein in cultured cells by the use of specific antibodies. We have used
indirect immunocytochemistry, where primary antibodies towards histone
H1.2, H1.3 and H1.5 have been used to detect the corresponding antigen,
with subsequent use of secondary antibodies labelled with the fluorochrome
Alexa594 to visualize the cellular location of H1.2, H1.3 and H1.5 using
confocal fluorescence microscopy. Immunocytochemistry was performed on
cultured fibroblasts after 1) paraformaldehyde (PFA) fixation, 2) methanol
fixation and permeabilisation, 3) blocking and permeabilisation, 4) primary
antibody incubation, 5) secondary antibody incubation, 6) rinsing and 7)
mounting on slides with a mounting medium containing the DNA binding
dye DAPI.
PFA fixation is presumed to stabilize proteins in their native locations, and
produces cross-links between proteins and DNA (Solomon and Varshavsky,
1985). Methanol is an organic solvent that disrupts cellular membranes and
denaturates proteins, which may increase antigen availability but also affect
histone H1 structure. All fixation and permeabilisation methods may result in
altered distribution and/or structure of epitopes and changes in cellular
structures. In one study, various fixation and permeabilisation techniques
applied resulted in changed distributions and differential extraction of soluble
proteins compared with in vivo distributions (Melan and Sluder, 1992).
MATERIAL AND METHODS | 65
Another aspect of possible side effects by fixation and permeabilisation, is the
preservation of the antigen conformation during these procedures (Montero,
2003).
The antibody specificity is another important aspect in interpreting
immunocytochemical experiments. In western blots presented in the antibody
specification sheets, the antibodies used in this study were found to
specifically recognize the recombinant H1 subtype expressed in yeast that they
were raised against, but no other recombinant H1 subtypes, indicating no
cross-reactivity of antibodies between subtypes. The H1.3 antibody has
previously been shown to display some cross-reactivity in chromatin
preparations to an unknown component according to the manufacturer.
With consideration taken to these aspects of immunocytochemistry, we
have chosen PFA fixation before permeabilisation to preserve antigen and
cellular structures and prevent loss of cytoplasmic H1 proteins, followed by
addition of methanol to increase chromatin accession for the antibodies used.
66 | MATERIAL AND METHODS
RESULTS AND DISCUSSION
The existence of H1 microsequence variants
Upon development of the HILIC technique, two microsequence variants,
H1.2Ala17Val and H1.4Lys173Arg were discovered. At the time this study
was initiated, H1 histones had no known sequence variants, except that the
H1.2Ala17Val variant had been detected in spleen on the protein level (Ohe et
al., 1989), without any genetic evidence. Since then, multiple single nucleotide
polymorphisms have been discovered in various H1 genes and included into
databases, for example NCBI Single Nucleotide Polymorphism
(SNP)(http://www.ncbi.nlm.nih.gov/SNP/). To my knowledge, no protein
data has been obtained on these sequence variations, so there is no data
available if these sequence variations are expressed in the populations
examined. The discovered sequence variations in histones H1.2, H1.3, H1.4
and H1.5 seem to be mostly either synonymous or very rare, with a few
exceptions. In H1.2, the genetic variant resulting in the Ala17Val variant is
common, especially in Asian populations (NCBI SNP database accession
rs2230653), and two less common variants, Gly124Ala (rs12111009) and
Ser113Ala (rs34810376) are also found. In H1.3, a rare substitution of
Glu75Lys (rs2050949) was found. H1.4 has been reported to have at least two
substitutions, Lys117Arg (rs36026202) and Lys152Arg (rs2298090) and H1.5
also contains at least two polymorphisms, Lys144Arg (rs11970638) and
RESULTS AND DISCUSSION | 67
Ala211Thr (rs34144478). The presence of all SNPs, except H1.2 Ala17Val
was below 6% according to NCBI SNP.
Therefore, we conclude that the H1.2 Ala17Val substitution is the only
common polymorphism known this far in H1.2-H1.5 genes. The H1.4
Lys173Arg has not been detected except in Raji cells, and is probably a
mutation in this cell line. Arginine substitution for lysines seems to be present
at additional sites in H1.4, and in one site in H1.5, suggesting that this
substitution is not essential for cellular function, at least not in these sites. The
Lys173Arg is situated in a phosphorylation motif, which may be of
importance for H1.4 phosphorylation.
When confirming the Ala17Val H1.2 substitution by DNA sequencing, I
discovered additional polymorphisms, one of which has not been submitted
to the NCBI SNP database. In the H1.2 gene of one normal blood donor, we
detected a heterozygous C to T substitution, encoding a Pro14Leu
substitution.
Since the functional role of H1 subtypes remains to be determined, the
relevance of H1 subtype microsequence variations is hard to predict. Also,
since the structure of H1 histones and their N- and C-terminal tails when they
are bound to chromatin is currently mostly unknown, the structural impact of
the microsequence variants on the H1 structure is difficult to predict. Once
these issues have been determined, the importance of the H1 sequence
variants may be determined.
Histone H1 and H1 phosphorylation in apoptosis
The function of H1 histones in apoptosis is still unclear, but many studies
indicate that H1 (Liu et al., 1999) and H1.2 (Konishi et al., 2003; Ruiz-Vela
and Korsmeyer, 2007) interacts with apoptotic factors and that H1 location
may be changed during apoptosis (Gabler et al., 2004; Ohsawa et al., 2008;
Wu et al., 2002), and that H1 modification patterns may be altered during
apoptosis (Th'ng, 2001) We show in Paper II, that a possible H1 function in
early apoptosis is not governed by phosphorylation or dephosphorylation in a
68 | RESULTS AND DISCUSSION
general way, since FAS-induced apoptosis occurs without major changes in
the phosphorylation pattern.
Many efforts have been made to determine the histone H1 phosphorylation
pattern in apoptosis (Th'ng, 2001). Altered H1 phosphorylation patterns in
apoptosis have been suggested to be related to H1 release from apoptotic
chromatin, apoptotic chromatin compaction, and apoptotic DNA
fragmentation. Many lines of evidence have suggested that dephosphorylation
occur after apoptosis induction (Kratzmeier et al., 2000; Th'ng, 2001). Since
none of these studies has addressed the possible cell cycle effects of the
apoptosis inducers used in these studies, our main goal in Paper II was to
separate these two features, and thereby investigate the H1 phosphorylation
pattern influenced solely by apoptosis. During the time this study was
performed, some of the authors from the study where rapid H1
dephosphorylation was correlated to apoptotic induction with topoisomerase
inhibitors in HL60 cells (Kratzmeier et al., 2000), published a paper where the
suggested dephosphorylation was concluded to be a result of cell cycle effects
promoted by the topoisomerase inhibitors, rather than of apoptosis induction
(Happel et al., 2005b). H1 dephosphorylation was also demonstrated to not be
coupled to DNA fragmentation (Goebel et al., 2007). This resulted in an
unclear view on the H1 phosphorylation pattern in apoptotic cells.
The results from paper II demonstrate that histone H1 dephosphorylation
is not a general apoptotic feature, at least not in early apoptosis, since FAS
ligation results in apoptosis without histone H1 dephosphorylation. In
apoptosis induced by camptothecin and γ-radiation, rapid Histone H1
dephosphorylation occurs, without pronounced cell cycle effects. Since these
apoptosis inducers both promotes apoptosis via the intrinsic pathway, histone
H1 dephosphorylation may be a consequence or a cause of apoptosis via this
pathway.
The actual effect of apoptosis induction on Histone H1 phosphorylation
can probably never be totally separated from cell cycle effects, even though
these were not detected in this study. Apoptosis signalling has been suggested
to be executed in close connection with cell cycle check points and therefore
RESULTS AND DISCUSSION | 69
conclusions on H1 dephosphorylation upon apoptosis induction may as well
be explained by here not detectable cell cycle effects. Therefore, the mapping
of H1 phosphorylation in the cell cycle, and elucidation of the function of H1
phosphorylation in the cell cycle is of major importance for determination of
the H1 phosphorylation pattern in apoptosis.
Many of the experiments on H1 phosphorylation have been made on
cancer cell lines. Many of these have disturbed cell cycle regulation and
progression, altered apoptotic responses and other deregulated mechanisms.
Therefore, experiments on normal human cells are a key issue in further
studies.
Histone H1 subtype distribution during cell division
In Paper III we have investigated the localization of three histone H1
subtypes during mitosis. We have found that Histones H1.2, H1.3 and H1.5
differ in their distribution in metaphase and anaphase of normal human
fibroblasts. H1.2 was located primarily to the cytoplasm during these mitotic
stages, while H1.5 was located both in chromatin and cytoplasm. H1.3 was
solely located to chromatin during all mitotic stages. Phosphorylated H1 was
located in both nuclei and cytoplasm during metaphase, anaphase and
telophase.
This study was initiated after observations of cytoplasmic H1.2 in
immunocytochemical preparations of normal human fibroblasts, initially
prepared for detection of H1.2 in apoptotic cells and in H1.2 knockdown
experiments. In metaphase cells, we could see a very distinct cytoplasmic pool
of H1.2. This was very surprising since studies have been done with H1.2GFP constructs where H1.2-GFP is associated with chromatin in metaphase
in different cell lines (Chen et al., 2005; Takata et al., 2007). Even so, the near
complete absence of H1.2 in fibroblast nuclei of metaphase and early
anaphase cells, and the presence of high amounts of H1.2 in the cytoplasm
disagree with the explanation of this phenomenon only being due to staining
artefacts. Since the same observation was made for H1.5 with the exception
that H1.5 was detected both in nuclei and cytoplasm during metaphase and
anaphase, a reasonable, but very far-fetched explanation for this phenomenon
70 | RESULTS AND DISCUSSION
would be that during metaphase and anaphase, an epitope will appear in the
cytoplasm that cross-reacts with both the H1.2 and H1.5 antibodies, but not
with H1.3, with substantially higher affinity than with chromatin bound H1.2
and H1.5. This epitope would in that case be present only in metaphase and
anaphase. Another possibility would be that the investigated H1 protein
distributions are affected by the fixation or permeabilisation procedures
(Melan and Sluder, 1992). A fact that argues against this possibility, is that
these procedures would need to affect the different H1 subtypes differently to
explain the differential H1 subtype distributions.
If we are proven right, thereby suggesting that H1.2-GFP would not
behave as native H1.2 during the cell cycle, our results will probably not be
easily accepted by histone H1 researchers. Another fact that disagrees with the
existence of cytoplasmic H1.2 and H1.5 during mitosis is that a cytoplasmic
pool of total H1 was not detected with the anti-H1 antibody AE-4. This
antibody has failed to bind to cytoplasmic H1 in other studies (Bleher and
Martin, 1999; Ohsawa et al., 2008), and may therefore not be suitable for
detection of cytoplasmic H1. In contrast, with other antibodies histone H1
has been detected in the cytoplasm on several other occasions (Parseghian and
Luhrs, 2006). We intend to extend Paper III by using an additional H1
antibody, in accordance with the reviewer’s request.
Another fact that has resulted in confusion regarding H1 phosphorylation
is that the antibody towards phosphorylated H1 histones has been detected in
the cytoplasm as well as in nuclei (Bleher and Martin, 1999; Boggs et al., 2000;
Lu et al., 1994), suggesting either presence of phosphorylated H1 in the
cytoplasm or cross-reactivity of the antibody. This discrepancy would be
nicely explained by the presence of phosphorylated H1 subtypes in the
cytoplasm. This antibody has been suggested to detect only phosphorylated
H1b (corresponding to human H1.5) (Chadee et al., 1995), which would
explain the presence of phosphorylated H1 in both nucleus and cytoplasm
detected both in this and other studies (Bleher and Martin, 1999; Boggs et al.,
2000; Lu et al., 1994).
RESULTS AND DISCUSSION | 71
A severe limitation to immunocytochemistry is the obstacle of determining
the relative amount of the antigen. A strong immunofluorescent signal may
correspond to relatively low amount of a particular subtype compared to the
other subtypes. HPCE data on the H1 subtype composition in human diploid
fibroblasts that has not been included in Paper III, show that these cells
contain approximately 15% H1.2, 10% H1.3, 30% H1.4 and 45% H1.5.
At present, we are trying to verify our data by other methods. We are very
limited in the choice of methods for verification since there are very few
metaphase and anaphase cells present in the cell population, possibly
eliminating western blotting, HPCE and HILIC as possible techniques to
detect cytoplasmic H1 subtypes. Also, the addition of colchemid to obtain
mitotically enriched cells without apoptosis induction is a difficult task.
A differential distribution of H1 subtypes in mitosis could be a key factor in
chromatin rearrangements during cell division and possibly explain the
differential function of the subtypes suggested by evolutionary data. Many
experiments suggest overlapping functions of the subtypes, but recent data
using inducible subtype knockouts indicate specific roles of H1 subtypes
(Sancho et al., 2008). In these experiments, H1.2 depletion caused G1-arrest
and decreased nucleosomal spacing (Sancho et al., 2008). In this view, the
H1.2 redistribution to the cytoplasm during mitosis may be an important step
in preparation for the subsequent G1 progression. Possibly, the presumed
translocations of certain H1 subtypes during mitosis may be a mechanism in
epigenetic reprogramming of cell nuclei, where H1 subtypes change location
in chromatin.
Histone H1 phosphorylation in the cell cycle
We have analysed the histone H1 phosphorylation pattern during the cell
cycle in activated T cells from normal human blood donors and in Jurkat T
lymphoblastoid cells. In human T cells, a serine phosphorylation pattern was
established in late G1/early S phase, with some additional phosphorylation in
S phase. In Jurkat G1 cells, this phosphorylation was more extended possibly
due to shorter cell cycling time of this cell line compared to Jurkat cells, or to
higher activity of a H1 kinase in the G1 phase. An increased Cdk2 activity and
72 | RESULTS AND DISCUSSION
H1 phosphorylation in G1 ras-transformed mouse fibroblasts has previously
been recognized, and these cells had less condensed chromatin than
untransformed cells (Chadee et al., 2002; Chadee et al., 1995). The relation
between H1 phosphorylation and chromatin condensation has been a matter
of debate, but many pieces of data indicate that H1 phosphorylation is related
to local chromatin decondensation (Alexandrow and Hamlin, 2005; Contreras
et al., 2003; Herrera et al., 1996). Possibly, the relaxed chromatin structure and
increased H1 phosphorylation is relevant for malignant transformation
(Chadee et al., 2002; Herrera et al., 1996). Especially, localization of H1
phosphorylation and Cdk2 activity seem relevant for chromatin
decondensation in replication foci (Alexandrow and Hamlin, 2005). If the
observation of higher G1 phosphorylation in Jurkat cells, compared to T cells
is a result of overactive Cdk2, a possible function of this may be local
chromatin decondensation for rapid progression into S-phase, resulting in
shorter cell cycling times. In line with this hypothesis, recent data on Physarum
polycephalum indicate that while unphosphorylated H1 inhibits chromatin
replication, phosphorylation of H1 histones may lead to H1 removal from
chromatin and replication competence (Thiriet and Hayes, 2008). Another
function may be that increased H1 phosphorylation results in deviant gene
expression (Chadee et al., 2002). It would be very interesting to investigate the
Cdk2 activity in sorted G1 cells of normal T cells and Jurkat cells, and also the
chromatin structure of both cell types.
Another aspect of this study is that the cell cycle phosphorylation of
histone H1 has often been considered to be a sequential event, with addition
of phosphate groups throughout the cell cycle (Gurley et al., 1975; Talasz et
al., 1996). The data from Paper IV indicate that instead of a sequential event, a
H1 serine phosphorylation pattern is established in late G1/early S phase, with
only some additional phosphorylation during S phase, possibly of newly
synthesized H1 histones. The phosphorylation patterns are similar in S- and
G2/M cells, implying that only some additional phosphorylation takes place
during G2/M. The mitotic up-phosphorylation detected in colchemid arrested
cells (Talasz et al., 1996) seem to be a very rapid event, as is the complete
dephosphorylation observed after mitosis, since we detect very low amounts
RESULTS AND DISCUSSION | 73
of the H1.5 p4 and p5 variants in the G2/M population. Further studies are
needed to clarify mitotic H1 phosphorylation.
The contradiction in H1 cell cycle phosphorylation between our and other
data (Gurley et al., 1975; Talasz et al., 1996), may be a result of cell
synchronisation and cell cycle arresting drugs used in other studies and the use
of cancer cell lines and hamster cell lines with one dominant H1 subtype,
while we have used cell sorting and normal human T cells. The presence of
only serine phosphorylations in sorted G1 and S phase populations are in
good agreement with the observed presence of solely serine phosphorylations
in interphase CEM cells and threonine phosphorylation in M phase (Sarg et
al., 2006).
With the data obtained during this thesis and the literature available, I make
the following suggestion about histone H1 distribution and phosphorylation:
During gene regulation and DNA replication, chromatin remodelling and
local chromatin decondensation is performed. In this processes, specific sites
on certain H1 subtypes are phosphorylated. This is probably performed by
Cdk2, possibly in addition with other, yet unidentified kinases and takes place
primarily on serine residues in SPXK motifs. This probably leads to structural
changes and, possibly, partial release of H1 phosphorylation motifs from
chromatin. During mitosis, the massive up-phosphorylation of primarily
threonine residues leads to further release of phosphorylation motifs from
chromatin, which may give access for chromatin condensing factors in
mitosis. At some stages during mitosis, certain H1 subtypes are released from
chromatin, either nearly completely (H1.2), in part (H1.5) or not at all (H1.3).
This may be explained by specific targeting of certain H1 subtypes, or binding
sites by specific chromatin condensing or remodelling factors. H1 histones
have been demonstrated and hypothesized to affect, interact and compete
with various chromatin binding factors (Bustin et al., 2005).
Since apoptosis induction in closely connected to cell cycle checkpoints,
dephosphorylation of H1 histones may be a consequence of early cell cycle
signalling.
74 | RESULTS AND DISCUSSION
CONCLUSIONS
Based on the results from Papers I-IV, the following conclusions can be
drawn:
- Even though H1 histones are relatively conserved proteins, we have
detected and confirmed sequence variations within the human H1.2 and H1.4
subtypes. One of these sequence variations is present in DNA from normal
blood donors, with an allele frequency of 6.8%. The H1.4 sequence variation
was concluded to be Raji specific
- Histone H1 dephosphorylation is not a general event in apoptosis, but
may be relevant in apoptosis induced via the intrinsic pathway, either as a
consequence of apoptotic induction or as a cause of early cell cycle effects.
- Certain H1 subtypes are located in chromatin during all stages of mitosis,
while others are located, nearly completely or partly, to the cytoplasm during
metaphase and early anaphase. This is a novel finding, and may be relevant in
epigenetic reprogramming
- Sequential addition of phosphates has been detected across the cell cycle
previously. In the cell cycle of activated human T cells, a H1 phosphorylation
pattern is established on serine residues in late G1/early S phase in the cell
CONCLUSIONS | 75
cycle of normal human T cells. Some additional phosphorylation takes place
during S-phase. Further up-phosphorylation was detected in the G2/M phase.
- The H1 phosphorylation pattern in G1cells was more extended in Jurkat T
lymphoblastoid cells than in activated T cells, which may be due to shorter
cell cycling times or increased H1 kinase activity in G1 Jurkat cells
76 | CONCLUSIONS
FUTURE ASPECTS
H1 histones are abundant proteins, and the existence of multiple subtypes a
conserved feature. Knockdown experiments have demonstrated redundancies
between the subtypes, but also specific roles of the subtypes and the need for
H1 histones in development. Since we have detected differences in the
localization of H1 subtypes during mitosis, it would be interesting to
investigate the physiological function of this relocalisation, and the
mechanism that promotes the relocalisation where other proteins that interact
with H1 histones can be identified.
Recent data indicate that H1 from Physarum polycephalum regulate the timing
of DNA replication and suggest that phosphorylation leads to replication
competency (Thiriet and Hayes, 2008). If this turns out to be a general
mechanism in DNA replication, it may explain the function of the established
phosphorylation pattern in late G1/S we detected in normal T cells. It would
be most interesting to establish the role of H1 subtypes and their
phosphorylation in DNA replication and determine the mechanism of the
proposed regulation of replication. Since we detect differences with more
extended H1 phosphorylation in G1 Jurkat cells compared to T cells, the
mechanism may be relevant in cell cycle control and thereby in carcinogenesis
and cancer progression.
FUTURE ASPECTS | 77
78 | FUTURE ASPECTS
ACKNOWLEDGEMENTS
The studies included in this thesis, as well as some others have been carried
out in the division of Cell Biology, Department of Clinical and Experimental
Medicine, Faculty of Health Sciences, Linköping University, Linköping,
Sweden. During the time I have spent here many people have contributed in
various ways to the work presented in this thesis. Some have shared their
knowledge and expertise in various fields of research and methods, while
others have others have contributed in creating a friendly atmosphere to work
in. Also, the work behind this thesis also includes many moments of joy, but
also of disappointment and stress. Thanks to all of you how have shared these
moments and supported me in god times as well as bad times. I would like to
thank all of you who have helped and supported me during this time,
especially:
My supervisor, Dr Ingemar Rundquist for accepting me into your lab. I
am most grateful to you for sharing your great knowledge, research skills and
many ideas with me, but also for your never ending patience, support and
belief in me. You are one of the most cheerful and generous people I have
ever known.
ACKNOWLEDGEMENTS | 79
Anita Lönn, for sharing your expertise in cell culturing and many other
things, but also for being friendly, nice, patient and above all, caring and
considerate. Thanks for all the fun moments, and for always being there.
Professor Peter Söderkvist, my second supervisor, for always taking the
time to help even in busy moments. Thanks for sharing your great knowledge
in the genetics field.
Professor Herbert Lindner, Innsbruck Medical University, , for expertise
in various techniques for H1 separations, and for taking the time for all sorts
of questions and discussions and for being a gracious host on our trips to
Austria
Dr Bettina Sarg, Innsbruck Medical University, for expert help with
various H1 separations and running many, many samples extracted in the lab
in Linköping with the greatest of patience. Also, for your nice company at
various conferences.
Professor Jean Thomas, Cambridge University, for accepting me as a
student in your lab in the beginning of my research on H1 histones. All the
things I learned from you were very valuable in completing this thesis.
Dr Elizabeth Andrews, for being a good friend and supervisor during my
research project at Cambridge University
Dr Jan-Ingvar Jönsson for your generosity, help and great knowledge of
knock-downs. Even though there is still some way to go on that project, I am
sure it will be finished
Dr Marie Larsson for sharing your expertise in T cell activation with me.
Your help was invaluable in completing Paper IV.
Professor Karin Öllinger, for sharing your knowledge in
immunocytochemistry and apoptosis, as well as your aid in finishing Paper III.
80 | ACKNOWLEDGEMENTS
Dr Kajsa Holmgren for your generous help with the confocal microscope
and your contribution in Paper III.
Dr Florence Sjögren for invaluable help with flow cytometry and flow
cytometers. Thanks for always taking the time for questions and requesting
service on various flow cytometers. I am happy that we are both still sane
after all the trouble we had with the FACSAria
Dr Uno Johansson for help with the flow cytometers, and for saving my
last experiment by rapidly changing parts in the FACSAria
Anette Molbæk, for your kindness and generosity, and for teaching me
DNA sequencing in various ways as well as various kinds of detection of
mutations.
Åsa Shippert, for teaching me real time PCR and other things
Dr Helena Loborg for help with various methods and things involving
H1 histones.
Dr Elisavet Koutzamani, my roommate, friend and colleague, most of all
for being such a kind, generous, supportive and loyal friend, and secondly for
teaching me about H1 histones, extraction and separation. I would share a
solution with you any day! And thanks for all the nice trips!
Cecilia Trinks, my roommate and friend. It has been a pleasure to work
with you, and I am looking forward to continue to share room with you. I will
stop piling up papers if you buy me ice-cream!
To everybody in Cell biology, floor 13, who have made it a very nice place
to work in. To Anita S, Kerstin W, Kristina, Emilie, Eva, Lotta B, Anna
M, Anders, Åke, Thomas, Lotta H, Annelie, Ingrid, Annette W,
Monika, Björn, Hanna and Maria for a friendly atmosphere often filled
with laughter and helping with all sorts of things!
ACKNOWLEDGEMENTS | 81
My two master students, Ulrika Genheden and Cecilia Helgesson for
their kindness and aid in various experiments
My friends, Karin, Anna, Lena and Helen who always listen, support and
help, for all the fun, laughter and tears we have shared during the years.
Elisabeth Hellberg, for being the best friend in the world through good
and bad times since we were children. You are just too good to be true, and
your friendship means everything to me.
My family for everything. Without you this thesis would never have been
written.
To my mother and father for your patience, love, help and support over
the years. Many of the experiments would never have been completed without
your help with the children.
My father, Hans for always being there, and for your patience, kindness
and consideration. Also for always being supportive and letting me make my
own decisions, and for being a fantastic grandfather
My mother, Ulla for always caring and helping, and for seeing me when
even I have forgotten that I exist in a chaos of sick children, work that needs
to be done and houses to look after. Also, for being an excellent grandmother
and for your love and care for Linnea and Julia, without you this thesis would
never been completed
My brother Lars, for being the best ‘lillebror’ in the world, and to my
sister-in-law Rebecka, for always helping out and loving Linnea and Julia
nearly as much as we do
The love of my life, Henrik, for your love, support and patience. Also, for
your help with flow cytometers, cell sorting and others things at work. You
are everything I have ever wished for with your kindness, intelligence, concern
and selflessness.
82 | ACKNOWLEDGEMENTS
To our wonderful daughters, Linnea and Julia, for teaching me what true
happiness is! You are the joy of my life, with your happiness, curiosity and
love.
ACKNOWLEDGEMENTS | 83
REFERENCES
Ajiro, K., Borun, T.W., and Cohen, L.H. (1981). Phosphorylation states of different
histone 1 subtypes and their relationship to chromatin functions during the HeLa S-3
cell cycle. Biochemistry 20, 1445-1454.
Alami, R., Fan, Y., Pack, S., Sonbuchner, T.M., Besse, A., Lin, Q., Greally, J.M.,
Skoultchi, A.I., and Bouhassira, E.E. (2003). Mammalian linker-histone subtypes
differentially affect gene expression in vivo. Proc Natl Acad Sci U S A 100, 5920-5925.
Albig, W., and Doenecke, D. (1997). The human histone gene cluster at the D6S105
locus. Hum Genet 101, 284-294.
Alekseev, O.M., Bencic, D.C., Richardson, R.T., Widgren, E.E., and O'Rand, M.G.
(2003). Overexpression of the Linker histone-binding protein tNASP affects
progression through the cell cycle. J Biol Chem 278, 8846-8852.
Alekseev, O.M., Widgren, E.E., Richardson, R.T., and O'Rand, M.G. (2005).
Association of NASP with HSP90 in mouse spermatogenic cells: stimulation of ATPase
activity and transport of linker histones into nuclei. J Biol Chem 280, 2904-2911.
Alexandrow, M.G., and Hamlin, J.L. (2005). Chromatin decondensation in S-phase
involves recruitment of Cdk2 by Cdc45 and histone H1 phosphorylation. J Cell Biol
168, 875-886.
Allan, J., Mitchell, T., Harborne, N., Bohm, L., and Crane-Robinson, C. (1986). Roles
of H1 domains in determining higher order chromatin structure and H1 location. J Mol
Biol 187, 591-601.
84 | REFERENCES
Arents, G., Burlingame, R.W., Wang, B.C., Love, W.E., and Moudrianakis, E.N.
(1991). The nucleosomal core histone octamer at 3.1 A resolution: a tripartite protein
assembly and a left-handed superhelix. Proc Natl Acad Sci U S A 88, 10148-10152.
Arents, G., and Moudrianakis, E.N. (1995). The histone fold: a ubiquitous architectural
motif utilized in DNA compaction and protein dimerization. Proc Natl Acad Sci U S A
92, 11170-11174.
Ausio, J. (2006). Histone variants--the structure behind the function. Brief Funct
Genomic Proteomic 5, 228-243.
Ausio, J., Abbott, D.W., Wang, X., and Moore, S.C. (2001). Histone variants and
histone modifications: a structural perspective. Biochem Cell Biol 79, 693-708.
Bacso, Z., Everson, R.B., and Eliason, J.F. (2000). The DNA of annexin V-binding
apoptotic cells is highly fragmented. Cancer Res 60, 4623-4628.
Belmont, A.S. (2006). Mitotic chromosome structure and condensation. Curr Opin Cell
Biol 18, 632-638.
Bharath, M.M., Ramesh, S., Chandra, N.R., and Rao, M.R. (2002). Identification of a
34 amino acid stretch within the C-terminus of histone H1 as the DNA-condensing
domain by site-directed mutagenesis. Biochemistry 41, 7617-7627.
Bhattacharjee, R.N., Banks, G.C., Trotter, K.W., Lee, H.L., and Archer, T.K. (2001).
Histone H1 phosphorylation by Cdk2 selectively modulates mouse mammary tumor
virus transcription through chromatin remodeling. Mol Cell Biol 21, 5417-5425.
Bleher, R., and Martin, R. (1999). Nucleo-cytoplasmic translocation of histone H1
during the HeLa cell cycle. Chromosoma 108, 308-316.
Blomen, V.A., and Boonstra, J. (2007). Cell fate determination during G1 phase
progression. Cell Mol Life Sci 64, 3084-3104.
Boggs, B.A., Allis, C.D., and Chinault, A.C. (2000). Immunofluorescent studies of
human chromosomes with antibodies against phosphorylated H1 histone. Chromosoma
108, 485-490.
Bolund, L.A., and Johns, E.W. (1973). The selective extraction of histone fractions
from deoxyribonucleoprotein. Eur J Biochem 35, 546-553.
Bradbury, E.M., Inglis, R.J., and Matthews, H.R. (1974). Control of cell division by
very lysine rich histone (F1) phosphorylation. Nature 247, 257-261.
Brown, D.T. (2003). Histone H1 and the dynamic regulation of chromatin function.
Biochem Cell Biol 81, 221-227.
REFERENCES | 85
Brown, D.T., Alexander, B.T., and Sittman, D.B. (1996). Differential effect of H1
variant overexpression on cell cycle progression and gene expression. Nucleic Acids
Res 24, 486-493.
Brown, D.T., Izard, T., and Misteli, T. (2006). Mapping the interaction surface of linker
histone H1(0) with the nucleosome of native chromatin in vivo. Nat Struct Mol Biol 13,
250-255.
Bustin, M., Catez, F., and Lim, J.H. (2005). The dynamics of histone H1 function in
chromatin. Mol Cell 17, 617-620.
Carruthers, L.M., Bednar, J., Woodcock, C.L., and Hansen, J.C. (1998). Linker histones
stabilize the intrinsic salt-dependent folding of nucleosomal arrays: mechanistic
ramifications for higher-order chromatin folding. Biochemistry 37, 14776-14787.
Catez, F., Ueda, T., and Bustin, M. (2006). Determinants of histone H1 mobility and
chromatin binding in living cells. Nat Struct Mol Biol 13, 305-310.
Chadee, D.N., Allis, C.D., Wright, J.A., and Davie, J.R. (1997). Histone H1b
phosphorylation is dependent upon ongoing transcription and replication in normal and
ras-transformed mouse fibroblasts. J Biol Chem 272, 8113-8116.
Chadee, D.N., Peltier, C.P., and Davie, J.R. (2002). Histone H1(S)-3 phosphorylation in
Ha-ras oncogene-transformed mouse fibroblasts. Oncogene 21, 8397-8403.
Chadee, D.N., Taylor, W.R., Hurta, R.A., Allis, C.D., Wright, J.A., and Davie, J.R.
(1995). Increased phosphorylation of histone H1 in mouse fibroblasts transformed with
oncogenes or constitutively active mitogen-activated protein kinase kinase. J Biol Chem
270, 20098-20105.
Chen, D., Dundr, M., Wang, C., Leung, A., Lamond, A., Misteli, T., and Huang, S.
(2005). Condensed mitotic chromatin is accessible to transcription factors and
chromatin structural proteins. J Cell Biol 168, 41-54.
Clark, D.J., Hill, C.S., Martin, S.R., and Thomas, J.O. (1988). Alpha-helix in the
carboxy-terminal domains of histones H1 and H5. EMBO J 7, 69-75.
Clarke, H.J., McLay, D.W., and Mohamed, O.A. (1998). Linker histone transitions
during mammalian oogenesis and embryogenesis. Dev Genet 22, 17-30.
Contreras, A., Hale, T.K., Stenoien, D.L., Rosen, J.M., Mancini, M.A., and Herrera,
R.E. (2003). The dynamic mobility of histone H1 is regulated by cyclin/CDK
phosphorylation. Mol Cell Biol 23, 8626-8636.
Crane-Robinson, C. (1999). How do linker histones mediate differential gene
expression? Bioessays 21, 367-371.
86 | REFERENCES
Cuisset, L., Tichonicky, L., and Delpech, M. (1999). Quantitative analysis of histone H1
degrees protein synthesis in HTC cells. Eur J Biochem 261, 593-599.
Daujat, S., Zeissler, U., Waldmann, T., Happel, N., and Schneider, R. (2005). HP1
binds specifically to Lys26-methylated histone H1.4, whereas simultaneous Ser27
phosphorylation blocks HP1 binding. J Biol Chem 280, 38090-38095.
Di Sabato, G., Hall, J.M., and Thompson, L. (1987). T cell mitogens and polyclonal B
cell activators. Methods Enzymol 150, 3-17.
Dimitri, P., Corradini, N., Rossi, F., and Verni, F. (2005). The paradox of functional
heterochromatin. Bioessays 27, 29-41.
Doenecke, D., Albig, W., Bode, C., Drabent, B., Franke, K., Gavenis, K., and Witt, O.
(1997). Histones: genetic diversity and tissue-specific gene expression. Histochem Cell
Biol 107, 1-10.
Doenecke, D., Albig, W., Bouterfa, H., and Drabent, B. (1994). Organization and
expression of H1 histone and H1 replacement histone genes. J Cell Biochem 54, 423431.
Dominski, Z., and Marzluff, W.F. (2007). Formation of the 3' end of histone mRNA:
getting closer to the end. Gene 396, 373-390.
Dou, Y., Bowen, J., Liu, Y., and Gorovsky, M.A. (2002). Phosphorylation and an ATPdependent process increase the dynamic exchange of H1 in chromatin. J Cell Biol 158,
1161-1170.
Dou, Y., and Gorovsky, M.A. (2000). Phosphorylation of linker histone H1 regulates
gene expression in vivo by creating a charge patch. Mol Cell 6, 225-231.
Dou, Y., Mizzen, C.A., Abrams, M., Allis, C.D., and Gorovsky, M.A. (1999).
Phosphorylation of linker histone H1 regulates gene expression in vivo by mimicking
H1 removal. Mol Cell 4, 641-647.
Drabent, B., Saftig, P., Bode, C., and Doenecke, D. (2000). Spermatogenesis proceeds
normally in mice without linker histone H1t. Histochem Cell Biol 113, 433-442.
Eirin-Lopez, J.M., Gonzalez-Tizon, A.M., Martinez, A., and Mendez, J. (2004). Birthand-death evolution with strong purifying selection in the histone H1 multigene family
and the origin of orphon H1 genes. Mol Biol Evol 21, 1992-2003.
Elmore, S. (2007). Apoptosis: a review of programmed cell death. Toxicol Pathol 35,
495-516.
Evans, T., Rosenthal, E.T., Youngblom, J., Distel, D., and Hunt, T. (1983). Cyclin: a
protein specified by maternal mRNA in sea urchin eggs that is destroyed at each
cleavage division. Cell 33, 389-396.
REFERENCES | 87
Fan, Y., Nikitina, T., Morin-Kensicki, E.M., Zhao, J., Magnuson, T.R., Woodcock,
C.L., and Skoultchi, A.I. (2003). H1 linker histones are essential for mouse
development and affect nucleosome spacing in vivo. Mol Cell Biol 23, 4559-4572.
Fan, Y., Nikitina, T., Zhao, J., Fleury, T.J., Bhattacharyya, R., Bouhassira, E.E., Stein,
A., Woodcock, C.L., and Skoultchi, A.I. (2005). Histone H1 depletion in mammals
alters global chromatin structure but causes specific changes in gene regulation. Cell
123, 1199-1212.
Fan, Y., Sirotkin, A., Russell, R.G., Ayala, J., and Skoultchi, A.I. (2001). Individual
somatic H1 subtypes are dispensable for mouse development even in mice lacking the
H1(0) replacement subtype. Mol Cell Biol 21, 7933-7943.
Finn, R.M., Browne, K., Hodgson, K.C., and Ausio, J. (2008). sNASP, a histone H1specific eukaryotic chaperone dimer that facilitates chromatin assembly. Biophys J 95,
1314-1325.
Franke, K., Drabent, B., and Doenecke, D. (1998). Expression of murine H1 histone
genes during postnatal development. Biochim Biophys Acta 1398, 232-242.
Gabler, C., Blank, N., Hieronymus, T., Schiller, M., Berden, J.H., Kalden, J.R., and
Lorenz, H.M. (2004). Extranuclear detection of histones and nucleosomes in activated
human lymphoblasts as an early event in apoptosis. Ann Rheum Dis 63, 1135-1144.
Gall, J.G. (2001). A role for Cajal bodies in assembly of the nuclear transcription
machinery. FEBS Lett 498, 164-167.
Godde, J.S., and Ura, K. (2008). Cracking the enigmatic linker histone code. J Biochem
143, 287-293.
Goebel, W., Obermeyer, N., Bleicher, N., Kratzmeier, M., Eibl, H.J., Doenecke, D., and
Albig, W. (2007). Apoptotic DNA fragmentation is not related to the phosphorylation
state of histone H1. Biol Chem 388, 197-206.
Green, A., Sarg, B., Koutzamani, E., Genheden, U., Lindner, H.H., and Rundquist, I.
(2008). Histone H1 dephosphorylation is not a general feature in early apoptosis.
Biochemistry 47, 7539-7547.
Gunjan, A., Alexander, B.T., Sittman, D.B., and Brown, D.T. (1999). Effects of H1
histone variant overexpression on chromatin structure. J Biol Chem 274, 37950-37956.
Guo, X.W., Th'ng, J.P., Swank, R.A., Anderson, H.J., Tudan, C., Bradbury, E.M., and
Roberge, M. (1995). Chromosome condensation induced by fostriecin does not require
p34cdc2 kinase activity and histone H1 hyperphosphorylation, but is associated with
enhanced histone H2A and H3 phosphorylation. EMBO J 14, 976-985.
88 | REFERENCES
Gurley, L.R., Valdez, J.G., and Buchanan, J.S. (1995). Characterization of the mitotic
specific phosphorylation site of histone H1. Absence of a consensus sequence for the
p34cdc2/cyclin B kinase. J Biol Chem 270, 27653-27660.
Gurley, L.R., Walters, R.A., and Tobey, R.A. (1975). Sequential phosphorylation of
histone subfractions in the Chinese hamster cell cycle. J Biol Chem 250, 3936-3944.
Hale, T.K., Contreras, A., Morrison, A.J., and Herrera, R.E. (2006). Phosphorylation of
the linker histone H1 by CDK regulates its binding to HP1alpha. Mol Cell 22, 693-699.
Han, J., Zhang, L., Shao, X., Shi, J., and Chi, C. (2006). The potent inhibitory activity
of histone H1.2 C-terminal fragments on furin. FEBS J 273, 4459-4469.
Happel, N., Doenecke, D., Sekeri-Pataryas, K.E., and Sourlingas, T.G. (2008). H1
histone subtype constitution and phosphorylation state of the ageing cell system of
human peripheral blood lymphocytes. Exp Gerontol 43, 184-199.
Happel, N., Schulze, E., and Doenecke, D. (2005a). Characterisation of human histone
H1x. Biol Chem 386, 541-551.
Happel, N., Sommer, A., Hanecke, K., Albig, W., and Doenecke, D. (2005b).
Topoisomerase inhibitor induced dephosphorylation of H1 and H3 histones as a
consequence of cell cycle arrest. J Cell Biochem 95, 1235-1247.
Hartman, P.G., Chapman, G.E., Moss, T., and Bradbury, E.M. (1977). Studies on the
role and mode of operation of the very-lysine-rich histone H1 in eukaryote chromatin.
The three structural regions of the histone H1 molecule. Eur J Biochem 77, 45-51.
Heitz, E. (1928). Das Heterochromatin der Moose, 1. Jahrb Wiss Botanik 69, 762-818.
Hengartner, M.O. (2000). The biochemistry of apoptosis. Nature 407, 770-776.
Herrera, R.E., Chen, F., and Weinberg, R.A. (1996). Increased histone H1
phosphorylation and relaxed chromatin structure in Rb-deficient fibroblasts. Proc Natl
Acad Sci U S A 93, 11510-11515.
Hohmann, P., Tobey, R.A., and Gurley, L.R. (1976). Phosphorylation of distinct regions
of f1 histone. Relationship to the cell cycle. J Biol Chem 251, 3685-3692.
Horn, P.J., Carruthers, L.M., Logie, C., Hill, D.A., Solomon, M.J., Wade, P.A.,
Imbalzano, A.N., Hansen, J.C., and Peterson, C.L. (2002). Phosphorylation of linker
histones regulates ATP-dependent chromatin remodeling enzymes. Nat Struct Biol 9,
263-267.
Iguchi, N., Tanaka, H., Yomogida, K., and Nishimune, Y. (2003). Isolation and
characterization of a novel cDNA encoding a DNA-binding protein (Hils1) specifically
expressed in testicular haploid germ cells. Int J Androl 26, 354-365.
REFERENCES | 89
Izzo, A., Kamieniarz, K., and Schneider, R. (2008). The histone H1 family: specific
members, specific functions? Biol Chem 389, 333-343.
Jaenisch, R., and Bird, A. (2003). Epigenetic regulation of gene expression: how the
genome integrates intrinsic and environmental signals. Nat Genet 33 Suppl, 245-254.
Jakel, S., Albig, W., Kutay, U., Bischoff, F.R., Schwamborn, K., Doenecke, D., and
Gorlich, D. (1999). The importin beta/importin 7 heterodimer is a functional nuclear
import receptor for histone H1. EMBO J 18, 2411-2423.
Jenuwein, T., and Allis, C.D. (2001). Translating the histone code. Science 293, 10741080.
Kaldis, P., and Aleem, E. (2005). Cell cycle sibling rivalry: Cdc2 vs. Cdk2. Cell Cycle
4, 1491-1494.
Kaludov, N.K., Pabon-Pena, L., Seavy, M., Robinson, G., and Hurt, M.M. (1997). A
mouse histone H1 variant, H1b, binds preferentially to a regulatory sequence within a
mouse H3.2 replication-dependent histone gene. J Biol Chem 272, 15120-15127.
Kerr, J.F., Wyllie, A.H., and Currie, A.R. (1972). Apoptosis: a basic biological
phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26, 239257.
Khochbin, S. (2001). Histone H1 diversity: bridging regulatory signals to linker histone
function. Gene 271, 1-12.
Khosravi-Far, R., and Esposti, M.D. (2004). Death receptor signals to mitochondria.
Cancer Biol Ther 3, 1051-1057.
Kim, K., Choi, J., Heo, K., Kim, H., Levens, D., Kohno, K., Johnson, E.M., Brock,
H.W., and An, W. (2008). Isolation and characterization of a novel H1.2 complex that
acts as a repressor of p53-mediated transcription. J Biol Chem 283, 9113-9126.
Konishi, A., Shimizu, S., Hirota, J., Takao, T., Fan, Y., Matsuoka, Y., Zhang, L.,
Yoneda, Y., Fujii, Y., Skoultchi, A.I., et al. (2003). Involvement of histone H1.2 in
apoptosis induced by DNA double-strand breaks. Cell 114, 673-688.
Kornberg, R.D. (1974). Chromatin structure: a repeating unit of histones and DNA.
Science 184, 868-871.
Kouzarides, T. (2007). Chromatin modifications and their function. Cell 128, 693-705.
Krammer, P.H. (2000). CD95's deadly mission in the immune system. Nature 407, 789795.
Kratzmeier, M., Albig, W., Hanecke, K., and Doenecke, D. (2000). Rapid
dephosphorylation of H1 histones after apoptosis induction. J Biol Chem 275, 3047830486.
90 | REFERENCES
Kurz, M., Doenecke, D., and Albig, W. (1997). Nuclear transport of H1 histones meets
the criteria of a nuclear localization signal-mediated process. J Cell Biochem 64, 573578.
Langan, T.A., Gautier, J., Lohka, M., Hollingsworth, R., Moreno, S., Nurse, P., Maller,
J., and Sclafani, R.A. (1989). Mammalian growth-associated H1 histone kinase: a
homolog of cdc2+/CDC28 protein kinases controlling mitotic entry in yeast and frog
cells. Mol Cell Biol 9, 3860-3868.
Lee, H., Habas, R., and Abate-Shen, C. (2004). MSX1 cooperates with histone H1b for
inhibition of transcription and myogenesis. Science 304, 1675-1678.
Lee, H.L., and Archer, T.K. (1998). Prolonged glucocorticoid exposure
dephosphorylates histone H1 and inactivates the MMTV promoter. EMBO J 17, 14541466.
Lennox, R.W., and Cohen, L.H. (1983). The histone H1 complements of dividing and
nondividing cells of the mouse. J Biol Chem 258, 262-268.
Lindner, H., Wurm, M., Dirschlmayer, A., Sarg, B., and Helliger, W. (1993).
Application of high-performance capillary electrophoresis to the analysis of H1
histones. Electrophoresis 14, 480-485.
Lindner, H.H. (2008). Analysis of histones, histone variants, and their posttranslationally modified forms. Electrophoresis 29, 2516-2532.
Liu, X., Zou, H., Widlak, P., Garrard, W., and Wang, X. (1999). Activation of the
apoptotic endonuclease DFF40 (caspase-activated DNase or nuclease). Oligomerization
and direct interaction with histone H1. J Biol Chem 274, 13836-13840.
Loborg, H., and Rundquist, I. (1997). DNA binding fluorochromes as probes for histone
H1-chromatin interactions in situ. Cytometry 28, 212-219.
Lu, M.J., Dadd, C.A., Mizzen, C.A., Perry, C.A., McLachlan, D.R., Annunziato, A.T.,
and Allis, C.D. (1994). Generation and characterization of novel antibodies highly
selective for phosphorylated linker histone H1 in Tetrahymena and HeLa cells.
Chromosoma 103, 111-121.
Lu, Z.H., Sittman, D.B., Romanowski, P., and Leno, G.H. (1998). Histone H1 reduces
the frequency of initiation in Xenopus egg extract by limiting the assembly of
prereplication complexes on sperm chromatin. Mol Biol Cell 9, 1163-1176.
Lu, Z.H., Xu, H., and Leno, G.H. (1999). DNA replication in quiescent cell nuclei:
regulation by the nuclear envelope and chromatin structure. Mol Biol Cell 10, 40914106.
REFERENCES | 91
Luger, K., Mader, A.W., Richmond, R.K., Sargent, D.F., and Richmond, T.J. (1997).
Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389, 251260.
Malumbres, M., and Barbacid, M. (2005). Mammalian cyclin-dependent kinases.
Trends Biochem Sci 30, 630-641.
Maresca, T.J., Freedman, B.S., and Heald, R. (2005). Histone H1 is essential for mitotic
chromosome architecture and segregation in Xenopus laevis egg extracts. J Cell Biol
169, 859-869.
Martianov, I., Brancorsini, S., Catena, R., Gansmuller, A., Kotaja, N., Parvinen, M.,
Sassone-Corsi, P., and Davidson, I. (2005). Polar nuclear localization of H1T2, a
histone H1 variant, required for spermatid elongation and DNA condensation during
spermiogenesis. Proc Natl Acad Sci U S A 102, 2808-2813.
Matsushime, H., Quelle, D.E., Shurtleff, S.A., Shibuya, M., Sherr, C.J., and Kato, J.Y.
(1994). D-type cyclin-dependent kinase activity in mammalian cells. Mol Cell Biol 14,
2066-2076.
Meergans, T., Albig, W., and Doenecke, D. (1997). Varied expression patterns of
human H1 histone genes in different cell lines. DNA Cell Biol 16, 1041-1049.
Melan, M.A., and Sluder, G. (1992). Redistribution and differential extraction of
soluble proteins in permeabilized cultured cells. Implications for immunofluorescence
microscopy. J Cell Sci 101, 731-743.
Meyerson, M., and Harlow, E. (1994). Identification of G1 kinase activity for cdk6, a
novel cyclin D partner. Mol Cell Biol 14, 2077-2086.
Mizzen, C.A. (2004). Purification and analyses of histone H1 variants and H1
posttranslational modifications. Methods Enzymol 375, 278-297.
Montero, C. (2003). The antigen-antibody reaction in immunohistochemistry. J
Histochem Cytochem 51, 1-4.
Moreno, S., and Nurse, P. (1990). Substrates for p34cdc2: in vivo veritas? Cell 61, 549551.
Murray, A.W. (2004). Recycling the cell cycle: cyclins revisited. Cell 116, 221-234.
Ohe, Y., Hayashi, H., and Iwai, K. (1989). Human spleen histone H1. Isolation and
amino acid sequences of three minor variants, H1a, H1c, and H1d. J Biochem 106, 844857.
Ohsawa, S., Hamada, S., Yoshida, H., and Miura, M. (2008). Caspase-mediated changes
in histone H1 in early apoptosis: prolonged caspase activation in developing olfactory
sensory neurons. Cell Death Differ 15, 1429-1439.
92 | REFERENCES
Ohsumi, K., Katagiri, C., and Kishimoto, T. (1993). Chromosome condensation in
Xenopus mitotic extracts without histone H1. Science 262, 2033-2035.
Olins, D.E., and Olins, A.L. (2003). Chromatin history: our view from the bridge. Nat
Rev Mol Cell Biol 4, 809-814.
Orrego, M., Ponte, I., Roque, A., Buschati, N., Mora, X., and Suau, P. (2007).
Differential affinity of mammalian histone H1 somatic subtypes for DNA and
chromatin. BMC biology 5, 22.
Parseghian, M.H., and Hamkalo, B.A. (2001). A compendium of the histone H1 family
of somatic subtypes: an elusive cast of characters and their characteristics. Biochem
Cell Biol 79, 289-304.
Parseghian, M.H., and Luhrs, K.A. (2006). Beyond the walls of the nucleus: the role of
histones in cellular signaling and innate immunity. Biochem Cell Biol 84, 589-604.
Paulson, J.R., Patzlaff, J.S., and Vallis, A.J. (1996). Evidence that the endogenous
histone H1 phosphatase in HeLa mitotic chromosomes is protein phosphatase 1, not
protein phosphatase 2A. J Cell Sci 109 ( Pt 6), 1437-1447.
Paweletz, N. (2001). Walther Flemming: pioneer of mitosis research. Nat Rev Mol Cell
Biol 2, 72-75.
Ponte, I., Vidal-Taboada, J.M., and Suau, P. (1998). Evolution of the vertebrate H1
histone class: evidence for the functional differentiation of the subtypes. Mol Biol Evol
15, 702-708.
Pozarowski, P., Huang, X., Halicka, D.H., Lee, B., Johnson, G., and Darzynkiewicz, Z.
(2003). Interactions of fluorochrome-labeled caspase inhibitors with apoptotic cells: a
caution in data interpretation. Cytometry A 55, 50-60.
Ramakrishnan, V. (1997a). Histone H1 and chromatin higher-order structure. Crit Rev
Eukaryot Gene Expr 7, 215-230.
Ramakrishnan, V. (1997b). Histone structure and the organization of the nucleosome.
Annu Rev Biophys Biomol Struct 26, 83-112.
Rasheed, B.K., Whisenant, E.C., Ghai, R.D., Papaioannou, V.E., and Bhatnagar, Y.M.
(1989). Biochemical and immunocytochemical analysis of a histone H1 variant from the
mouse testis. J Cell Sci 94, 61-71.
Robinson, P.J., and Rhodes, D. (2006). Structure of the '30 nm' chromatin fibre: a key
role for the linker histone. Curr Opin Struct Biol 16, 336-343.
Roque, A., Ponte, I., Arrondo, J.L., and Suau, P. (2008). Phosphorylation of the
carboxy-terminal domain of histone H1: effects on secondary structure and DNA
condensation. Nucleic Acids Res 36, 4719-4726.
REFERENCES | 93
Roth, S.Y., and Allis, C.D. (1992). Chromatin condensation: does histone H1
dephosphorylation play a role? Trends Biochem Sci 17, 93-98.
Rozen, S., and Skaletsky, H. (2000). Primer3 on the WWW for general users and for
biologist programmers. Methods Mol Biol 132, 365-386.
Ruiz-Vela, A., and Korsmeyer, S.J. (2007). Proapoptotic histone H1.2 induces CASP-3
and -7 activation by forming a protein complex with CYT c, APAF-1 and CASP-9.
FEBS Lett 581, 3422-3428.
Saha, A., Wittmeyer, J., and Cairns, B.R. (2006). Chromatin remodelling: the industrial
revolution of DNA around histones. Nat Rev Mol Cell Biol 7, 437-447.
Sancho, M., Diani, E., Beato, M., and Jordan, A. (2008). Depletion of human histone
H1 variants uncovers specific roles in gene expression and cell growth. PLoS genetics
4, e1000227.
Sanger, F., Nicklen, S., and Coulson, A.R. (1977). DNA sequencing with chainterminating inhibitors. Proc Natl Acad Sci U S A 74, 5463-5467.
Sarg, B., Green, A., Soderkvist, P., Helliger, W., Rundquist, I., and Lindner, H.H.
(2005). Characterization of sequence variations in human histone H1.2 and H1.4
subtypes. FEBS J 272, 3673-3683.
Sarg, B., Helliger, W., Talasz, H., Forg, B., and Lindner, H.H. (2006). Histone H1
phosphorylation occurs site-specifically during interphase and mitosis: identification of
a novel phosphorylation site on histone H1. J Biol Chem 281, 6573-6580.
Scaffidi, C., Fulda, S., Srinivasan, A., Friesen, C., Li, F., Tomaselli, K.J., Debatin,
K.M., Krammer, P.H., and Peter, M.E. (1998). Two CD95 (APO-1/Fas) signaling
pathways. EMBO J 17, 1675-1687.
Schnitzler, G.R. (2008). Control of nucleosome positions by DNA sequence and
remodeling machines. Cell Biochem Biophys 51, 67-80.
Sekeri-Pataryas, K.E., and Sourlingas, T.G. (2007). The differentiation-associated linker
histone, H1.0, during the in vitro aging and senescence of human diploid fibroblasts.
Ann N Y Acad Sci 1100, 361-367.
Seyedin, S.M., and Kistler, W.S. (1980). Isolation and characterization of rat testis H1t.
An H1 histone variant associated with spermatogenesis. J Biol Chem 255, 5949-5954.
Shechter, D., Dormann, H.L., Allis, C.D., and Hake, S.B. (2007). Extraction,
purification and analysis of histones. Nat Protoc 2, 1445-1457.
Shen, X., and Gorovsky, M.A. (1996). Linker histone H1 regulates specific gene
expression but not global transcription in vivo. Cell 86, 475-483.
94 | REFERENCES
Shen, X., Yu, L., Weir, J.W., and Gorovsky, M.A. (1995). Linker histones are not
essential and affect chromatin condensation in vivo. Cell 82, 47-56.
Sirotkin, A.M., Edelmann, W., Cheng, G., Klein-Szanto, A., Kucherlapati, R., and
Skoultchi, A.I. (1995). Mice develop normally without the H1(0) linker histone. Proc
Natl Acad Sci U S A 92, 6434-6438.
Sivolob, A., and Prunell, A. (2003). Linker histone-dependent organization and
dynamics of nucleosome entry/exit DNAs. J Mol Biol 331, 1025-1040.
Solomon, M.J., and Varshavsky, A. (1985). Formaldehyde-mediated DNA-protein
crosslinking: a probe for in vivo chromatin structures. Proc Natl Acad Sci U S A 82,
6470-6474.
Song, X., and Gorovsky, M.A. (2007). Unphosphorylated H1 is enriched in a specific
region of the promoter when CDC2 is down-regulated during starvation. Mol Cell Biol
27, 1925-1933.
Stavreva, D.A., and McNally, J.G. (2006). Role of H1 phosphorylation in rapid GR
exchange and function at the MMTV promoter. Histochem Cell Biol 125, 83-89.
Staynov, D.Z. (2008). The controversial 30 nm chromatin fibre. Bioessays 30, 10031009.
Stein, P., and Schultz, R.M. (2000). Initiation of a chromatin-based transcriptionally
repressive state in the preimplantation mouse embryo: lack of a primary role for
expression of somatic histone H1. Mol Reprod Dev 55, 241-248.
Stoldt, S., Wenzel, D., Schulze, E., Doenecke, D., and Happel, N. (2007). G1 phasedependent nucleolar accumulation of human histone H1x. Biol Cell 99, 541-552.
Swank, R.A., Th'ng, J.P., Guo, X.W., Valdez, J., Bradbury, E.M., and Gurley, L.R.
(1997). Four distinct cyclin-dependent kinases phosphorylate histone H1 at all of its
growth-related phosphorylation sites. Biochemistry 36, 13761-13768.
Takata, H., Matsunaga, S., Morimoto, A., Ono-Maniwa, R., Uchiyama, S., and Fukui,
K. (2007). H1.X with different properties from other linker histones is required for
mitotic progression. FEBS Lett 581, 3783-3788.
Talasz, H., Helliger, W., Puschendorf, B., and Lindner, H. (1996). In vivo
phosphorylation of histone H1 variants during the cell cycle. Biochemistry 35, 17611767.
Tanaka, M., Kihara, M., Meczekalski, B., King, G.J., and Adashi, E.Y. (2003a). H1oo:
a pre-embryonic H1 linker histone in search of a function. Mol Cell Endocrinol 202, 59.
REFERENCES | 95
Tanaka, Y., Kato, S., Tanaka, M., Kuji, N., and Yoshimura, Y. (2003b). Structure and
expression of the human oocyte-specific histone H1 gene elucidated by direct RTnested PCR of a single oocyte. Biochem Biophys Res Commun 304, 351-357.
Testa, U. (2004). Apoptotic mechanisms in the control of erythropoiesis. Leukemia 18,
1176-1199.
Th'ng, J.P. (2001). Histone modifications and apoptosis: cause or consequence?
Biochem Cell Biol 79, 305-311.
Th'ng, J.P., Guo, X.W., Swank, R.A., Crissman, H.A., and Bradbury, E.M. (1994).
Inhibition of histone phosphorylation by staurosporine leads to chromosome
decondensation. J Biol Chem 269, 9568-9573.
Th'ng, J.P., Sung, R., Ye, M., and Hendzel, M.J. (2005). H1 family histones in the
nucleus. Control of binding and localization by the C-terminal domain. J Biol Chem
280, 27809-27814.
Thiriet, C., and Hayes, J.J. (2008). Linker histone phosphorylation regulates global
timing of replication origin firing. J Biol Chem, in press.
Thomas, J.O. (1999). Histone H1: location and role. Curr Opin Cell Biol 11, 312-317.
Travers, A. (1999). The location of the linker histone on the nucleosome. Trends
Biochem Sci 24, 4-7.
Twomey, C., and McCarthy, J.V. (2005). Pathways of apoptosis and importance in
development. J Cell Mol Med 9, 345-359.
Wallace, P.K., Tario, J.D., Jr., Fisher, J.L., Wallace, S.S., Ernstoff, M.S., and Muirhead,
K.A. (2008). Tracking antigen-driven responses by flow cytometry: monitoring
proliferation by dye dilution. Cytometry A 73, 1019-1034.
van Holde, K. (1989). Chromatin. Springer, New York.
Watrin, E., and Legagneux, V. (2003). Introduction to chromosome dynamics in
mitosis. Biol Cell 95, 507-513.
Weintraub, H. (1984). Histone-H1-dependent chromatin superstructures and the
suppression of gene activity. Cell 38, 17-27.
Vila, R., Ponte, I., Jimenez, M.A., Rico, M., and Suau, P. (2002). An inducible helixGly-Gly-helix motif in the N-terminal domain of histone H1e: a CD and NMR study.
Protein Sci 11, 214-220.
Vincze, T., Posfai, J., and Roberts, R.J. (2003). NEBcutter: A program to cleave DNA
with restriction enzymes. Nucleic Acids Res 31, 3688-3691.
Vindelov, L.L., Christensen, I.J., and Nissen, N.I. (1983). A detergent-trypsin method
for the preparation of nuclei for flow cytometric DNA analysis. Cytometry 3, 323-327.
96 | REFERENCES
Wisniewski, J.R., Zougman, A., Kruger, S., and Mann, M. (2007). Mass spectrometric
mapping of linker histone H1 variants reveals multiple acetylations, methylations, and
phosphorylation as well as differences between cell culture and tissue. Mol Cell
Proteomics 6, 72-87.
Wolffe, A.P., Khochbin, S., and Dimitrov, S. (1997). What do linker histones do in
chromatin? Bioessays 19, 249-255.
Volz, A., Albig, W., Doenecke, D., and Ziegler, A. (1997). Physical mapping of two
histone gene clusters on human chromosome 6p22.1-22.2. DNA Seq 8, 173-179.
Woodcock, C.L., Skoultchi, A.I., and Fan, Y. (2006). Role of linker histone in
chromatin structure and function: H1 stoichiometry and nucleosome repeat length.
Chromosome Res 14, 17-25.
Wu, D., Ingram, A., Lahti, J.H., Mazza, B., Grenet, J., Kapoor, A., Liu, L., Kidd, V.J.,
and Tang, D. (2002). Apoptotic release of histones from nucleosomes. J Biol Chem 277,
12001-12008.
Xiao, W., and Oefner, P.J. (2001). Denaturing high-performance liquid
chromatography: A review. Hum Mutat 17, 439-474.
Yamamoto, T., and Horikoshi, M. (1996). Cloning of the cDNA encoding a novel
subtype of histone H1. Gene 173, 281-285.
Yan, W., Ma, L., Burns, K.H., and Matzuk, M.M. (2003). HILS1 is a spermatid-specific
linker histone H1-like protein implicated in chromatin remodeling during mammalian
spermiogenesis. Proc Natl Acad Sci U S A 100, 10546-10551.
Yoon, Y.S., Kim, J.W., Kang, K.W., Kim, Y.S., Choi, K.H., and Joe, C.O. (1996).
Poly(ADP-ribosyl)ation of histone H1 correlates with internucleosomal DNA
fragmentation during apoptosis. J Biol Chem 271, 9129-9134.
Yu, B., Sawyer, N.A., Chiu, C., Oefner, P.J., and Underhill, P.A. (2006). DNA mutation
detection using denaturing high-performance liquid chromatography (DHPLC). Curr
Protoc Hum Genet, 7.10.11-7.10.14.
Zlatanova, J., Caiafa, P., and Van Holde, K. (2000). Linker histone binding and
displacement: versatile mechanism for transcriptional regulation. FASEB J 14, 16971704.
Zougman, A., and Wisniewski, J.R. (2006). Beyond linker histones and high mobility
group proteins: global profiling of perchloric acid soluble proteins. J Proteome Res 5,
925-934.
REFERENCES | 97