© The Authors Journal compilation © 2010 Biochemical Society
Essays Biochem. (2010) 48, 45–61; doi:10.1042/BSE0480045
3
Epigenetic markers and their
cross-talk
Stefan Winter1 and Wolfgang Fischle1
Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077
Göttingen, Germany
Abstract
Post-translational modifications of histone proteins in conjunction with DNA
methylation represent important events in the regulation of local and global
genome functions. Advances in the study of these chromatin modifications
established temporal and spatial co-localization of several distinct ‘marks’ on
the same histone and/or the same nucleosome. Such complex modification
patterns suggest the possibility of combinatorial effects. This idea was
originally proposed to establish a code of histone modifications that regulates
the interpretation of the genetic code of DNA. Indeed, interdependency of
different modifications is now well documented in the literature. Our current
understanding is that the function of a given histone modification is influenced
by neighbouring or additional modifications. Such context sensitivity of the
readout of a modification provides more flexible translation than would be
possible if distinct modifications function as isolated units. The mechanistic
principles for modification cross-talk can originate in the modulation of the
activity of histone-modifying enzymes or may be due to selective recognition
of these marks via modification of specific binding proteins. In the present
chapter, we discuss fundamental biochemical principles of modification
cross-talk and reflect on the interplay of chromatin marks in cellular signalling,
cell-cycle progression and cell-fate determination.
1Correspondence
may be addressed to either author (email stefan.winter@
mpibpc.mpg.de or [email protected]).
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Introduction
The basic unit of eukaryotic chromatin is the nucleosome. The core nucleosome
consists of 146 bp of DNA wrapped around an octameric histone protein
complex [1], which contains a central histone (H3/H4)2 tetramer and two
histone H2A/H2B dimers. Eukaryotic histones have two domains, a central
globular histone fold domain and less structured N-terminal tail regions.
In addition, histones H2A and H2B contain unstructured C-terminal
tails. The histone fold domains are the major structural components of the
nucleosome core. The tail regions, in contrast, protrude out of the nucleosome.
There they are targets of various PTMs (post-translational modifications) that
are important for the regulation of chromatin function.
The overall arrangement of chromatin in a cell is not uniform but is
ordered into particular structures and superstructures [2,3]. In the simplest
classification, chromatin is categorized into heterochromatin and euchromatin.
The latter is enriched for active genes and characterized by low compaction
as well as the presence of certain PTMs and non-canonical histone variants
associated with active transcription (reviewed in [4]). In contrast, more compacted and transcriptionally less or inactive genomic regions constitute heterochromatin, which is also associated with a different set of PTMs and histone
variants. Heterochromatin is further categorized as facultative or constitutive.
Transcriptional suppression in facultative heterochromatin is directed by different signals such as from extracellular stimuli during cell-cycle progression
or stages of development. In contrast, constitutive heterochromatin (e.g. pericentromeres, telomeres) is found at all times throughout cellular differentiation. It frequently contains highly repetitive elements such as pericentromeric
satellite repeats.
Epigenetic markers and combinatorial readout
For this chapter ‘epigenetic’ will be used to describe all processes that regulate
functional states of chromatin – although the exact definition of this term
comprises only heritable changes in gene expression profiles that do not
involve alterations of DNA sequence. Factors that contribute to epigenetic
mechanisms in this sense are an incorporation of non-canonical histone
variants, chromatin remodelling, non-histone protein components, DNA
methylation, DNA-binding proteins, RNAs and histone modifications
(Figure 1). In this chapter we will focus our discussion on histone
modifications and DNA methylation. We will mainly consider a framework
for epigenetic phenomena that comprises cellular signalling, cell-cycle
progression and cell-fate determination (Figure 1).
Histone and DNA modifications have been designated ‘marks’ owing
to the observation that their presence can correlate with a certain chromatin condition such as transcriptional activation or repression. The colocalization of several distinct modifications on the same histones (cis) and
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Figure 1. Factors that regulate chromatin structure and function
Processes that have a direct effect on chromatin composition and biology are shown (coloured
ellipses). In this chapter we focus on the interplay of several of these factors in cellular signalling,
cell-cycle-specific events and during cell-fate determination.
on different histones within a nucleosome or defined stretches of chromatin
(trans) was suggested to constitute a ‘histone code’ [5–7]. According to
this hypothesis, the different modifications exert their function in a combinatorial manner, implying that the placing and/or the effect of one PTM
can be positively or negatively influenced by additional modifications.
Diverse and context-specific functions that are observed for one and the
same histone modification have been used to argue in favour of such a code.
There are many different ways that such modification cross-talk can occur.
Combinatorial effects for several histone PTMs are now well documented in
the literature, however, evidence for a universal ‘histone code’ is still lacking
[5,8–10]. We therefore suggest that modification cross-talk may be considered as a context-dependent readout.
Different PTM systems target histone molecules
The multitude of different chemical groups added to histones includes mono-,
di- and tri-methylation (me1–3), acetylation (ac) and ubiquitination (ub)
of specific lysine residues, monomethylation and symmetric- (symme2) or
asymmetric (asymme2) dimethylation of arginine residues and phosphorylation
(ph) of serine, threonine and tyrosine residues. These modifications are placed
and removed by enzymes that are downstream from signalling cascades. We
will not discuss all of the different histone PTMs, but will focus on examples
where cross-talk with other modifications is well documented. Reversible
acetylation of lysine residues has been mainly linked to transcriptionally active
regions of the genome. Lysine acetylation is considered to be highly dynamic
and is governed by HATs (histone acetyltransferases; also known as KATs)
[11] and HDACs (histone deacetylases). HATs require acetyl-coenzyme A
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for their activity and frequently associate with transcriptional co-activators.
Nevertheless, they are also involved in cell-cycle progression or dosage
compensation and acetylation of various non-histone proteins [12,13]. HDACs
remove acetyl groups and are common components of multiprotein complexes
that mediate transcriptional repression.
Mono-, di- or tri-methylation (Kme1–3) of specific histone lysine residues
is catalysed by HMTs (histone methyltransferases; also known as KMTs) that
use the coenzyme SAM (S-adenosyl methionine) as the methyl donor [14].
Different degrees of methylation have different functional implications in a
wide range of biological processes including transcriptional activation, elongation or repression, imprinting, DNA replication and DNA-damage repair
[15–17]. For example, methylation of histone H3 at lysine 9 (H3K9) or H3K27
has mainly been linked to transcriptionally repressive chromatin. In contrast,
H3K4 methylation is found at transcriptionally active sites. Correspondingly,
HMTs display much higher substrate specificity than HATs. In mammals the
two Suv39h1/h2 isoform enzymes, for example, mediate H3K9me2/3 in pericentromeric heterochromatin, whereas H3K9me1/2 in euchromatic regions
is placed by the G9a–GLP complex. EZH2, another HMT, trimethylates
H3K27. Histone KDMs (lysine demethylases) comprise two major groups, the
flavin-dependent amine oxidases such as LSD1/2 that cannot process trimethylated substrates, and JMJDs (JmjC-domain-containing demethylases) that
require Fe(II) and α-oxoglutarate as cofactors [18].
Phosphorylation of serine and threonine residues was found in different
histones and has been associated with a multitude of biological processes such
as apoptosis, mitosis/meiosis, DNA-damage repair, transcriptional induction,
dosage compensation and heterochromatin formation in post-mitotic cells
[19–26]. Histone phosphorylation is highly transient and requires the stable
presence of kinases due to the constant action of phosphatases [27,28]. Many
distinct kinases have been implicated in phosphorylation of histones [25].
Transcription-related recruitment of kinases appears to be mediated mainly
via sequence-specific DNA-binding proteins. In contrast, targeting of phosphatases is not well understood [29]. The genome-wide distribution of histone
phosphorylation marks differs considerably between diverse eukaryotic lineages and even during different stages of the cell cycle, indicating that it may
be frequently involved in combinatorial modification readout (reviewed in
[19,25]).
Several histones were found to be ubiquitinated on lysine residues with
diverse biological functions [30]. Mono-ubiquitination of yeast histone
H2BK123 (H2BK120 in mammals) is conserved throughout eukaryotic organisms and is pivotal in regulation of gene transcription. In yeast, the modification is placed via sequential activity of E1-activating, E2-conjugating (RAD6)
and E3-ligase (BRE1) enzymes. Dynamic regulation of histone H2B ubiquitination is essential for gene transcription and requires the activity of the isopeptidase Ubp8, which removes ubiquitin [30].
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Methylation of genomic DNA has been reported in numerous organisms [17]. In mammals, genomic DNA can be methylated at CpG dinucleotides via DNMTs (DNA methyltransferases). These enzymes are grouped
into three separate classes: Dnmt1, Dnmt2 and Dnmt3. Dnmt1 is the major
enzyme required for maintenance of DNA methylation profiles. Dnmt2 has
been implicated in DNA and RNA methylation. However, the functional
significance of this enzyme is not well understood. Dnmt3a and Dnmt3b are
required for de novo DNA methylation, and Dnmt3L does not possess enzymatic activity, but is required for proper DNA methylation.
How do chromatin modifications have an impact on chromatin
structure and thereby regulate its function?
Histone PTMs might direct chromatin conformation in two not necessarily
exclusive ways. First, histone modifications can directly influence the
compaction status of chromatin by altering inter- or intra-nucleosomal
interactions. This scenario has been most extensively discussed for lysine
acetylation and serine/threonine phosphorylation, as these modifications
may serve to reduce net positive charge of the histone N-terminal tails and
may therefore interrupt interactions with negatively charged DNA [31].
Furthermore, acetylated nucleosomes may be inhibitory to the formation of
internucleosomal contacts [32]. One notable modification, H4K16ac, was
demonstrated to inhibit 30 nm chromatin fibre folding, demonstrating that
a single acetylation signal could have an impact on chromatin higher-order
structure [33].
In the second effector-mediated readout mode, PTMs are specifically
recognized by proteins that either directly alter the function of chromatin or
recruit additional protein complexes that initiate further modification steps [6].
Multiple protein-interaction domains that recognize particular modifications
have now been extensively characterized, providing a comprehensive understanding of the molecular basis for selective PTM binding (Table 1) [17,34].
Modification cross-talk can occur in different modes
There is expanding experimental evidence that histone PTMs are interdependent,
and a particular modification may provide the context for additional modification
events. Modification cross-talk can be best understood in the effector-mediated
readout mode, but interdependency may also be relevant for modifications that
directly alter chromatin structure.
Different histone modifications can influence each other either in a positive
or negative manner (Figure 2A). If the interacting modifications are located on the
same histone molecule we refer to this as cross-talk in cis, whereas trans cross-talk
occurs if the modifications are on different histones or nucleosomes (Figure 2A).
The simplest form of cross-talk is the exclusion of modifications that target the
same residues, such as histone H3K9 methylation and acetylation. Different correlations between distinct modifications (Figure 2A) are the observable result
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Table 1. Binding domains for different histone PTMs
Domain
PHD finger
Modification recognized
Example
H3K4me3, H3K4me0, H3K9me3, ING2, BPTF, Yng1p, BHC80
H3K36me3
WD40 repeat
H3R2me2, H3K4me2
Royal Superfamily
Chromodomain
H3K9me2/3, H3K27me2/3
HP1, CDY, Pc
Double Chromodomain
H3K4me1/2/3
CHD1
Chromo barrel
H3K36me1-3, H3K4me
Eaf3, MOF
Tudor
Rme2s
Tandem / Double Tudor
H3K4me3, H3K4me0,
JMJD2A, 53BP1
H4K20me1/2, p53K370
MBT
H4K20me1/2, H1K26me1/2,
L3MBTL1
H3K4me1, H3K9me1/2
Bromodomain
Kac, H4K16ac
PCAF/Gcn5
Double bromodomain
Kac, H4K5acK12ac,
hTAF1
H4K8acK16ac(?)
Tandem bromodomain
H3K14ac
Rsc4p,
Multivalent bromodomain
H4K5/K8ac
Brdt
14-3-3
H3S10ph, H3S28ph
BRCT
H2A.XS139ph
of cross-talk on two levels: the enzymatic systems that place or remove the
modifications (Figure 2B) and the readout of PTMs by modification-dependent
binding proteins (Figure 2C).
Pre-placed modifications can directly stimulate or block the enzymatic
activity of secondary chromatin modifiers (Figure 2Bi and 2Bii). Cross-talk
on this level can occur in cis and in trans. For example, H3K9me demethylases
LSD1 and JMJD2C are inhibited by H3S10ph (cis) and histone H2BK120ub
stimulates the Dot1 HMT enzyme to methylate histone H3K79 (trans) [35–
37]. Correspondingly, removal of a blocking modification by one factor may
Figure 2. Different modes of modification cross-talk
(A) Two or more different histone modifications can display positive or negative correlation.
If the interacting modifications are on the same histone, they function in cis. In contrast,
modifications from different histones, nucleosomes or DNA methylation cross-talk in trans.
(B) The cross-talk between different modifications can originate from the effect of one
modification on the activity or localization of the enzyme that places or removes a second
modification. Modifying enzymes can be directly stimulated (i and ii, left-hand side) or inhibited
(i and ii, right-hand side) by already existing modifications. The impact of a modification on
the activity of an enzyme can also be indirect, if the modification serves to recruit the enzyme
via a PTM-dependent interaction domain (iii). This can happen in cis (not shown) or in trans.
Enzymes can also be indirectly recruited to chromatin as components of multiprotein complexes
(iii, right-hand side). In this case one subunit of the complex may recognize a particular
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modification and thereby recruit the enzymatic activity. Some enzymes may also cross-talk with
histone modifications based on turnover of their substrate. For example, enzymes that place
or remove modifications on multiple sites of a histone may do this in a certain order (iv) (e.g.
from the N-terminal to the C-terminal end), which might be manifested in a positive cross-talk
between the preceding and the following modification (processivity). (C) Cross-talk is also
realized on the level of modification-dependent binding proteins. Combinatorial modification
patterns can either inhibit (i) or enhance (ii) the binding of proteins. If an interaction protein or
a protein complex contains more than one modification-binding domain, it can also recognize
modifications that are on separate histone tails or even distinct nucleosomes (iii and iv).
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relieve repulsion of another factor that can now bind or modify a particular
site (Figure 2Bi). Also, complex modification patterns can be placed by the
sequential activity of enzymes, such as the initial phosphorylation of histone
H3S10 that stimulates the HAT Gcn5 to acetylate H3K9/14 [38] (Figure 2Bi).
Histone PTMs can also indirectly mediate cross-talk by PTM-dependent
enzyme recruitment (Figure 2Biii). This form of cross-talk can involve a single
factor that recognizes a modification directly via a PTM-dependent interaction
domain (Figure 2Biii). For example, the HMT G9a can bind histone H3K9me2
via ankyrin repeats and might consecutively methylate nearby nucleosomes,
thereby propagating the H3K9me2 on the chromatin fibre [39]. In addition, indirect enzyme recruitment can involve the assembly of multiprotein
complexes. In this case the modification is recognized by a subcomponent of
the complex and not by the enzyme directly (Figure 2Biii). For example the
PHD-finger containing ING (inhibitor of growth) proteins can bind histone
H3K4me3 and associate with HAT or HDAC enzymes to mediate acetylation
or deacetylation of nearby histones [40]. Some enzymes may have to place or
remove modifications in a consecutive order as has been suggested for HATs
and HDACs (Figure 2Biv) [41].
More indirect cross-talk is established on the level of modification readout via effector protein binding. Modification patterns can form co-operative
or repulsive binding sites for PTM-dependent factors. For example, HP1
(heterochromatin protein 1) proteins are removed from H3K9me2/3 binding
by adjacent H3S10ph. Synergistic binding has been observed for 14-3-3 proteins to H3S10phK9/14ac [42,43] (Figure 2Ci and 2Cii). Binding of multiple
modifications by one factor or a multiprotein complex can also occur in trans
and mediate interaction between different histone tails or even nucleosomes
(Figure 2Ciii and 2Civ). For example, Brd4 can simultaneously bind acetylated
lysine residues on histones H3 and H4 via its two bromodomains (Figure
2Ciii) [44]. The interaction between different nucleosomes is particularly
interesting, as this would enable trans-fibre interactions that may be crucial
for chromatin structural organization. Some PTM-dependent binding proteins
such as HP1 can form dimers or multimers and therefore have the potential to
mediate such trans-fibre interactions (Figure 2Civ).
Our current understanding suggests that certain (if not all) histone PTMs
are interconnected and the impact of particular modifications on chromatin
structure is only comprehensible on the basis of the overall PTM content of
a chromatin region. So far we have discussed general modes of modification
cross-talk. In the following sections we will have a more detailed look at
examples of cross-talk in concrete cellular situations (see Figure 1).
Modification cross-talk during transcription
Transcription constitutes one of the most complex cellular processes and
encompasses initiation, reinitiation, elongation and termination. Modification
of chromatin is a potent platform for the regulation of transcription.
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Trans-modification cross-talk during transcription initiation
Analysis of yeast strains displaying defects in histone H2BK123 ubiquitination
revealed a strong reduction of histone H3K4 and H3K79 di- and tri-methylation,
which corresponds to a positive trans cross-talk system (Figure 2A). This
modification cross-talk is quite complex and involves the HMT enzymes Dot1
and Set1 that methylate histone H3K79 and H3K4 respectively. Histone H2B
ubiquitination is not required for the recruitment of Set1 and Dot1, but directly
stimulates their processivity in trans (Figure 2Bii) [36,45]. Besides regulating
H3K4/K79 methylation, histone H2BK123ub also recruits two additional
proteins Rpt4 and Rpt6, which may support the localization of the SAGA–
HAT protein complex, corresponding to an indirect recruitment of enzymatic
activity (Figure 2Biii). The SAGA complex not only has HAT activity, but also
contains the ubiquitin protease Ubp8 that removes ubiquitin from H2BK123,
a step that is essential for productive elongation. In addition, the CHD1
subunit of SAGA contains a double chromodomain that has been suggested
to bind H3K4me2/3 and thereby might support recruitment of the complex
(Table 1). This complicated reciprocal cross-talk system also demonstrates that
sequentially placed histone modifications regulate transcription in a dynamic
manner.
Cis-modification cross-talk during transcription
Serine/threonine phosphorylation of histone H3 displays opposite patterns in
mitosis and interphase. It correlates with chromosome condensation on one
side and with gene transcription on the other side [25]. Histone H3S10ph has
been implicated in gene activation in response to MAPK (mitogen-activated
protein kinase) or nuclear hormone receptor signalling [25] and appears to
positively cross-talk with H3K9/K14ac in cis (Figure 2A) [46]. Numerous
H3S10 kinases were described, including RPS6K (ribosomal S6 kinase),
MSK1/2 (mitogen- and stress-activated kinase 1/2), JIL1 and PIM1. These
enzymes appear to be recruited to their target regions via sequence-specific
DNA-binding proteins. So far it is not known whether H3S10ph has a major
impact on chromatin higher-order structure [47].
Phosphorylation of H3S10 relieves HP1 from its H3K9me2/3-binding
site and generates a low-affinity interaction site for 14-3-3 proteins [43,48,49].
These factors are then more stably bound upon additional H3K9ac or
H3K14ac [50]. Therefore this modification can participate in both repulsion
and enhancement of protein binding depending on the context of interacting
proteins and additional modifications (Figure 2Ci and 2Cii). 14-3-3 proteins
were demonstrated to recruit chromatin remodelling complexes to promoter
regions [51]. Another function of 14-3-3 binding to histone H3 appears to be
cross-talk with histone H4K16ac via recruitment of the MOF acetyltransferase
(Figure 2Biii) [44]. The resulting acetylations of H3K9 and H4K16 constitute
a binding platform for the double bromodomains of Brd4 (Figure 2Ciii). Brd4
in turn recruits p-TEFb that is required for RNA polymerase II elongation
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[44]. Interestingly, co-operative binding of two acetyl groups (H4K5ac/K8ac)
can also be mediated by a single bromodomain, as has been demonstrated for
the mouse TAF1 homologue Brdt [52]. This observation suggests that acetylation-dependent protein recruitment can occur in a threshold-dependent
manner. Another example of phosphorylation-dependent cross-talk includes
reduced target binding of the HAT Gcn5 initiated by loss of H3T11ph at
DNA-damaged chromatin regions [53]. The regulatory examples demonstrate
that gene transcription is strongly effected by modification cross-talk, thereby
setting up a (sometimes gene-specific) cause-and-effect chain that serves to
fine-tune the complex process of transcription.
Cell cycle dependent cross-talk
Mitosis constitutes a special section of the cell cycle, with major alterations
in chromatin conformation. Several histone PTMs are subjected to cyclical
patterns of appearance and removal during mitotic progression, including
methylation, acetylation and phosphorylation [54]. Indeed, massive
phosphorylation of several serine residues in histone H3 constitutes a hallmark
of M-phase chromatin. Aurora B and NIMA kinases phosphorylate histone
H3S10 and H3S28 whereas Haspin modifies H3T3 [25]. Furthermore, mitotic
phosphorylation of H3T11 has been described. Although the exact function of
these simultaneous phosphorylation events is still not fully clear, it is tempting
to speculate that these are causal for the displacement of a large number of
factors involved in chromatin functional regulation from mitotic chromosomes
including HATs, HDACs and HP1. In addition, transcription factors such
as Sp1/3, E2F1, TFIIB/D and RNA polymerase II are released at the onset of
M-phase.
What is the purpose of these massive chromatin alterations? The major
function of mitotic chromatin modifications is most likely to generate a
chromatin conformation that is compatible with chromosome segregation.
However, at the same time it is necessary to propagate epigenetic markers
throughout cell division to re-establish parental chromatin states in daughter
cells. Accordingly, not all genes are completely silenced during mitosis and
some chromatin-associated proteins such as CTCF (CCCTC-binding factor),
TBP (TATA-binding protein) or Brd4 remain bound to mitotic chromosomes,
but in a locally restricted manner. These factors are thought to mark genes for
re-establishing gene expression after mitosis [55]. The mitotic marking function of Brd4 is particularly interesting, as binding of this factor may require
simultaneous histone H3 and H4 acetylation. Mitotic chromatin is globally
hypoacetylated, nevertheless Brd4 is selectively bound to genes that are immediately expressed after mitosis [55]. This observation indicates that during
mitosis a particular pathway locally maintains histone H3/H4 acetylation to
regulate marking via Brd4.
Conversely, negative cross-talk of modifications has been described at the
onset of mitosis for H3S10ph at silenced pericentromeric heterochromatin
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that directly interrupts the association of HP1 proteins with H3K9me2/3 [42].
It was shown in fission yeast that displacement of the HP1 homologue Swi6
results in transcription of centromeric repeats. These transcripts are further
processed into siRNAs (small interfering RNAs) that redirect H3K9me2/3
and restoration of heterochromatin [56]. Paradoxically the propagation of
constitutive heterochromatin is therefore dependent on its transcription [57].
Consequently, H3S10ph may function as a signal to direct either gene transcription in interphase (see above) or heterochromatin transcription in mitosis
to mediate epigenetic heterochromatin preservation. Together, the addition or
removal of histone PTMs and chromatin-associated proteins may support the
propagation of gene expression while at the same time these mechanisms allow
chromosome compaction and chromatid separation.
Cross-talk in cell-fate decisions
The switch from cellular pluripotency to lineage-committed or terminally
differentiated cells involves major alterations in chromatin structure, including
DNA and histone methylation. These epigenetic adaptations are critical for
the maintenance of the identity of a cell. Conversely, cellular transformation
entails faulty epigenetic changes that complement genetic alterations in the
progression of cancer [58,59] (Figure 3).
DNA methylation and histone methylation cross-talk
DNA methylation constitutes a central parameter in the establishment of
cell-type-specific chromatin states. Cross-talk of DNA methylation with
histone modifications, especially histone H3K9me2/3, has been observed in
different model systems [17]. The mammalian Suv39h1 enzyme methylates
histone H3K9 and this modification was shown to mediate localization of
the DNMT Dnmt3b and accordingly DNA methylation to pericentromeric
repeats. In contrast, binding of Dnmt3L to the histone H3 N-terminal tail is
inhibited by H3K4 methylation in vitro [60]. In this way de novo methylation
via Dnmt3a/b may be positively and negatively regulated by pre-placed
histone modifications. Another histone H3K9 methyltransferase, G9a/GLP,
is required to direct DNA methylation at euchromatic regions (reviewed in
[17]). Both systems appear to cross-talk in a reciprocal manner, as Dnmt1 and
Dnmt3b mutant cells also show changes in the H3K9 methylation profile [61].
In addition, histone H3S10 phosphorylation appears to be involved in
this system. Loss of the phosphatase PP1 results in increased histone H3S10ph
in Neurospora crassa concomitant with loss of histone H3K9me3 and DNA
methylation [62]. In Drosophila, histone H3S10ph counteracts heterochromatin formation and has an impact on chromatin structural organization [63].
These findings place H3S10ph upstream from histone and DNA methylation,
thereby preventing transcriptionally repressive heterochromatin formation.
Nevertheless, histone H3S10 phosphorylation by Aurora B together with
H3K9me3 was reported to mark heterochromatin in the course of terminal
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Figure 3. Chromatin structure and its role in cell-fate decision
The model depicts the ‘fate’ of chromatin at a particular gene locus during the undifferentiated
state, cellular differentiation, epigenetic propagation and cellular transformation (nucleosomes
are drawn as barrels). In the undifferentiated cell state ‘A’ the locus is bivalently marked by
H3K4me3 and H3K27me3. During differentiation the locus becomes active in cell state ‘B’ but
not in cell state ‘C’ where the gene is maintained in a repressed state. This step is mediated by
removal of either H3K27me3 (state ‘B’) or H4K3me3 (state ‘C’) by lysine demethylases. DNA
methylation at CpG dinucleotides locks the repressed state. These conditions are propagated to
consecutive generations via epigenetic mechanisms. Cellular transformation can occur if tumour
suppressor genes are erroneously silenced (loss-of-function), which involves DNA methylation
and repressive histone modifications, or if oncogenes become activated after spurious loss of
repressive chromatin marks (gain-of-function).
differentiation [26]. Furthermore, DNA methylation has been implicated in
Aurora-B-mediated histone H3S10ph at pericentromeric heterochromatin [64].
These contradictory observations indicate that this cross-talk system could be
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used in different manners in distinct organisms. Alternatively, additional modifications and factors that have not yet been described may participate in the
cross-talk between DNA and histone methylation and phosphorylation.
Opposing histone modifications mark genes in ESCs
(embryonic stem cells)
Another interesting example of chromatin cross-talk in cell-fate decision
involves histone H3K4 and H3K27 methylation in ESCs. Histone H3K27 is
methylated by the EZH2 methyltransferase subunit of the PRC2 (Polycomb
repressive complex 2). It recruits the PRC1 complex for repression of major
developmental regulator genes (reviewed in [65]). In ESCs, H3K27me3 covers
a large portion of the genome, but surprisingly the majority of these regions
are also marked with histone H3K4me3 that is normally found at sites of
active transcription [66]. The sites of co-existence of these two opposing
histone modifications have been termed ‘bivalent domains’ [66] as they may
poise a gene for either transcriptional activation or repression, depending on
the particular fate of the ESC (Figure 3). This means that upon differentiation
into a particular cell lineage, a gene whose regulatory region is marked in a
bivalent manner will either become transcriptionally active and will lose the
repressive H3K27me3 mark, or will maintain this modification, but will lose
H3K4me3 (reviewed in [65]). Two additional factors demonstrate cross-talk
with this system. Recruitment of the PRC1 complex mediates histone H2AK119
ubiquitination via its RING1 component, which is crucial to keep RNA
polymerase II located at the bivalently marked genes in a non-elongating
form [65]. In addition, a specialized histone variant, H2A.Z that was shown
to negatively correlate with DNA methylation is located at these regions.
Together these mechanisms appear to ensure that differentiation-specific
genes are not stably silenced in ESCs, but at the same time they inhibit
erroneous expression, which would interfere with the pluripotent state.
It is not yet clear if or to what extent histone H3K4 and K27 methylation
exert cross-talk, or even if they occur on the same histone. However, the
resolution of these domains into transcriptional active sites may require the
activity of H3K27-specific demethylases along with H3K4 methyltransferases.
Accordingly, the stable silencing of these domains may require H3K4-specific
demethylase together with H3K27me3-mediated polycomb silencing and DNA
methylation. Perhaps both systems exert cross-talk to exclude the opposing
state. Indeed, recruitment of H3K27 demethylases via H3K4 methylation in the
course of cell differentiation might block reconversion into a bivalent state [67].
Conclusions
Histone PTMs are important regulators during all processes that involve
alteration of chromatin behaviour, such as transcription, DNA-damage repair,
cell-cycle progression and apoptosis. Experimental evidence that chromatin
modifications are interdependent is continuously increasing. In this chapter
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we have discussed examples of cross-talk between histone modifications and
between histone PTMs and DNA methylation in different cellular processes.
Cross-talking systems offer the advantage of increased flexibility and functional
control due to combinatorial modification usage. Despite clear examples for an
interconnection between different histone modifications, our understanding of
this issue is far from complete. Clearly, there is much more to be discovered in
the near future. We would like to encourage the interested reader to take the
cited literature as a starting point for further and continuous reading.
Summary
•
•
•
•
•
•
Histone PTMs serve as marks for particular epigenetic states.
Histone modifications have an impact on chromatin structure either
directly, by altering internucleosomal or nucleosome–DNA interactions, or by recruiting modification-dependent binding factors (effector
proteins).
Multiple histone modifications do not function as isolated signals but
cross-talk with additional marks to mediate a particular biological readout.
Modification cross-talk occurs on different levels. First, PTMs have a
direct impact on the activity of enzymes that place additional modifications and second, they modulate effector protein binding (Figure 2).
Cross-talk can occur either in cis, if modifications on the same histone
tail are involved or in trans, if cross-talk involves modifications on different histone tails, different nucleosomes or DNA methylation. The
readout of combinatorial modification patterns can be either positive or
negative.
The combinatorial readout of histone modifications is essential for multiple cellular processes such as transcription, mitosis or cellular differentiation.
We apologize to many colleagues whose work could not be cited owing to space
limitations. We are grateful to Kathy Gelato for comments on the manuscript.
Work in the Fischle laboratory is funded by the Max Planck Society, Deutsche
Forschungsgemeinschaft (DFG) and the European Union (EU, FP6). Stefan Winter is
supported by the EMBO long-term fellowship programme.
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