A glimpse into the epigenetic landscape of gene regulation
Introduction
Histone proteins are subject to four major post-translational modifications: methylation of arginine and lysine residues, serine phosphorylation, lysine acetylation and lysine ubiquitylation [1, 2]. Early studies in Drosophila, budding yeast and fission yeast gave the first indication, now confirmed in mammals, that lysine (K) methylation (me) and acetylation (ac) show functionally relevant and distinct associations with repressed or active chromatin. This review explores genome-wide distributions for these modifications associated with repressed or active chromatin in pluripotent and differentiated mammalian cells.
Section snippets
Techniques to study histone modifications
Two techniques are used to characterise histone modifications: mass spectrometry and chromatin immunoprecipitation (ChIP). Mass spectrometry of histone proteins can be used to detect novel modifications, to determine whether two modifications occupy the same histone tail [3], to show interdependency between modifications [4] and to compare the range of modifications in different organisms [5]. Mass spectrometry, however, gives no information on where a modified histone is found in the genome.
Lysine acetylation defines promoters, enhancers and active chromatin
Acetylation neutralises the positive charge on the ɛ amino group of lysine and acts as a binding site for proteins containing a bromodomain [6]. Although the data on genome-wide distributions for individual acetylated lysine residues lags far behind that for lysine methylation, acetylation is generally associated with active or decondensed chromatin [7, 8, 9]. In human T-cells, there are over 45 000 acetylation islands rich in H3K9acK14ac, many of which correspond to transcriptional regulatory
Lysine methylation
Patterns of lysine methylation are more complex than acetylation. Each of the three different methylated forms of any particular lysine (me1, me2 and me3) is a functionally distinct mark capable of specific interactions with domains such as PHD, chromo, MBT or Tudor on histone-binding proteins [15]. These interactions play a key role in transducing the pattern of modifications into a functional outcome [6].
Lysine methylation at enhancers and promoters
There are clear functional links between methylation and acetylation [3, 16, 17]. Acetylated nucleosomes at promoters and enhancers in mammalian cells are also methylated at several positions. H3K4me1 was the first methyl mark shown to be enriched at enhancers in HeLa cells [13•]. ChIP seq in human T-cells has allowed this initial observation to be refined and reveals a general pattern of methylation at enhancers, DNAase I hypersensitive sites, CTCF insulators and some chromosome breakpoints [18
Lysine methylation and repressed chromatin
There are three modifications, K9me3, K20me3 and K27me3, which are associated with repressed chromatin in many organisms. High-resolution ChIP seq in different mammalian cells gives a genome-wide snapshot of the modifications and reveals distinct patterns that reflect chromosome organisation [18•, 19••] (Figure 2).
H3K9me3 and H4K20me3 mark silent imprinted genes, silent clustered gene repeats, non-expressed pseudogenes, centromeres and telomeres
High levels of H3K9me3 and H4K20me3 show strong enrichment at telomeric, satellite and long terminal repeats (LTRs) as well as clustered silent genes such as the ZNF repeats [18•, 19••] (Figure 2a). H3K27me3 is excluded from these regions. H3K9me3 and H3K20me3 also appear together as localised foci on the promoters of silent imprinted genes and non-expressed pseudogenes within a large cluster of imprinted genes found within a 250 kb region in mouse embryo fibroblasts [14•] (Figure 2d). Thus, the
Tissue-specific and developmentally regulated silent genes are marked with H3K27me3
In mouse embryonic fibroblasts, the Igf2r imprinted gene cluster, marked with H3K9me3 and H3K20me3 is interspersed with a large domain of tissue-specific silent genes and several expressed genes [14•]. The domain of tissue-specific silent genes is marked with H3K27me3 alone, while the expressed genes are marked, as expected, at promoters and regulatory sequences with H3K4me3, H3K4me2 and H3K9ac. Interestingly, there are smooth transitions between the different chromatin marks with no evidence
Non-coding RNAs are implicated in silencing within discrete domains
ncRNAs are implicated in silencing both H3K9me3/H3K20me3 marked chromatin and H3K27me3 marked chromatin [22, 23, 24•]. ncRNA-based sequence-specific interactions in cis and even in trans could demarcate silent and active chromatin [25•, 26, 27]. A ncRNA-based system could discriminate different modifying activities and limit their site of action to homologous sequences. ncRNAs in association with the PRC2 complex will deposit H3K27me3 at homologous sequences, while Ago-containing complexes such
Both active (H3K4me3) and repressive marks (H3K9me3) at gene loci define imprinted regions or regulatory non-coding transcripts
One of the most puzzling observations to appear in the literature over the past year or so is that of genes marked with both activating (H3K4me3) and repressing modifications (either H3K9me3 with K20me3 or H3K27me3). One explanation for this is allelic difference, for example, an imprinted locus showing differential expression where one allele is repressed and the other active [19••, 28] (Figure 2d). In the example illustrated in Figure 2d both repressive and active marks show discrete foci on
Both active (H3K4me3) and repressive marks (H3K27me3) define poised but silent genes with initiated RNA polymerase II at the promoter
Genes marked with H3K4me3 and H3K27me3 (the so-called ‘bivalent’ modification) have been reported by a number of groups [3, 11, 18•, 30, 31•, 32•, 33•, 34]. This type of bivalent mark is particularly associated with the pluripotency of embryonic stem cells [31•, 32•, 33•, 34] and the PRC2 H3K27me3 HMTase complex [35], but is also found in differentiated cells [11, 18•, 30] and ciliates [3]. Genes carrying bivalent marks often have complex expression patterns and include key developmental
Retention and resolution of the bivalent mark during differentiation
A detailed analysis correlating the bivalent mark with gene expression in mouse stem cells, neural progenitors, mouse embryonic fibroblasts and adult cells derived from these lineages, revealed lineage-specific resolution and retention of the bivalent mark [19••]. Genes not destined to be expressed in these cell types resolve to the silent state marked with either H3K27me3 or no mark (Figure 2c). By contrast, genes actually expressed resolve to H3K4me3 alone while those destined to be expressed
DNA sequence at promoters correlates with the nature of the epigenetic mark
The nature of the underlying DNA sequence also influences how a gene is epigenetically marked [19••]. Genes with promoters rich in CG dinucleotides (CpG-rich) are almost always marked with H3K4me3 whether they are expressed or not and are enriched in the class of genes showing the bivalent mark and control at the post-initiation stage of transcription. CpG-poor promoters in contrast are only marked with H3K4me3 when expressed, whatever the cell type, and virtually none have the bivalent mark.
Bivalent marks are resolved using H3K27me3 demethylases
This new work relating the epigenome to expression profiles predicts that genes with the bivalent mark will require an H3K27me3 demethylase for expression (Figure 2e). Several recent reports link H3K27me3 demethylases such as UTX and JMJD3 to demethylation of HOX genes during the transition from a pluripotent state to a differentiated state or activation of macrophages by inflammatory stimuli [36•, 37•, 38•, 39•]. In both examples, the H3K27me3 demethylase that resolves the bivalent state is
Conclusions and perspectives
The work reviewed here shows clearly that post-recruitment or post-initiation regulatory mechanisms, maintained epigenetically through many generations, are likely to be as important as activation-coupled recruitment of RNA polymerase II in controlling gene expression. It is difficult to gauge the generality of post-initiation control of gene transcription in other eukaryotes. There are, however, reports that many thousands of genes in Drosophila have poised polymerase at their promoters [40].
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgement
Work in the author's laboratory is supported by The Wellcome Trust.
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