Grasping trimethylation of histone H3 at lysine 4 - Future Medicine

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Grasping trimethylation of histone H3 at lysine 4 Post-translational modifications of chromatin have become a ‘booming’ area of biomedical research. One particularly interesting modification that is important for eukaryotic gene expression is trimethylation of histone H3 lysine 4 (H3K4me3), which is almost exclusively associated with active promoters of RNA polymerase II. In this article, we highlight the recent progress related to the biochemistry and biology of this histone mark, including its relevant ‘writers’ and ‘readers’. We also outline the complex regulatory mechanisms that are involved in establishing H3K4me3 in health and disease. Further understanding of H3K4me3 regulation will offer both more insight into chromatin-based mechanisms of gene regulation and provide opportunities for epigenetic intervention of the diseased state. KEYWORDS: chromatin modifications n epigenetics n gene regulation n histone methylation n TFIID

Michiel Vermeulen1 & HT Marc Timmers†1 Department of Physiological Chemistry, Cancer Genomics Centre & Netherlands Proteomics Centre, University Medical Centre Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands † Author for correspondence: Tel.: +31 887 568 981 Fax: +31 887 568 101 [email protected] 1

Chromatin, post-translational modification of histones & gene expression The direct involvement of chromatin in the regulation of gene expression has become apparent by the identification of chromatin-modifying activities in many transcriptional cofactors. This stresses the fact that eukaryotic genes are under tight control of chromatin-dependent mechanisms, which involve changes in chromatin organization and post-translational modifications. Whereas chromatin control acts on all DNAdependent processes in eukaryotes, its regulatory effects on gene expression have been studied in the most detail. Assembly of genes into chromatin involves wrapping of the DNA fiber around a set of highly conserved basic histone proteins, which together form the nucleosomal core particle. Nucleosomes represent the fundamental unit of chromatin and they consist of approximately 147 bp of DNA wrapped almost two-times around an octamer of the four core histones (H3, H4, H2A and H2B) [1,2] . The nucleosome is not a static entity in time, but displays dynamic behavior with respect to interactions with DNA and to composition of the histone component [3,4] . Within the octamer, specific histones can be replaced by histone variants, such as H2A.Z, macroH2A or H2A.X for canonical H2A and CENP-A or H3.3 for H3 [5,6] . Nucleosomes incorporating such histone variants are found at specific regions of the genome. For regulation of transcription the H3.3 and H2A.Z variants are most relevant, since H3.3

has been linked to actively transcribed genes and H2A.Z is primarily found at 5´-end of genes [7,8] . Besides the subunit dynamics of nucleosomes, a variety of chromatin remodeling complexes have been identified, which employ ATP-hydrolysis to alter nucleosome structure and position relative to the DNA fiber [9] . Obviously, such alterations are not stably transmitted through cell divisions, but the continuous presence of such remodeling activities can dictate stable chromatin states [10] . Elucidation of the nucleosomal structure demonstrated that the unstructured N-terminal tails of histones project outward from nucleosome core particles [2] . These tails can interact with neighboring nucleosomes and/or form binding sites for regulatory protein complexes. Histones and, in particular the histone tails, are subjected to extensive covalent modifications, which include acetylation, methylation, phosphorylation and ubiquitination [11] . Although histone modifications have been associated with epigenetic processes, the majority of histone modifications are biochemically reversible. The particular combination of post-translational modifications on a nucleosomal particle has been proposed to constitute the ‘histone code’, which acts to control accessibility and expression of genes via direct binding of effector proteins [12] . The N-terminal tail of histone H3 is subjected to many post-translational modifications. Whereas acetylation of H3 is mostly involved in transcriptional activation, methylation of histones can be associated both with gene expression and with gene silencing, which depends on the specific residue in the histone

10.2217/EPI.10.11 © 2010 Future Medicine Ltd

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tail [13,14] . Methylation can occur on arginine and lysine side chains and both can bear multiple methyl groups. Different methylation states of lysines are associated with distinct chromatin functions. Trimethylation of lysine 4, lysine 36 and lysine 79 (H3K4, H3K36 and H3K79, respectively) are enriched at transcribed regions of genomes, whereas trimethylation of H3K9 and H3K27 generally correlates with inactive genes. Genes involved in regulation and recognition of lysine-methylated histones have been found to be either mutated or overexpressed in human developmental and cancer syndromes (e.g., [15–19]). This underscores the importance of lysine methylation of histones as a versatile regulatory tool and its reversibility offers unique possibilities for therapeutic interventions. In this article, we will focus on trimethylation of lysine 4 of histone H3 (H3K4me3) and its involvement in the regulation of transcription. We include new insights in the recruitment of the enzymes responsible for deposition of this mark, in effectors of H3K4 methylation, and in the crosstalk and co-occurrence with other chromatin modifications. For other aspects on methylation of H3K4 we refer to several excellent reviews, which have been published recently [17,20–26] .

Establishing H3K4 methylation patterns in yeast All three methylation states of H3K4 in Saccharomyces cerevisiae depend on a single histone lysine methyltransferase (KMT) enzyme, Set1p [27] . The enzymatic core of this ‘histone code writer’ is embedded in the Set domain, which is the hallmark domain of the KMT group of enzymes. The yeast genome encodes at least two other Set-domain proteins: Set2p is responsible for H3K36 methylation and is associated with transcription elongation [28] and Set3p, which lacks KMT activity and functions during meiosis [29,30] . The conserved Dot1p is a non-Set-domain KMT, which is responsible for methylation of H3K79 located in the globular part of the nucleosome [21,31] . The positive correlation between H3K4me3 and gene activity was first recognized in yeast [27] . Subsequent genome-wide mapping by chromatin immunoprecipitation with microarray technology (ChIP–chip) approaches revealed that H3K4me3 peaks at the 5´-ends of active genes [32] . Furthermore, the H3K4me3 peak correlates with increased H3K9, H3K14 and H4 acetylation. This study also revealed that intergenic regions display a lower nucleosome density 396

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compared with transcribed parts of genes. The patterns of the other methylation states of H3K4 are quite distinct from H3K4me3. H3K4me2 levels peak downstream of H3K4me3 and the monomethyl mark is dispersed throughout the transcribed region [32] . This differential distribution of H3K4 methylation is remarkable, since the Set1p enzyme is responsible for all three methylation states. The molecular mechanisms involved in this have been a subject of intense investigation. The strict substrate specificity of the KMTs is in sharp contrast to histone acetyltransferase enzymes. Processivity of the KMTs to advance from the mono- to the tri-methyl state is dependent on the catalytic center within the Set domain [33,34] , but it is also regulated via other histone modifications or via associated proteins. Purification of Set1p showed that it is part of a protein complex of eight subunits, which form the Set1C or Complex Proteins Associated with Set1 (COMPASS) complex [35,36] . Assembly of Set1p into this complex is essential for its enzymatic activity toward H3K4, which occurs in a nonprocessive manner. Ubiquitination of histone H2B is required for activation of H3K4 methylation [37] and this activation involves monoubiquitination and recruitment of specific subunits to the Set1C/COMPASS complex [38–40] . It is interesting to note that several Set1C/COMPASS subunits harbor protein domains capable of interacting with (modified) histone tails. These observations are probably relevant in light of the distinct genomic patterns of the three H3K4 methylation states in yeast.

Distribution of H3K4 methylation in mammalian cells Global analysis of H3K4me distribution in mammalian cells yields a similar but not identical picture compared with yeast. Whereas H3K4me3 and H3K4me2 clearly peak at promoters of the 5´-end of genes correlating with their transcriptional output [41–43] , H3K4me1 marks enhancer elements distant to the transcription start site and H3K4me3 is conspicuously absent from such genomic loci [43,44] . Particularly interesting is the co-occurrence of the activating H3K4me3 with the repressive H3K27me3 mark in pluripotent embryonic stem cells (ESCs) on silent transcription regulators important for ESC differentiation [45,46] . These ‘bivalent’ domains consist of peaks of H3K4me3 coinciding with regions of H3K27me3. It is important to note that bivalent domains have also been observed in other cell types like future science group

Function & occurrence of H3K4me3 modification

primary human T cells [47] . Bivalent domains are assumed to dictate a ‘poised state’ for transcription with K4me3 serving for rapid activation upon removal of the repressive H3K27me3 mark. In line with this hypothesis, it has been shown that that removal of the PcG proteins responsible for maintaining H3K27 methylation results in loss of pluripotency of ESCs [48] . PcG binding also coincides with H2A.Z on develop mental genes [49] . While this histone variant is not important for the pluripotent state, H2A.Z depletion results in impaired differentiation of ESCs. Whereas RNA polymerase II (pol II) promoters in general carry nucleosomes marked by H3K4me3, the transcribed regions of active genes are also enriched for the H3K36me3 mark [50] . In fact, the simultaneous presence of H3K4me3 and H3K36me3 on genes has been used to discover new transcriptional units [51,52] . Taken together, these observations indicate that not only the presence of the H3K4me3 mark itself but also the modification status and structure of the surrounding chromatin are relevant for H3K4me3 function in gene expression. The Set-domain family of KMT proteins has expanded during evolution to higher eukaryotes. In mammalian cells seven Set-domain subfamilies have been identified and each contains multiple members [53] . To date at least eight different Set-domain proteins capable of methylating (‘writing’) H3K4 are known [21] . The best studied are the Set1p-like proteins bearing similarity to the mixed-lineage leukemia (MLL) proteins: MLL and MLL2–4 (Table 1) . The MLL gene is a frequent translocation partner in a unique group of acute leukemias, which are associated with a poor prognosis [18] . The resulting MLL fusion proteins have lost KMT activity and they can induce the dedifferentiation of myeloid cells, and activation of their self-renewal properties to yield the cancerous state. Besides the MLLs, the human hSet1A and hSet1B proteins can also methylate H3K4. All MLL and hSet1A/B proteins reside in large multisubunit complexes similar to the yeast Set1C/COMPASS complex [54–58] . The core of the complexes is formed by the hAsh2L, RBBP5, WDR5 and hDPY-30 subunits. The MLL and hSet1 complexes are distinguished by the presence of menin and HCF1 in MLL/MLL2 [54,56] , PTIP, PA1, NCOA6, and UTX in MLL3/ MLL4 [57,59] or Wdr82, HCF1 and CXXC1 in hSet1A/B complexes [58] . Both the Set1/MLL enzymes and their associated subunits contain chromatin-interaction domains, which may anchor these complexes to specific genomic future science group

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Table 1. Subunit composition of the mammalian hSet1 and mixed-lineage leukemia complexes. Complex

Enzymatic subunit

Core subunits† Complex-specific subunits‡

hSet1A/B

hSet1A/hSet1B

MLL/MLL2

MLL/MLL2

Ash2L RBBP5 WDR5 hDPY30 Ash2L RBBP5 WDR5 hDPY30 Ash2L RBBP5 WDR5 hDPY30

MLL3/MLL4 MLL3/MLL4

CXXC1 Wdr82 HCF1 Menin HCF1/2 LEDGF PTIP PA1 NCOA6 UTX

These complexes can convert histone H3K4 to a mono-, di- and tri-methyl states. It is important to point out that the methylase functions are not redundant during mouse embryonic development. † The core of these H3K4 methylase enzymes is formed by the four subunits. ‡ The complex specific subunits may play a role in recruitment (Wdr82, Menin, HCF1/2 and PTIP) or contain additional enzymatic activities (UTX). MLL: Mixed-lineage leukemia.

loci. In addition, several subunits link transcription activators to H3K4 methylation (see later). Together, the different subunit compositions and interaction partners for MLL/hSet1 complexes suggests a functional specification for these H3K4 methyltransferases.

Recruitment of H3K4 methylation activities An important issue in understanding the link between gene activity and H3K4 methylation is whether the deposition of this modification mark is a cause or consequence of pol II transcription. Experiments in yeast have provided support for the latter as Set1p has been shown to co-immunoprecipitate with the initiating form of pol II [60] . During the transcription cycle of pol II its C-terminal domain (CTD) undergoes a cycle of phosphorylation with serine 5 and serine 7 associated with the initiation and serine 2 with the elongation phase [61] . Yeast Set1p co-immunoprecipitates with Ser5and not with Ser2-phosphorylated CTD [60] . This interaction depends on the TFIIH kinase responsible for Ser5/Ser7 phosphorylation of the CTD [61] . In addition, Set1p occupancy correlates strongly with yeast promoters [60] . A similar picture was initially obtained in mammalian cells by ChIP–chip mapping of MLL distribution [62] . However, it is now believed that MLL complexes play a gene-selective role and that the hSet1A/B complexes are instead responsible for maintaining global H3K4 methylation [63] . This is supported by findings that the hSet1A/B-specific subunit Wdr82 in www.futuremedicine.com

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conjunction of the RNA-recognition motif (RRM) of hSet1A can interact with the Ser5phosphorylated CTD peptides. Wdr82 is crucial for recruitment of these complexes to transcription start sites [64] . Interestingly, yeast Set1p also contains an RRM domain and its deletion only affects global H3K4me3 and not H3K4me2 levels [65] . By contrast, MLL proteins do not bear an RRM domain, which suggests that hSet1A/B complexes are the equivalent of the yeast Set1C/COMPASS complex and that deposition of H3K4me3 by this complex is a consequence of transcription. The gene-selective function of MLL complexes was first indicated by induced expression of the Hoxa cluster in myeloid cells transformed by MLL fusions [66] . Analysis of MLL -/- mouse embryonic fibroblasts also indicated that MLL is required for the expression of a subset of genes [67] . The Hox gene clusters contain large regions of H3K4 methylation, which coincides with functional MLL binding [50,67,68] . How is MLL recruited in a gene-selective manner? Analysis of mRNA profiles in mouse embryonic fibroblasts suggests that the menin subunit of MLL/MLL2 complexes is involved in this [54,67] . Menin is encoded by the MEN1 tumor suppressor gene and genetic inactivation of this gene is causative to multiple endocrine neoplasia type 1 [24,69] . Menin can act as a transcriptional cofactor for activated nuclear hormone receptors, such as the estrogen receptor-a and PPAR-g receptors and direct H3K4me3 to their target genes [70–72] . The MLL2 enzyme itself can also directly interact with activated estrogen receptor-a [73] . Similarly, E2F and LEF-1/TCF can recruit MLL/MLL2 complexes and H3K4 trimethylation via its HCF1 subunit and the b-catenin coactivator, respectively, to target promoters [74,75] . These observations indicate that recruitment of MLL/MLL2 complexes and the subsequent H3K4 methylation is a cause for transcription and these complexes serve as direct transcriptional coactivators. MLL3/MLL4 complexes can also be recruited to hormone- or p53-activated promoters [59,76,77] . Taken together it has become clear that the specific H3K4 KMT complexes in mammals are dedicated to specific and nonredundant roles in linking H3K4 methylation to gene regulation.

Stability of histone methylation The discovery of histone lysine demethylase (KDM) enzymes revealed that the methylation state is biochemically dynamic [78] . Similar to 398


the KMTs, the family of demethylases (the socalled ‘erasers’) displays an exquisite substratespecificity and has also expanded significantly during evolution (F igur e 1) [17,79] . The mammalian LSD1 (KDM1A) and AOF1/LSD2 (KDM1B) proteins can use H3K4me2 and H3K4me1 as substrates, whereas the Jumonjidomain containing Jarid1/KDM5 family can employ H3K4me3. The Jarid1/KDM5 family consists of Rbp2 (Jarid1A/KDM5A), Plu-1 (Jarid1B/KDM5B), SMCX (Jarid1C/KDM5C) and SMCY (Jarid1d/KDM5D) [79] . As was expected, Jarid1 family members are involved in transcriptional repression [80–84] . The in vitro activity of KDMs in general seems rather low [85] and this raises the question of the in vivo turnover of histone methyl marks. In yeast, it has been shown that the H3K4me3 mark persists after GAL gene shutoff with a half-life of approximately 60 min [60] . This is consistent with recent genome-wide mapping of H3K4me3 during progression of the yeast cell cycle, which indicated contributions of both active removal by the Jhd2p demethylase and of dilution through DNA replication in loss of H3K4me3 [86] . Recently, a quantitative mass spectrometry approach was developed to study histone methylation kinetics in mammalian cells [87] , which indicated that mono-, di- and tri-methylated residues have progressively slower formation rates on bulk histones. Turnover rates of the methyl groups at H3K9, H3K27 and HK36 lie in the range of hours or even days. Unfortunately, stability of H3K4me3 could not be determined owing to the low abundance of this mark. Besides its turnover at the bulk nucleosome level, it would be interesting to determine turnover of histone methylation and of H3K4me3, in particular, at the genespecific level. This question can be addressed in detail by combining specific inhibitors for the MLL/Set KMTs and Jarid1 KDMs with ChIP or by advanced quantitative mass spectro metry methods to follow methylated peptides at specific genomic loci. It is interesting to point out that histone acetylation on a subset of nucleosomes is very labile with half-lives ranging between 1 and 5 min. This high turnover is targeted to H3 tails, which also carry the H3K4me3 mark [88] . In line with high turnover rates of acetyl groups are genome mapping results, which demonstrated that both acetyltransferases and deacetylases colocalize at active human genes [89] . Their presence allows for dynamic and rapid alterations in histone acetylation upon future science group


intracellular and extracellular cues. We expect that the H3K4me3 mark will also display a significant turnover at (a subset of) promoters. In fact the colocalization of Rbp2/Jarid1A with H3K4me3 already hints in this direction [90] . Linkage of H3K4 methylation with inducible gene expression would also be consistent with a rapid turnover of H3K4me3 at active promoters.

H3K4me3 readers: functional diversity & recruitment specificity Characterization of the entire repertoire of effector proteins (‘readers’) that can bind to H3K4me3 is key towards a full understanding of this histone modification mark, since the effector proteins determine the functional outcome of H3K4me3. Thus far, a large number of readers for H3K4me3 have been described in mammalian cells. Reader proteins typically contain plant homeo-domain (PHD), Tudor or chromodomains (Figure 2) [22,91] and the list of H3K4me3 readers is most likely not complete yet. As discussed below the interaction with H3K4me3 is not the only ‘chromatin anchor’ for most of the H3K4me3 readers. The active pursuit of the different contributions to H3K4me3 binding can be undertaken through single-step purifications in combination with stable isotope labeling with amino acids in cell culture-based quantitative proteomics [92] . From our experience the stable isotope labeling with amino acids in cell culture ratios obtained for H3K4me3 binders are corresponding remarkably well with the relative affinities of these proteins for H3K4me3 as determined in equilibrium-based assays like isothermal calorimetry and tryptophan fluorescence [91,93] . We expect that a further application of quantitative proteomics [94] will yield further insights into the highly regulated spatio- temporal recruitment of H3K4me3 readers to specific chromatin loci in eukaryotic cells. A surprising observation is that the different H3K4me3 readers known to date display quite diverse biological functions. Their activities range from activation of transcription (TFIID, Jmjd2A), chromatin remodeling (BPTF, CHD1 and ISWI) and histone acetylation (ING4/5), to transcriptional repression (ING1/2, Rbp2/Jarid1a, Jmjd2A) and splicing efficiency (CHD1) [90,91,93,95–99] . Given this functional diversity an important question is how recruitment specificity is achieved, since obviously not all H3K4me3 readers are binding to the same promoters at the same time. Part of this specificity comes from cell type-specific expression as exemplified by the H3K4me3-binding RAG2 future science group

Writers MLL MLL2 MLL3 MLL4 hSet1A hSet1B

Readers BPTF INGs JMJD2A RAG2 TAF3 CHD1

Review

Erasers Jarid1A-D LSD1/KDM1A (only me2/1) LSD2/KDM1B (only me2/1)

MM M

A R T K Q T A R K S - H3 M

MP

Figure 1. Transactions at the N-terminal tail of histone H3. The first residues of histone H3 are given in the single letter code for amino acids. Depicted are the mammalian MLL/Set1 lysine methyltransferases (KMTs) for H3K4 as ‘writers’, a selection of proteins known to selectively bind to H3K4me3-modified H3 tails as ‘readers’ and the KDMs for methylated H3K4. Please note that the Ash1, Set7/9 and SMYD proteins can also direct H3K4 methylation [21] . The Jarid1/KDM5 proteins and LSD1 proteins can use K4me3 and K4me2 or K4me2 and K4me1 as substrates, respectively. Also indicated are methylation at H3R2 and phosphorylation of H3T3, which depend on the PRMT6 or haspin and VRK1 kinases, respectively. KDM: Lysine demethylase; MLL: Mixed-lineage leukemia.

protein, which is expressed only in lymphocytes that undergo active rearrangements of antigen receptor genes [100] . But the general presence of H3K4me3 on virtually all transcriptionally active pol II promoters indicates that recruitment specificity is also achieved by other means. Specificity can be achieved through interactions with DNA-sequence specific transcription factors that can recruit H3K4me3 readers to target genes. For example, the TATA binding proteinassociated factor (TAF) subunits of TFIID are known to interact with a variety of transcriptional activators [101,102] . Similarly, Ing1 interacts with the p53 protein, which is required for inhibition of cellular proliferation [103] . Presumably, this interaction stabilizes the mSin3A-Ing1 histone deacetylase complex on promoters to be repressed by p53. A third mechanism to achieve recruitment specificity lies in the direct interaction of the H3K4me3 reader complex with DNA. Again TFIID serves as a good example as it can interact via specific subunits to DNA sequences like the TATA-box (TATA binding protein), the Inr and downstream core element (TAF1/2) and the downstream promoter element (TAF6/9) [104] . These core promoter elements are present in different combinations and thus, the presence of the H3K4me3 can have promoter-specific effects on TFIID binding and activity [91] . It is important to note that the major class of mammalian promoters are represented by CpG islands, which lacks these core promoter www.futuremedicine.com

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TFIID, transcription TAF3-PHD

mRNA splicing CHD1-chromo

NuRF, chromation remodeling BPTF-PHD

H3K4me3

H3K9 and K36 demethylase JMJD2a-Tudor

Sin3/HDAC, repression ING2-PHD

HBO1, histone acetylation ING4-PHD H3K4me3

H3K9me2 demethylase PHF8-PHD

V(D)J recombination RAG2-PHD H3K4me3 demethylase Jarid1a-PHD

Figure 2. ‘Readers’ of the H3K4me3 mark. Depicted are mammalian proteins and protein complexes binding to the H3K4me3 modification. Their molecular function and domain of the protein (subunit), which directly binds to the H3K4me3 mark, is indicated. H3K4me3: Trimethylation of histone H3 lysine 4; NuRF: Nucleosome-remodeling factor; PHD: Plant homeo-domain; TAF: TATA binding protein-associated factor.

elements altogether [105] and we propose that the H3K4me3 mark is particularly important for TFIID recruitment to CpG-island promoters. The Rbp2 (Jarid1A/KDM5A) protein represents another example for contributions of DNA sequence in recruitment. The AT-rich interaction domain of this H3K4me3 KDM has a preference for the CCGCCC sequence and this domain is essential for the transcription regulatory properties of Rbp2 [106] . Differential affinities of reader modules for H3K4me3 represent a rather obvious determinant for recruitment specificity. The published interactors bind to H3K4me3 with distinct affinities ranging from approximately 160 nM (TAF3 of TFIID) to approximately 10 µM (the double tudor domain of JMJD2A/KDM4A) [26,91,107] . With a few exceptions (the PHD of yeast Set3 [108]) the affinity of a reader for H3K4me3 is four- to tenfold higher than for H3K4me2 [22] . This large range of different affinities is relevant for competitive binding to H3K4me3, at least for common target genes. A last mechanism important in recruitment specificity is the co-occurrence of other histone modifications with H3K4me3, which is discussed later.

Fine-tuning of H3K4me3 binding through chromatin crosstalk The histone code hypothesis postulates that the combination of marks is crucial for ‘reading by other proteins to bring about distinct 400


downstream events’ [12] . Several approaches indicate that certain histone marks are likely to co-occur [32,43,45,109] . For several H3K4me3 readers additional marks have been shown to be relevant for their recruitment to target genes. For example, both the TFIID and bromodomain and PHD domain transcription factor (BPTF) complexes carry bromodomains that act agonistically with the PHD fingers of TAF3 and of BPTF, respectively, to stably anchor these proteins on active promoters carrying K4me3 with K9ac and K14ac on histone H3. Given the general co-occurrence of these marks on active genes it is therefore perhaps not surprising that complexes containing H3K4me3 readers have evolved to acquire binding domains for both of these marks. In addition to crosstalk with acetylation, the interaction with H3K4me3 can also be fine-tuned through other modifications on the N-terminus of H3. In this context asymmetric dimethylation of histone H3 arginine 2 (H3R2me2a) mediated by the arginine methyl transferase PRMT6 has been of particular interest [110–113] . H3R2me2a differentially affects the binding of readers to H3K4me3. For example, the interaction between the TAF3 PHD finger and H3K4me3 is eight- to 20-fold lower when H3R2 is asymmetrically dimethylated (H3R2me2a), whereas the interaction between the ING2 and BPTF PHD finger and H3K4me3 seems less affected [91,113] . In yeast, the interaction future science group


of the PHD finger of the Spp1p subunit of the Set1C/COMPASS complex with H3K4me3 is severely affected by H3R2me2a. Therefore, H3R2me2a prevents the transition of H3K4me2 to H3K4me3 by the Set1 complex, which further illustrates the antagonistic effect of H3R2me2a on H3K4me3 [114] . In contrast to these inhibitory effects the PHD of RAG2 seems to prefer a doubly modified R2me2a/K4me3 tail of H3 [115] . Adjacent to H3K4 is a threonine residue, which can be phosphorylated during mitosis by the haspin and VRK1 kinases [116,117] . Many lysine residues on histone H3 are flanked by serine or threonine residues. These phosphorylations can negatively affect the binding of proteins to adjacent methyl lysine residues. For example, it was shown that during mitosis HP1 is destabilized from chromatin by H3S10 phosphorylation [118] , although another study showed that in fact HP1 binding to H3K9me3 is not negatively affected by H3S10 phosphorylation [119] . Similar to H3K4me3 the genomic distribution of the histone variant H2A.Z indicates a strong association with pol II promoter regions. H2A.Z enrichment is particularly evident for the first nucleosome (+1) of the transcribed region [41,120,121] . H2A.Z function has been linked to transcription regulation in mammals [49] , but the molecular function of this histone variant is not entirely clear. The N-terminal tail of H2A.Z is subjected to modification by acetylation and replacement of the canonical H2A by H2A.Z is achieved by specialized ATPdependent chromatin-remodeling complexes: the Swr1 complex in yeast or the SRCAP and p400 complexes in mammals [122] . How these remodelers are targeting H2A.Z to the +1 nucleosome is not clear yet. Also, specialized readers of H2A.Z or its acetylated isoforms remain to be identified. Based on similarities in the distribution of H3K4me3 and H2A.Z we speculate that these chromatin marks can act to cooperate in the regulation of gene expression. However, it is important to stress that similar genomic distributions of histone modifications and/or histone variants does not prove that these also co-occur at the single-nucleosome level. A clear illustration of this is represented by the genomic distributions of the H3K4me3 and H3K4ac marks [109] . Although they may co-occur on the same nucleosomal particle, these marks are clearly mutually exclusive on a single H3 tail. By displaying genomic distributions of histone marks by gene averaging [32,41,109] the gene-specific features of these marks are lost. In analogy of H3K4me3/H3K27me3 and H2A.Z/PcG future science group

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distributions in ESCs [41,49] , it may well be that the combination of H3K4me3 with H3K4ac and/or acetylated H2A.Z marks a subset of gene promoters of distinct regulatory properties in certain cell types. Co-occurence at the singlenucleosome level has been demonstrated for the H3.3/H2A.Z variants by sequential immunoprecipitations using epitope-tagged histones [8] and this can also be applied to other combinations of (modified) histones. In addition, development of enrichment procedures for specifically marked nucleosomes in combination with advanced mass spectrometry methods [123,124] is required to determine which chromatin marks actually co-occur at the single-nucleosome level. Together with novel biochemical procedures for preparing homogenously modified nucleosomes for in vitro studies [125,126] , many of the questions with regard to the crosstalk of H3K4me3 can be solved in the future. We also expect that further developments in the isolation of small molecules interfering with histone methylation pathways [127–129] will soon yield effective compounds that can target the H3K4me3 mark. These developments will deepen our understanding of the role of H3K4 methylation in gene regulation and cellular functions and at the same time provide intervention strategies in disease situations. With all these experimental tools the future looks very bright for grasping H3K4me3 function.

Conclusion In this article, we outlined the recent progress regarding the histone mark H3K4me3 and its role in regulating transcription of pol II-transcribed genes. Whereas the association of this mark with active pol II promoters is undisputed, the exact mechanisms surrounding the distribution of H3K4me3 to promoters remain rather elusive. The functional diversity of the regulatory proteins that are involved in H3K4me3 deposition and function implies a complex biology that we are only beginning to unravel. The association of both writers and erasers of H3K4me3 with transcriptional activators and repressors may hint towards a cyclical mechanism as proposed for histone acetylation by histone acetyltransferase and histone deacetylases for proper expression of RNA pol II genes [89] . To address this it will be important to determine the turnover of the H3K4me3 mark at specific genomic loci. To further gain insights into regulatory mechanisms involving H3K4me3, the crosstalk between H3K4me3 and other histone marks needs to be addressed www.futuremedicine.com

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in detail. This will require technologies for unbiased screening [130] of known and novel histone modifications co-occurring with H3K4me3 on histone H3. Apart from a fundamental scientific interest, the biology surrounding H3K4me3 is highly relevant from a clinical perspective as indicated by the involvement of several writers and readers of H3K4me3 in human disease.

Future perspective In the coming years, we expect to witness significant progress in deciphering the molecular mechanism responsible for recruiting the H3K4me3 writers to specific locations in the genome and for H3K4me3-mediated effects on gene expression. We speculate that the role of allosteric mechanisms in the deposition of the

Executive summary Chromatin, post-translational modification of histones & gene expression Epigenetic controls, or the effects on chromatin structure and gene expression that are not related to direct recognition of the underlying DNA sequence, have received a lot of attention in recent years. One particularly interesting epigenetic mark is trimethylation on lysine 4 of histone H3 (H3K4me3). This mark is almost exclusively associated with active RNA polymerase II (pol II) genes and forms the focus of this article. Establishing H3K4 methylation patterns in yeast In yeast, mono-, di-, and tri-methylation of histone H3K4 is catalyzed by Set1p, which assembles into the larger Set1C/complex proteins associated with Set1 (COMPASS) complex. H3K4me3 correlates with active transcription and can be found at the 5´-end of active genes; by contrast, H3K4me2 and H3K4me1 display markedly different genomic distributions. Distribution of H3K4 methylation in mammalian cells The global distribution of H3K4me3 in mammalian cells is similar to that observed in yeast, while H3K4me1 marks distal enhancers in higher eukaryotes. In embryonic stem cells, H3K4me3 in found in bivalent domains with the repressive methylation mark H3K27me3 on developmentally relevant genes that are silent but ‘poised’ for activation. There are at least eight different Set domain proteins known to ‘write’ H3K4me3. The best studied are the mixed-lineage leukemia and hSet1A/B family of proteins. Each of these different enzymes is specifically recruited in large multiprotein complexes to target genes in a highly regulated manner. Recruitment of H3K4 methylation activities Experiments in yeast showing that Set1p binds to the initiating form of RNA polymerase II suggests that H3K4me3 is a consequence rather than a cause of transcription. Mammalian hSet1A/B seem to have the similar function in mammals and interact with the C-terminal domain of pol II through their RNA-recognition motif domain and the associated Wdr82 protein. In contrast, mixed-lineage leukemia proteins lack the RNA-recognition motif domain, and these proteins seem to direct H3K4me3 modification to activate transcription. Stability of histone methylation The discovery of histone lysine demethylase enzymes targeting methylated H3K4 indicates that histone methylation states are biochemically dynamic. At least in vitro, the catalytic activity of these enzymes is rather poor. Lysine demethylase enzymes for H3K4me3 have been found on active promoters indicating that methylation of H34me3 on active genes may display a cyclic behavior. H3K4me3 readers: functional diversity & recruitment specificity Numerous proteins that can bind to methylated H3K4 have been identified. These ‘reader’ proteins typically contain plant homeodomain, tudor or chromodomains. These reader proteins display quite diverse biological functions ranging from activation of transcription and chromatin remodeling to histone acetylation and transcriptional repression. Given these diverse biological activities, deciphering the specific recruitment of all these readers to H3K4me3 marked promoters is an important task that lies ahead. Fine-tuning of H3K4me3 binding through chromatin crosstalk The interaction between H3K4me3 and its readers can be either positively or negatively fine-tuned through adjacent modifications on the same histone H3 tail. H3K9 and K14 acetylation have been shown to act agonistically with H3K4me3 to anchor TFIID and BPTF complexes on the histone H3 tail. Asymmetric dimethylation of H3R2 and possibly phosphorylation of H3T3 negatively affect the binding of readers to H3K4me3. Conclusion The association of both writers and erasers of H3K4me3 with transcriptional activators and repressors may hint towards a cyclical methylation/demethylation mechanism necessary for the proper expression of target genes. Several writers and readers of H3K4me3 have been linked to pathologies including cancer. Given the reversible nature of epigenetic modifications this implies a huge therapeutic potential to develop so-called ‘epidrugs’.

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Acknowledgements The authors thank Radhika Warrier, Petra de Graaf and Pim Pijnappel for critical reading and constructive comments of this manuscript.

Financial & competing interests disclosure The work of Marc Timmers is supported by grants from the Netherlands Organization for Scientific Research (NWO-TOP #700.57.302) and the European Union (EUTR ACC LSHG-CT-2006 - 037445). Michiel Vermeulen is supported by a grant from the Netherlands Genomics Initiative /Netherlands Organization for Scientific Research. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

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