Int. J. Dev. Biol. 53: 335-354 (2009) DEVELOPMENTALTHE INTERNATIONAL JOURNAL OF doi: 10.1387/ijdb.082717ph BIOLOGY www.intjdevbiol.com Mechanisms of transcriptional repression by histone lysine methylation PHILIP HUBLITZ#, MAREIKE ALBERT## and ANTOINE H.F.M. PETERS* Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland ABSTRACT During development, covalent modification of both, histones and DNA contribute to the specification and maintenance of cell identity. Repressive modifications are thought to stabilize cell type specific gene expression patterns, reducing the likelihood of reactivation of lineage-unrelated genes. In this report, we review the recent literature to deduce mechanisms underlying Polycomb and H3K9 methylation mediated repression, and describe the functional interplay with activating H3K4 methylation. We summarize recent data that indicate a close relationship between GC density of promoter sequences, transcription factor binding and the antagonizing activities of distinct epigenetic regulators such as histone methyltransferases (HMTs) and histone demethylases (HDMs). Subsequently, we compare chromatin signatures associated with different types of transcriptional outcomes from stable repression to highly dynamic regulated genes, strongly suggesting that the interplay of different epigenetic pathways is essential in defining specific types of heritable chromatin and associated transcriptional states. KEY WORDS: transcriptional control, histone lysine methylation, methyltransferase, demethylase, polycomb Genetic and epigenetic mechanisms ensure that complex devel- transcription factor occupancy in relation to transcriptional ON opmental programs are correctly executed. One important post- and OFF states. We mainly focus on the dynamics of activating translational modification that regulates transcriptional outcomes, H3K4 and repressive H3K27 methylation marks at promoter genome integrity and cellular identity is histone lysine methyla- sequences. The genomic data serve as a foundation to under- tion. Defined methylation patterns are related to distinct functional stand the interrelationship between transcription factor and chro- readouts of chromosomal DNA. The initial discoveries of histone matin based pathways. We in-depth review the responsible modifying enzymes lead to the postulation of the “histone code” classes of enzymes mediating those modifications and report on hypothesis, whereby defined histone modifications, acting in a their functional importance. In a second part, we review two combinatorial or sequential fashion on one or multiple histone scenarios of dynamically controlled transcriptional systems, termini, specify the transcriptional state of a gene by recruitment nuclear hormone receptor signaling, whose repression is largely of regulatory proteins. In this review, we discuss the indexing based on H3K9 methylation, and the regulation of the cell division potential of histone lysine methylation in the light of how histone cycle, where senescence and proliferation are controlled by H3K9 methyltransferases (HMTs) and histone demethylases (HDMs) and H3K27 methylation. Both systems are especially well charac- are targeted to given promoter contexts, how the GC content of terized in terms of recruitment of histone modifying enzymes and target promoters influences the regulatory response, and how the functional interplay between HMTs and HDMs ultimately defines Abbreviations used in this paper: AR, androgen receptor; CpG, cytosine- transcriptional states. For in-depth discussions of the develop- guanidine dinucleotide; ER, estrogen receptor; HDM, histone demethylase; mental functions of individual HMTs and HDMs, we refer the HMT, histone methyltransferase; NHR, nuclear hormone receptor; PcG, reader to recent reviews (Cloos et al., 2008; Martin and Zhang, polycomb group; PTM, post translational modification; RNAPII, DNA- 2005) (Fig. 1). We first summarize new insights gained by the dependent RNA-polymerase II; TrxG, trithorax group; TSS, transcriptional recent epigenomic profiling studies of histone methylation and start site. *Address correspondence to: Dr. Antoine H.F.M. Peters. Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058 Basel, Switzerland. Tel: +41-61-697-8761. Fax: +41-61-697-3976. e-mail: [email protected] # Note: Current address is EMBL Monterotondo, Mouse Biology Unit, Via E. Ramarini 32, 00015 Monterotondo, Italy. ## Note: This author is formerly known as Mareike Puschendorf. Current address is: Biotech Research and Innovation Centre. University of Copenhagen, Ole Maaloes Vej 5, 2200 Copenhagen, Denmark. Published online: 28 April 2009. ISSN: Online 1696-3547, Print 0214-6282 © 2009 UBC Press Printed in Spain 336 P. Hublitz et al. in combining several epigenetic pathways to achieve the desired et al., 2006b) showed that core components of the Polycomb transcriptional outcome. Repressive Complex PRC1 (Rnf2, Phc1), PRC2 (Eed, Suz12) and H3K27me3 do not only colocalize to classical target genes Transcriptional repression by H3K27 methylation (such as the four Hox clusters) but also to many other loci. Particularly genes coding for developmental regulators including Bivalency of H3K4 and H3K27 methylation homeodomain (Dlx, Irx, Lhx, Pou, Pax, Six) and other transcrip- In mammals, recent genome-wide mapping studies of Polycomb tion factors (such as Fox, Sox, Gata and Tbx) were highly Group (PcG) and Trithorax Group (TrxG) proteins and their overrepresented among PcG target genes. These proteins serve marks, classically associated with repressed and active transcrip- master regulatory functions in organogenesis and morphogen- tional states, respectively, revealed many novel target genes. esis, pattern specification, cell differentiation, embryonic devel- These studies provide the framework to delineate mechanisms of opment and cell fate commitment (Boyer et al., 2006; Lee et al., targeting and gene regulation mediated by the different epige- 2006b). PcG targets are generally repressed in ES cells and are netic modifiers. Three initial ChIP-chip studies in human and preferentially activated during ES cell differentiation. In Eed and mouse ES cells (Bernstein et al., 2006a; Boyer et al., 2006; Lee Suz12 gene deficient ES cells that lack detectable amounts of PRC2 complexes and H3K27me3, tran- script levels of most PcG target genes were increased (Boyer et al., 2006; Lee et al., A Mll1-4 KMT2A-D 2006b). These data suggested that Set1a/b KMT2F/G Ash2L Nsd1 Polycomb complexes are required for main- me KMT2H (H3K4me2/3 only) me KMT3B Set7/9 KMT7 Nsd2/Whsc1 taining ES cell pluripotency and plasticity me me me me Meisetz Smyd2 KMT3C during embryonic development. me me me me me me Smyd1/Bop1 HypB While studying genomic regions that Smyd3 harbor highly conserved non-coding ele- Whistle ments, Bernstein and colleagues made a A remarkable observation. They noticed that ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVS KKP H3 4 36 extended regions marked by H3K27me3 me me were also marked by H3K4me3, a histone Lsd1 KDM1* me modification normally associated with tran- scriptional activity (Bernstein et al., 2006a). me me me Jhdm1a/b KDM2A/B* me me me Jhdm1a/b KDM2A/B* Sequential ChIP experiments confirmed that me me Jarid1a/Rbp2 KDM5A me me Jmjd2a KDM4A* identical alleles were labeled by both “re- me Jarid1b/c/d KDM5B/C/D pressive” and “activating” chromatin modi- fications, resulting in the term “bivalent do- mains”. Genes within bivalent domains were B largely repressed, to almost the same ex- G9a KMT1C tent as genes that were marked by me Glp me me KMT1D H3K27me3 alone. In contrast, genes only Riz1 KMT8 me Ezh1 me me Eset KMT1E me me Fig. 1. Overview of histone methylation pro- Ezh2 KMT6 me me me Suv39h1/2 KMT1A/B me me me cesses. (A) Methylation of lysines H3K4 and H3K36 is generally correlated with transcriptional ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPA H3 S activity, and demethylation of H3K4 is required 9 27 for effective silencing. The specificities of H3K4 me me me me me Utx KDM6A and H3K36 HMTs are not unambiguously clear Lsd1 + AR KDM1* me me me Jmjd3 KDM6B yet. (B) Methylation of H3K9 and H3K27 are hallmarks of transcriptional repression, and the me me me Jmjd1a KDM3A antagonizing HDMs are categorized as transcrip- me me Jmjd2a/b KDM4A/B tional co-activators. H3K9me3 is a hallmark of me Jmjd2c/Gasc1 KDM4C constitutive heterochromatin; H3K27me3 is the readout of PcG mediated silencing. (C) In the tail of histone H4, only K20 is targeted by HMTs. CDH4K20me1 correlates with ongoing transcrip- me PrSet7 KMT5A me tion, whereas H4K20me3 is an integral part of me me me me heterochromatin mediated silencing. (D)Dot1L is Suv4-20h1/2 KMT5B/C Dot1L KMT4 me me me me me me the only HMT known to target H3K79 in the globular domain of H3. No HDM is known to K target either H4K20 or H3K79. HMTs are indi- ...GAKRHRKVLRDN H4 H3 cated above the histone tails, HDMs are below, 20 and hexagons represent the respective methyla- 79 tion status. Asterisks (*) indicate enzymes that target multiple lysine residues. Transcriptional repression by histone lysine methylation 337 associated with H3K4me3 were highly expressed (Bernstein et coding elements (Bejerano et al., 2004; Nobrega et al., 2003) and al., 2006a) (Fig. 2A). These results indicate that in bivalent being devoid of retrotransposons (Bernstein et al., 2006a; Simons domains the repressive H3K27me3 state generally overrules the et al., 2006; Tanay et al., 2007). As such, Bernstein et al. may thus activating effect