Epigenetic Regulation

Some of the key epigenetic regulatory mechanisms appeared early during evolution, and the acquisition of novel epigenetic regulators apparently facilitated certain evolutionary transitions. In this short review we focus mainly on the major epigenetic mechanisms that control chromatin structure and accessibility in mammalian cells. The enzymes methylating CpG dinucleotides and those involved in the active demethylation of 5-metylcytosine (5mC) are outlined together with the members of the methyl binding protein (MBP) family that bind to and “interpret” the 5mC mark. The enzymes involved in reversible, covalent modifi cations of core histone proteins that affect chromatin structure are also described briefly. Proteins that build up Polycomb group (PcG) and Trithorax group (TrxG) protein complexes may also modify histones. By establishing heritable chromatin states, PcG and TrxG complexes contribute – similarly to cytosine methylation – to the transmission of cell type-specific gene expression patterns from cell generation to cell generation. Novel players involved in epigenetic regulation, including variant histones, pioneer transcription factors, long noncoding RNA molecules and the regulators of long-distance chromatin interactions are introduced as well, followed by the characterization of various chromatin types.

1 Epigenetic Regulation Janos Minarovits, Ferenc Banati, Kalman Szenthe, and Hans Helmut Niller Abstract Some of the key epigenetic regulatory mechanisms appeared early during evolution, and the acquisition of novel epigenetic regulators apparently facilitated certain evolutionary transitions. In this short review we focus mainly on the major epigenetic mechanisms that control chromatin structure and accessibility in mammalian cells. The enzymes methylating CpG dinucleotides and those involved in the active demethylation of 5-metylcytosine (5mC) are outlined together with the members of the methyl binding protein (MBP) family that bind to and “interpret” the 5mC mark. The enzymes involved in reversible, covalent modifications of core histone proteins that affect chromatin structure are also described briefly. Proteins that build up Polycomb group (PcG) and Trithorax group (TrxG) protein complexes may also modify histones. By establishing heritable chromatin states, PcG and TrxG complexes contribute – similarly to cytosine methylation – to the transmission of cell type-specific gene expression patterns from cell generation to cell generation. Novel players involved in epigenetic regulation, including variant histones, pioneer transcription factors, long noncoding RNA molecules and the regulators of long-distance chromatin interactions are introduced as well, followed by the characterization of various chromatin types. J. Minarovits (*) Department of Oral Biology and Experimental Dental Research, Faculty of Dentistry, University of Szeged, Tisza Lajos krt. 64, H-6720 Szeged, Hungary e-mail: minimicrobi@hotmail.com F. Banati • K. Szenthe RT-Europe Nonprofit Research Center, Pozsonyi út 88, H-9200 Mosonmagyarovar, Hungary H.H. Niller Institute for Medical Microbiology and Hygiene, University of Regensburg, Regensburg, Germany e-mail: hans-helmut.niller@klinik.uni-regensburg.de © Springer International Publishing Switzerland 2016 J. Minarovits, H.H. Niller (eds.), Patho-Epigenetics of Infectious Disease, Advances in Experimental Medicine and Biology 879, DOI 10.1007/978-3-319-24738-0_1 1 2 J. Minarovits et al. Keywords DNA methylation • Histone modifications • Polycomb and Trithorax complexes • Variant histones • Pioneer transcription factors • Long noncoding RNAs • Chromatin loops 1.1 Introduction: Epigenetic Regulation in the Domains of Life Epigenetic regulatory mechanisms ensure the heritable alterations of cellular states without affecting the nucleotide sequence of the DNA. In multicellular organisms belonging to the taxon Eukarya or Eukaryota, epigenetic control of transcription forms the basis for the phenotypic and functional diversity of various cell types that carry identical, or nearly identical genomes. Epigenetic regulators interact with a DNA protein complex, called chromatin, that is organized into repeating units (Kornberg 1974). The basic chromatin unit is the nucleosome composed of a segment of 147 bp long DNA wrapped around a core of eight histone proteins (Richmond et al. 1984). Nucleosomes are connected by free linker DNA segments of 20–60 bp. The histone octamer is formed by the core histones, H2A, H2B, H3 and H4, each in two copies. In many eukaryotes, a single molecule of the linker histone, histone H1 stabilizes the nucleosome and plays a role in the formation of a higher-order chromatin structure by binding to the linker DNA (Harshman et al. 2013). The nucleosome and the linker histone form the chromatosome and resembles a “beads-on-a-string” structure. It is worthy to note that the eukaryotic genes coding for the core (invariant) histones were apparently generated by gene duplication events from archaeal genes (see below; Volle and Dalal 2014). Epigenetic regulators affect chromatin structure and promoter activity by depositing stable but reversible marks on DNA or DNA-associated proteins. Such epigenetic marks ensure faithful transmission of gene expression patterns to each progeny cell upon division (epigenetic memory) (Strahl and Allis 2000; Jenuwein and Allis 2001; Bird 2002; Rohlf et al. 2012). In addition, incorporation of variant histone molecules into the nucleosomes in selected chromatin regions or high-affinity binding of certain nonhistone proteins to the mitotic chromatin may “bookmark” the genes to be expressed or silenced in the daughter cells (Zaret et al. 2008; Kelly et al. 2010; Zaidi et al. 2011; Follmer et al. 2012). Thus, there are distinct forms of epigenetic memory that do not necessarily rely on the covalent modifications of chromatin constituents. Even more, the accessibility of distinct chromatin domains to transcription factors, RNA polymerases and recombinases depends on their location to various nuclear subcompartments as well, and switching of the nuclear position from one compartment to another may change the epigenetic marks and activity of coregulated promoters (Gyory and Minarovits 2005). Such a complexity of epigenetic regulatory mechanisms is characteristic for multicellular eukaryotes (Aravind et al. 2011; Jin et al. 2011; Jurkowski and Jeltsch 2011; Jeltsch 2013). Epigenetic modifications are also well documented, however, in unicellular eukaryotes. They are of clinical importance, because protozoan pathogens control the expression of virulence genes and differentiation related gene sets by epigenetic regulators that can be targeted by epigenetic drugs (epigenetic therapy) (Aravind et al. 2011; Fisk and Read 2011; Coyne et al. 2012; Hoeijmakers et al. 2012; Niller et al. 2012). Most eukaryote genomes encode DNA methyltransferases (DNMTs) that recognise CpG dinucleotides and modify the C5 position of cytosine within that sequence. The wide distribution of DNA-(cytosine C5)-methyltransferases in eukaryote species suggests that they appeared early during evolution and that the genome of the last eukaryotic common ancestor (LECA) possibly encoded at least one DNA-(cytosine C5)methyltransferase (Jurkowski and Jeltsch 2011). 1 Epigenetic Regulation Certain proteins encoded by a single gene in invertebrate genomes and by several genes in vertebrates preferentially bind to DNA sequences containing one or more symmetrically methylated CpG dinucleotides (reviewed by Hendrich and Tweedie 2003). In vertebrates, such methyl-CpG binding domain (MBD) proteins may increase the fidelity of DNA methylation mediated gene silencing by “reading” the methylation mark and “interpreting” it via their association with histone deacetylases, nucleosome remodelling complexes and histone methyltransferases (reviewed by Hendrich and Tweedie 2003; Van Emburgh and Robertson 2008; Hashimoto et al. 2010). Phylogenetic analysis of the gene coding for the histone methyltransferase enzyme, H3K9 HMTase Su(var)3-9, suggests that histone modification also appeared early during evolution: the HMTase coding sequence fused to a functionally unrelated gene, the γ subunit of the translation initiation factor eIF2 approximately 400 myears ago (Krauss et al. 2006). Acquisition of novel genes coding for epigenetic regulators possibly facilitated evolutionary transitions. It was suggested that the horizontal transfer of a histone methyltransferase gene from an animal host enabled the free living ancestor of the malaria parasite Plasmodium falciparum and other apicomplexans to change life style and evolve to an obligate parasite (Kishore et al. 2013). Acquisition of the novel regulator presumably allowed epigenetically controlled, coordinated expression of immune-evasion genes. The social amoeba Dictyostelium discoideum but not other amoebae, also acquired a histone methyltransferase gene from animals, an event possibly related to the appearance of important characteristics of D. discoideum, i.e. complex cellular communication and differentiation (Kishore et al. 2013). Similarly to eukaryotes, genetically identical unicellular organisms belonging to the domain Bacteria may also undergo differentiation or display heritable phenotypic heterogeneity. It is well documented that these morphological and physiological changes are frequently controlled by epigenetic regulatory mechanisms, especially DNA methylation (reviewed by Casadesus and Low 2013). 3 Putative chromatin modifying enzymes, related to the epigenetic regulators encoded by eukaryote genomes were also detected in organisms of the domain Archaea (Manzur and Zhou 2005; Niu et al. 2013). One of the two main archaeal phyla, Euryarchaea synthesize tetrameric histone homologues, and archeal histone genes were detected in certain Crenarchaea, too (Reeve 2003; Cubonova et al. 2005). These data suggested that histones appeared quite early during evolution, probably after the divergence of Bacteria – that lack histone-like sequences – and Archaea, but before the separation of Archaea and Eukarya. In addition, the demonstration of acetylated and methylated nucleoid proteins or archeal chromatin proteins raised the idea that in Archaea, similarly to Eukarya, chromatin modification may play an active role in transcriptional silencing (Bell et al. 2002; Wardleworth et al. 2002; Reeve 2003). 1.2 Epigenetic Regulatory Mechanisms Epigenetic marks determine chromatin structure and accessibility by interacting with “reader” factors including methylcytosine (mC) binding proteins, transcription factors, and chromatin remodeling complexes that affect and regulate transcription (Jin et al. 2011). Active promoters are usually located to domains of open chromatin or euchromatin, whereas silent promoters are typically situated in closed, condensed chromatin domains or heterochromatin (Fig. 1.1). 1.2.1 CpG Methylation In Eukarya, DNA methyltransferases are epigenetic regulators that typically methylate the C-5 position of cytosines within CpG dinucleotides. With some exceptions, CpG methylation is associated with promoter silencing: it was observed that in vertebrate cells the control regions of inactive promoters are frequently methylated and located to “closed” chromatin domains suppressing transcription (reviewed by Robertson 2001). In addition, in somatic cells, a high level of CpG 4 J. Minarovits et al. Fig. 1.1 Chromatin states and promoter activity. Nucleosomes are formed by stretches of DNA (indicated by orange threads) wrapped around histone octamers (depicted as gray cylinders). In the heterochromatic state (top) the chromatin is condensed; a silent promoter located to heterochromatin is indicated by an arrow with a red X. The regulatory regions of silent promoters are frequently hypermethylated as shown by filled lollipop symbols corresponding to methylated CpG dinucleotides. In the euchromatic state (bottom) the chromatin is loose, decondensed; an active promoter located to euchromatin is indicated by an arrow. The regulatory regions of active promoters are frequently unmethylated or hypomethylated as shown by open lollipop symbols corresponding to unmethylated CpG dinucleotides methylation was typically observed at retrotransposons, including endogenous retrovirus genomes flanked by long terminal repeat (LTR) sequences and other repetitive elements lacking LTRs (e.g. long interspersed nuclear elements, LINEs) (reviewed by Ooi et al. 2009). Tandem repetitive sequences (e.g. pericentromeric minor and major satellite sequences), were also found to be densely methylated, similarly to the silent X chromosome in females (reviewed by Ooi et al. 2009). In humans, cytosine methylation patterns are maintained by DNA methyltransferase 1 (DNMT1) that has a high affinity to hemimethylated DNA substrates generated during semiconservative DNA replication (Van Emburgh and Robertson 2008, Table 1.1, Fig. 1.2). DNMT1 restores the methylation pattern of the parental DNA strands on the initially unmethylated daughter strands by transferring a methyl group from the universal methyl donor S-adenosyl-Lmethionine to the 5-position of cytosine. There are five conservative motifs in the C-terminal catalytic domain of DNMT1 that are also present in the prokaryotic DNA-(cytosine-C5) methyltransferases involved in restriction/modification phenomena (Posfai et al. 1989; Bestor 2000). In contrast, there are no significant sequence homologies with bacterial DNA methyltransferases at the N-terminal domain: thus, the origin of that regulatory domain is unknown at present. It is 1 Epigenetic Regulation 5 Table 1.1 Human DNA methyltransferases and their relatives lacking DNA methyltransferase activity Enzyme DNMT1 DNMT2 DNMT3A DNMT3B DNMT3L Function Maintenance methylase Lacks DNA methyltransferase activity; tRNA methyltransferase (?) De novo methylase development De novo methylase Lacks DNA methyltransferase activity Maintenance methylation M M CG GC CG GC M CG GC CG GC Critical for embryonic development Critical for embryonic development Sequence similarity with bacterial DNMTs; enhances the activity of de novo DNMTs; forms repressive complexes with HDACs CG GC M M M Note Critical for embryonic development Sequence similarity with bacterial DNMTs CG GC CG GC CG GC M M M M CG GC CG GC M CG GC M M M CG GC CG GC M CG GC CG GC M Fig. 1.2 Maintenance DNA methylation. A stretch of double stranded DNA with CpG dinucleotides (CG) either methylated (M, blue) or unmethylated on both strands is shown (top). During DNA replication (arrows), two hemi- methylated DNA molecules are generated (one strand methylated, the other unmethylated) (middle). Finally, the original methylation pattern is restored (arrows) by DNMT1 (bottom) apparently unique to eukaryotes and it is involved in the nuclear import of the enzyme. In addition, the N-terminal regulatory domain targets DNMT1 and the related murine enzyme, Dnmt1, to the replication foci during S phase and coordinates DNA replication with CpG methylation (reviewed by Bestor 2000). Maintenance DNA methyltransferases are targeted to replication foci by interacting with PCNA (proliferating cell nuclear antigen) and UHRF1 (ubiquitin-like protein containing PHD and RING finger domains 1); alternatively, they could be recruited by a series of transcription factors, too (Araujo et al. 2001; Iida et al. 2002; Spada et al. 2007; Hervouet et al. 2010, 2012). The SRA (SET and RING-associated) domain of UHRF1 contacts both the major and minor grooves of DNA containing a hemimethylated CpG site – where only one DNA strand is methylated – by two loops. Both loops penetrate into the middle of the DNA helix that causes flipping out of the parental strand 5-methylcytosine (Hashimoto et al. 2008). The SRA-DNA interaction serves as an anchor keeping UHRF1 at the hemimethylated CpG site. By contacting UHRF1, DNMT1 is guided to the opposite unmethylated CpG dinucleotide located in the daughter strand. Recruitment of DNMT1 by UHRF1 is followed 6 J. Minarovits et al. by the transfer of a methyl group to the unmethylated cytosine of the target sequence (Hashimoto et al. 2010). DNMT2, a protein that shares conserved motifs with bacterial DNA methyltransferases, has no DNA methyltransferase activity and it is not known to be involved in the regulation of CpG methylation in mammals (Defossez 2013, Table 1.1). DNMT2 can function, however, as a tRNA methyltransferase (Goll et al. 2006). De novo DNA methyltransferases (DNMT3A, DNMT3B in humans) are involved in the establishment of DNA methylation patterns during embryonic development. They act preferentially on completely unmethylated DNA strands. In addition, by anchoring strongly to nucleosomes in highly methylated regions of the genome DNMT3A and DNMT3B play a role in maintenance DNA methylation as well, via de novo methylating the sites missed by DNMT1 in densely methylated, heterochromatic domains (Jones and Liang 2009; Gatto et al. 2012, Table 1.1). Similarly to DNMT1, the C-terminal catalytic domain of DNMT3A and DNMT3B also harbour conserved methyltransferase domains. However, the N-terminal regulatory domain differs from that of DNMT1: it consists of a cysteine-rich region, the ADD (ATRX/DNMT3/ DNMT3L) domain and a proline-tryptophan motif (PWWP domain, reviewed by Gatto et al. 2012). These regions mediate interactions with a series of transcription factors, histone-modifying proteins, histone H3 tails with unmodified lysine 4 residues (H3K4me0) and histone H3 trimethylated at lysine 36 (H3K36me3). Both DNMT3A and DNMT3B form tetrameric complexes with DNMT3L molecules. The latter do not have enzymatic activity, but stimulate DNMT3A and DNMT3B function (reviewed by Hashimoto et al. 2010; Gatto et al. 2012, Table 1.1). Methylated DNA sequences are specifically recognized by methyl-CpG binding proteins that are also referred to as members of the methyl binding protein (MBP) family (reviewed by Parry and Clarke 2011). MBPs “read”, i.e. selectively bind methylated CpG dinucleotides (mCpGs) and “interpret” this epigenetic mark by recruiting histone-modifying enzymes that favour the establishment of a repressive chromatin structure silencing promoter activity. MeCP2 (methyl CpG binding protein 2), a chromosomal protein, contains a methyl-CpG binding domain (MBD) of 85 amino acids that recognizes a single symmetrically methylated CpG dinucleotide and a transcriptional-repression domain associating with mSin3A, a transcriptional repressor, and histone deacetylases (Nan et al. 1993, 1998; Free et al. 2001, Fig. 1.3). In addition, MeCP2 may Fig. 1.3 Recruitment of histone deacetylase to methylated DNA sequences. The left side of the figure shows a nucleosome formed by a stretch of DNA (indicated by an orange thread) wrapped around a histone octamer (depicted as a gray cylinder) modified by acetylation (Ac, green). A DNA methyltransferase (DNMT, red) adds methyl groups (M, blue) to CpG dinucleotides. The right side of the figure shows that after binding of MBD (methyl-CpG-binding domain) family proteins (MBD, blue) to the methylated DNA, histone deacetylase enzymes (HDAC, orange) are recruited to the region and stabilize the heterochromatic state by removing the acetyl moieties from histones 1 Epigenetic Regulation reinforce transcriptional repression by tethering a histone methyltransferase activity to methylated DNA sequences (Fuks et al. 2003; Subbanna et al. 2014). In addition to MeCP2, other nuclear proteins (MBD1 to MBD6) also contain the MBD domain. MBD1, 2, 3 and 4 associate with repressor complexes. MBD4 that has a glycosylase activity interacts with the DNA repair machinery as well (reviewed by Parry and Clarke 2011). MBD 5 and 6 do not bind to methylated DNA, but interact with PcG proteins (Baymaz et al. 2014). MBD1 forms a stable complex with SETDB1 (SET domain, bifurcated 1), a histone H3-K9 methylase at DNA replication foci, coupling CpG methylation to histone methylation and heterochromatin formation (Sarraf and Stancheva 2004). Non-methylated DNA sequences are specifically recognized by a family of proteins containing a zink finger-CxxC domain (ZF-CxxC domain) (Long et al. 2013). ZF-CxxC proteins may contribute to the maintenance of euchromatic histone marks at CpG islands and may prevent de novo CpG methylation by recruiting protein complexes with histone H3K4 methyltransferase (see Sect. 1.2.2.2) and DNA demethylase activities (see below), respectively. CFP1 (CxxC finger protein 1), a component of the mammalian SETD1 (SET domain 1) complex involved in histone H3K4 methylation, regularly associates with CpG islands (Thomson et al. 2010). There is a rigid CpG recognition loop within the CFP1 CxxC domain, consisting of a three amino acid residues (IRQ), which is unable to accomodate methylated CpG, but allows the binding of the non-methylated CpG dinucleotide (Xu et al. 2011). In addition, the H3K4 methyltransferases MLL1 and MLL2 also contain a zink finger-CxxC domain (reviewed by Long et al. 2013). It was also observed that TET2 and TET3 proteins that maintain the unmethylated state of CpG islands by converting 5-methylcytosine to 5-hydroxymethylcytosine, co-localize with HCF1 at active promoters and promote binding of the SET1 H3K4 methyltransferase complex to chromatin (Deplus et al. 2013). Cytosine methylation is reversible, it can be removed by active or passive “eraser” mechanisms. The Tet (10–11 translocation) family of 7 dioxygenases converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) followed by base excision repair (active demethylation; reviewed by Wu and Zhang 2011). Passive demethylation may occur when the recruitment of maintenance DNA methyltransferase DNMT1 is inefficient or the activity of the enzyme is inhibited during DNA replication (reviewed by Smith and Meissner 2013). Tet proteins belong to the oxoglutarate and iron-dependent dioxygenase enzymes that are encoded by Tet1, Tet2 and Tet3 genes, derivatives of an ancestral gene of jawed vertebrates (Iyer et al. 2009). In addition to oxoglutarate and iron binding domains, the human TET1 and TET3 proteins contain a zink finger (CxxC) DNA binding domain as well that can recognize unmodified, methylated and hydroxymethylated sequences (reviewed by Branco et al. 2012). Dependence of Tet proteins on oxoglutarate and oxygen suggested that they may function as sensors of metabolic and oxidative cell states (Chia et al. 2011). During development, Tet proteins are involved in global DNA demethylation events that result in epigenetic reprogramming in the early zygote and later in the migrating primordial germ cells (Hill et al. 2014). The rapid loss of 5-methylcytosine is attributed to Tet-mediated iterative oxidation followed by excision and repair in both cases. Although most of the CpG dinucleotides are methylated in mammalian genomes, certain regions called CpG islands are devoid of methylation due to high levels of a modified histone (H3K4me3, histone H3 trimethylated at lysine 4; see Sect. 1.2.2) preventing the recruitment of de novo DNA methyltransferases. In addition, binding of Tet1 to unmethylated CpG rich sequences may also contribute to the protection of CpG islands from stochastic, aberrant methylation by converting the newly methylated 5mC to 5hmC (Williams et al. 2011, 2012). 5hmC may interfere with DNMT1, the maintenance methyltransferase, resulting in passive demethylation, or it may be converted to higher oxidative products targeted by thymine-DNA glycosylase. The lesion created 8 J. Minarovits et al. by the removal of 5fC and 5caC is processed through the base excision repair pathway (BER), restoring the unmethylated state (Williams et al. 2012; Hill et al. 2014). Thus, Tet proteins may function as the “guardians of CpG islands” (Williams et al. 2012). It is also worthy to note that conversion of 5mC to 5hmC may affect the association of 5mC-binding proteins and methylation-sensitive transcription factors with their recognition sequences, influencing thereby promoter activity (Williams et al. 2011). In addition, Tet proteins interact with SIN3A, a co-repressor complex, and OGT (O-linked N-acetylglucosamine transferase), an enzyme promoting transcription by glucosylating histone H2B (Williams et al. 2011; Vella et al. 2013; Dehennaut et al. 2014; Hill et al. 2014). 1.2.2 Histone Modifications Two molecules each of the core histone proteins, histone H2A, H2B, H3 and H4 assemble to an octamer around which a stretch of 146 bp DNA is wrapped, forming a nucleosome which is the structural and functional unit of chromatin. Core histone proteins are characterized by a central α-helix flanked by shorter loops and helices and an N-terminal tail which is an unstructured region frequently undergoing chemical modifications (reviewed by Horikoshi 2013). Various types of covalent modifications affecting the core histone molecules constitute epigenetic marks that influence the structure and accessibility of chromatin as well as promoter activity. Mitotically heritable histone marks may be deposited by histone acetylases, protein arginine methyltransferases and histone lysine methyltransferases. Histone lysine methyltransferases are components of Polycomb group (PcG) and Trithorax group (TrxG) multiprotein complexes as well (reviewed by Jin et al. 2011). It is important to note that certain TrxG and PcG proteins not only covalently modify histones but also remain associated with mitotic chromatin and influence the activity of neighbouring promoters in postmitotic cells (Aoto et al. 2008; Blobel et al. 2009). Similarly to DNA methylation, histone modifications are reversible: they are removed by histone deacetylases, histone lysine demethylases or the histone arginine demethylase JMJD6 (jumonji domain-containing 6 protein) (Cloos et al. 2008; Haberland et al. 2009). It is noteworthy that both demethylation of lysine and active demethylation of cytosine depends on oxygenases (reviewed by Jeltsch 2013). Thus, Jeltsch argued that the major reversible epigenetic systems could possibly appear only in the Cambrian period, after an increase in atmospheric oxygen. He suggested that such a change could have been a precondition for the generation of different classes of oxygenases, the enzymes permitting the use of stable but reversible covalent chromatin modifications for gene regulation, a key factor in the evolution of multicellular organisms (Jeltsch 2013). In addition to epigenetic regulation, histone modifications play an important role in DNA damage responses and chromatin restoration, too (reviewed by Zhu and Wani 2010). The various types of histone modifications are listed in Table 1.2. All of them may affect the structure of the chromatin. It was suggested that histone acetylation and methylation may contribute to epigenetic inheritance. 1.2.2.1 Histone Acetylation Acetylated histones are regularly associated with active promoters. Histone acetyltransferases (HATs) that are frequently associated with multiprotein complexes in the nuclei, transfer an acetyl group from acetyl CoA to the ε-amino group of lysine side chains (reviewed by Bannister and Kouzarides 2011; Horikoshi 2013, Table 1.3). This modification results in weakening of histone-DNA interaction as well as in the formation of potential binding sites for bromodomain proteins that specifically recognize acetylated lysine residues and mediate transcription and anti-silencing functions. In addition to histones, non-histone proteins are also modified by HATs. Histone acetylation at the ε-amino group of lysine residues is reversible, the acetyl groups are removed by histone deacetylases (HDACs) 1 Epigenetic Regulation 9 Table 1.2 Histone modifications Type Acetylation Methylation Phosphorylation Ubiquitination SUMOylation ADP ribosylation Deimination Proline isomerisation O-GlcNAcylation Crotonylation Amino acid modified Lysine Lysine Arginine Serine Threonine Tyrosine Lysine Lysine Glutamate Arginine (to citrulline) Proline Serine Threonine Lysine Writer HATs HKMTs PRMTs Ser/Thr kinase family Tyrosine kinase Reader Eraser Brd2, Brd4 HDACs CHD1, HP1, PC KDMs JMJD6 14-3-3 Phosphatase enzymes Ubiquitin ligase ? ? ? PARP1 Peptidylarginine deaminase Proline isomerase OGT, O-linked β-D-Nacetylglucosamine transferase ? DUBs SENP1 ? ? Proline isomerase OGA, O-GlcNAc- ase ? ? Based on Bannister and Kouzarides (2011), Tan et al. (2011), and Xu et al. (2014) Table 1.3 Histone acetyltransferases, acetylated histone-binding proteins and histone deacetylases in mammals Protein GNAT family MYST family P300/CBP Function Histone acetylation Histone acetylation Histone acetylation Basal transcription factor family Nuclear receptor cofactor family Brd2 Histone acetylation Histone acetylation Reader Brd4 Reader HDAC Class I Class II Class III Class IV Histone deacetylation Note C-terminal bromodomain Chromodomain, zinc fingers Bromodomain, zinc fingers, binding of transcription factors, co-activator function Related to TAFII250 Two bromodomains binding histone H4K12ac; co-activator Two bromodomains, co-activator, chromatin insulator Constitutive expression Tissue specific expression Sirtuins, NAD+ dependence Tissue specific expression Based on Roth et al. (2001), Yang (2004), Bannister and Kouzarides (2011), Josling et al. (2012), and Bassett and Barnett (2014) Abbreviations: GNAT Gcn5-related N-acetyltransferase, Gcn5 general control non-derepressible 5 (yeast), MYST named for its founding members: MOZ, Ybf2/Sas3, Sas2, and Tip60, p300 E1A-associated 300 kDa protein, CBP CREB-binding protein, TAFII250 TBP-associated factor of 250 kDa, TBP TATA-binding protein, Brd2 bromodomaincontaining protein 2, Brd4 bromodomain-containing protein 4, NAD+ nicotinamide adenine dinucleotide, oxidized (Yang and Seto 2008; Bagui et al. 2013; Lakshmaiah et al. 2014, Table 1.3). Whereas active promoters are typically located to acetylation islands, i.e. chromatin regions enriched in acetylated histones, deacetylated histones are usually located to transcriptionally repressed, heterochromatic domains. Thus, HDACs function as transcriptional corepressors and they are 10 J. Minarovits et al. divided into four classes based on sequence homology. Class I (HDACs 1, 2, 3, 8), class II (HDACs 4, 5, 6, 7, 9, 10), class III (the sirtuins) and Class IV (HDAC11) (Yang and Seto 2008). Because the level of histone acetylation decreases during the mitosis, and most of the acetylated histone-binding proteins are not associated with mitotic chromatin, histone acetylation was not considered as a putative mechanism transmitting epigenetic information from cell generation to cell generation. Recent data however suggest that certain gene loci remain associated with acetylated histones bound by a bromodomain protein in mitotic chromatin ((Shibata and Nishiwaki 2014), see Sect. 1.2.3). Thus, acetylated chromatin domains may also play a role in epigenetic memory. 1.2.2.2 Histone Methylation Methylation of lysine (K) or arginine (R) residues of histone H3 or H4 may activate or repress the activity of nearby promoters. Conserved sequence motifs of histone lysine methyltransferases (HKMTs) bring S-adenosyl-L-methionine (SAM) in the proximity of the ε-amino group of the target lysine residue and create mono-, di-, or trimethylated lysines. Depending on the position of the methylated lysine residue, such modifications may favour the formation of euchromatin, a loose chromatin structure favouring promoter activity, or may result in a more condensed, repressive chromatin structure (heterochromatin). A typical euchromatic mark around active promoters is histone H3K4me3 (histone H3 trimethylated on lysine 4). In contrast, histone H3K9me3 and histone H3K27me3 (histone H3 trimethylated on lysine 9 and lysine 27, respectively) usually associate with silent promoters. Lysine-specific histone methyltransferases typically contain a SET domain of approximately 130 amino acids that forms a catalytic core observed in the prototypical HKMTs Su(var)3-9, Enhancer of Zeste and Trithorax (reviewed by Dillon et al. 2005, Table 1.4). The non-SET domain containing DOT1 (disruptor of telomeric Table 1.4 Human histone lysine methyltransferases (HKMTs) Enzyme (A) SET domain HKMTs EZH1 (PRC2 member) EZH2 (PRC2 member) G9a GLP1 (EuHMT1) MLL1 (ALL-1, HRX) MLL2 (ALR-1) MLL3 (HALR) SET1 SET7/9 SET8 SETDB1 SUV4-20H1 SUV4-20H2 SUVAR39H1 SUVAR39H2 (B) Non-SET domain HKMT DOT1 and DOT1L Based on Dillon et al. (2005) Specificity Target residue Function Histone H3 Histone H1 Histone H3 Histone H3 Histone H3 Histone H3 Histone H3 Histone H3 Histone H3 Histone H3 Histone H4 Histone H3 Histone H4 Histone H4 Histone H3 Histone H3 K27 K26 K27 K9 K9 K4 K4 K4 K4 K4 K20 K9 K20 K20 K9 K9 Transcriptional silencing Transcriptional silencing Transcriptional silencing Transcriptional silencing Transcriptional silencing Transcriptional activation Transcriptional activation Transcriptional activation Transcriptional activation Transcriptional activation Transcriptional silencing Transcriptional silencing Transcriptional silencing Transcriptional silencing Transcriptional silencing Transcriptional silencing Histone H3 K79 Demarcation of euchromatin 1 Epigenetic Regulation 11 Table 1.5 Protein arginine methyltransferases (PRMTs) targeting histone tails Enzyme PRMT1 PRMT6 PRMT4 (CARM1) PRMT5 PRMT6 PRMT7 Targeted histone Histone H4 Histone H2A Histone H4 Histone H2A Histone H3 Histone H3 Histone H3 Histone H4 Histone H2A Histone H3 Histone H4 Histone H2A Histone H3 Histone H4 Histone H2A silencing) and DOT1L (DOT1-Like) proteins target the globular domain (K79) of histone H3 and may play a role in the demarcation of euchromatic domains and transcriptional elongation (reviewed by Dillon et al. 2005; Nguyen and Zhang 2011). Protein arginine N-methyltransferases (PRMTs) also use SAM as a methyl donor to mono- or dimethylate the guanidinium side chain of arginine. Dimethylation can be either asymmetrical (N,N-dimethylation) or symmetrical (N,N′-dimethylation) (Kouzarides 2007, Table 1.5). Such modifications targeting histone H3 and H4 may either activate or repress transcription. PRMTs modify, however, a series of nonhistone proteins as well at glycine- and arginine-rich patches within their substrates (Bedford 2007). Only a subset of protein arginine methyltransferases (PRMTs) modify the tails of histone H3, H4 or H2A (reviewed by Di Lorenzo and Bedford 2011). PRMT1 and PRMT6 generate transcriptional activation marks by methylating histone H4 and H2A at arginine 3 (R3me2a; a: asymmetric) whereas PRMT4 (CARM1) targets histone H3 producing the euchromatic marks H3R17me2a and H3R26me2a. In contrast, PRMT5 and PRMT6 leave heterochromatic marks on histone H3 (H3R8me2s and H3R2me2a, respectively; s: symmetric) (Di Lorenzo and Bedford 2011). Methylation mark R3me2a R3me2a R3me2a R3me2a R2me2a R17me2a R26me2a R3me2s R3me2s R8me2s R3me2a R3me2a R2me2a R3me2s R3me2s Function Trancriptional activation Trancriptional activation Trancriptional activation Trancriptional activation Transcriptional repression Trancriptional activation Trancriptional activation Transcriptional repression Transcriptional repression Transcriptional repression Trancriptional activation Trancriptional activation Transcriptional repression Transcriptional repression Transcriptional repression PRMT5 and PRMT7 target arginine 3 of histone H4 and H2A yielding R3me2s marks repressing transcription. The chromodomain protein CHD1 (chromodomain helicase DNA binding protein 1), an ATP-dependent chromatin remodeler, binds to the euchromatic histone mark H3K4me3 whereas methylated H3K9me3 and H3K27me3 associate with other chromodomain proteins HP1 (heterochromatin protein 1) and PC (Polycomb), respectively (Cao et al. 2002). PC is a component of the polycomb repressor complex 1 (PRC1) involved in transcriptional repression and in the establishment of three dimensional, long-range chromatin interactions (reviewed by Schwartz and Pirrotta 2007; Lanzuolo et al. 2012; Cheutin and Cavalli 2014). Histone lysine demethylases (KDMs) are either amine oxidases that may associate with a repressor complex and utilize FAD (flavin adenine dinucleotide) as a co-factor (e.g. LSD1, lysine-specific demethylase 1, also known as KDM1A), or they are Jumonji C (JMJC)-domain containing proteins that use Fe(II), α-ketoglutarate and molecular oxygen as co-factors, similarly to the Tet family of dioxygenases that target 5mC (Labbe et al. 2013). The only arginine demethylase described so far is JMJD6 (Jumonji domaincontaining 6 protein). JMJD6 acts as a 12 dioxygenase and demethylates histone H3 dimethylated at arginine 2 (H3R2me2) and histone H4 dimethylated at arginine 3 (H4R3me2) (Chang et al. 2007). It also acts on methylated estrogen receptor alpha (metERα) and affects the estrogen-activated signal transduction cascades outside of the nucleus by reducing methylation at the asymmetrically dimethylated arginine R260 (Poulard et al. 2014). 1.2.2.3 Histone Phosphorylation The genome-wide histone phosphorylation resulting in chromosome condensation during mitosis is mediated by the serine/threonine kinase Aurora-B targeting histone H3 at serines 10 and 28 (Goto et al. 2002; Sugiyama et al. 2002). It was observed that Aurora B was also active in the late G2 phase of the cell cycle when it was localized to the heterochromatin and phosphorylated serine 10 of histone H3 (Goto et al. 2002). During interphase, histone H3 phosphorylation by signal transduction kinases ensures rapid transcriptional induction of immediate early genes, including cfos and c-jun, that respond to mitotic signals (reviewed by Sawicka and Seiser 2012). In addition, histone H3 phosphorylation mediates induction of a wide variety of genes upon stimulation with extracellular signals in different cell types and may play a role in transient derepression of promoters silenced by histone H3K9me3 (Sawicka and Seiser 2012). Histone H3 phosphorylation at serine 10 (H3S10ph) recruits members of the 14-3-3 protein family to the chromatin, especially when the neighbouring K9 or K14 lysine residue is acetylated, and such a dual modification ensures full transcriptional activation of the Hdac1 and p21 genes (reviewed by Sawicka and Seiser 2014). In resting B lymphocytes Aurora B phosphorylates histone H3S28 at active promoters (Frangini et al. 2013). This modification may block the E3 ubiquitin ligase activity of Ring1B, a Polycomb protein which is deposited to the same set of promoters (Frangini et al. 2013). Histone phosphorylation can be reverted by histone phosphatases. Histone H3S10ph is dephosphorylated by protein phosphatase 1 (PP1) (Murnion et al. 2001). J. Minarovits et al. 1.2.2.4 Histone Ubiquitination Ubiquitin, a 8.5 kDa polypeptide of 76 amino acids is added to proteins by the concerted action of ubiquitin activating, conjugating and ligating enzymes. Ubiquitination affects the cellular location and interactions of proteins and typically signals their proteasomal degradation. Histones are the most abundant ubiquitinated proteins: the core histones, especially histone H2A and H2B, but also histone H1, the linker histone can be mono- or polyubiquitinated (Cao and Yan 2012). Polyubiquitination of histones is usually induced by DNA damage, and polyubiquitinated histones provide binding sites for the mediators of the DNA damage response. Both histone ubiquitination and deubiquitination were implicated in silencing of HOX genes (homeotic genes) by Polycomb group (PcG) proteins (reviewed by Schuettengruber and Cavalli 2010; see also Sect. 1.2.3). The PcG protein RING1B monoubiquitinates histone H2A at lysine 119, whereas other PcG proteins, RING1B and BMI1 upregulate the activity of RING1B (Cao and Yan 2012). Histone H2AK119ub1 is involved in Polycomb-mediated silencing (Wang et al. 2004). Other members of the PcG complex contain a H2A deubiquitinase (DUB) activity, that cleaves the isopeptide bond between ubiquitine and lysine (reviewed by Schuettengruber and Cavalli 2010 ). Monoubiquitination of histone H2B at lysine 120 occurs during transcription and stimulates histone H3K4 di- and trimethylation, i.e. the establishment of an euchromatic mark (Kim et al. 2009). This suggests that histone ubiquitination, indirectly, may play a role in the transmission of epigenetic information from cell generation to cell generation. RNF8 is a RING-finger ubiquitin ligase that facilitates the assembly of DNA repair proteins at DNA double-strand breaks (Mailand et al. 2007). Recently it was demonstrated that RNF8mediated ubiquitination of histone H2A on the sex chromosomes during meiosis is necessary for dimethylation of histone H3 at lysine 4 (H3K4me2), an epigenetic mark that persists throughout meiotic division and may serve as the epigenetic memory in post-meiotic spermatids 1 Epigenetic Regulation where further RNF8-dependent epigenetic modifications occur (Sin et al. 2012). These include histone H3K4me3, histone crotonylation and deposition of the variant histone H2A.Z at the promoters of “escape” genes to be activated on inactive sex chromosomes in post-meiotic, round spermatids (Sin et al. 2012). Thus, histone ubiquitination may ensure the transmission of epigenetic information from meiotic cells to post-meiotic cells. 1.2.2.5 Histone SUMOylation Small ubiquitin-like modifiers (SUMO proteins) are 92–97 amino acid long, approximately 10 kDa molecules that share structural similarities with ubiquitin. Their conjugation to protein targets is mediated by an enzymatic machinery resembling the one involved in the ubiquitination pathway: so called E1, E2 and E3 enzymes perform activation, conjugation and finally ligation of SUMO proteins to core histones at lysine residues (Gareau and Lima 2010). There are multiple E3 ligases involved in the last step, the formation of an isopeptide bond between the carboxy-terminal carboxyl group of SUMO and an ε-amino group of a substrate acceptor Lys residue (Yang and Chiang 2013). SUMOylation could possibly block the establishment of other covalent modifications including acetylation and ubiquitination. It was demonstrated that SUMOylation of histone H4 repressed promoter activity in cultured cells by the recruitment of histone deacetylase and heterochromatin protein 1 (HP1) (Shiio and Eisenman 2003). SUMOylation appears to be an important modulator of transcription factor function (Yang and Chiang 2013). It was observed that SUMOylated nonhistone proteins were associated with the transcription start site on many of the most active housekeeping genes in HeLa cells, and SUMO-1 distribution correlated with that of H3K4me3, an euchromatic mark (Liu et al. 2012). In contrast, SUMOylation of the transcription factor Elk-1 repressed Elk-1 regulated promoters by the recruitment of histone deacetylase (Yang and Sharrocks 2004). SUMO isoforms may be removed by enzymes belonging to the sentrin-specific protease (SENP) family. 13 The role of SUMOylation in epigenetic memory remains to be established. 1.2.2.6 Histone ADP Ribosylation ADP-ribosyltransferases modify glutamate residues of histones by the transfer of ADP-ribose from NAD+, a process reverted by the action of poly-ADP-ribose polymerases (PARPs, reviewed by Bannister and Kouzarides 2011). The contribution of ADP ribosylation to the transfer of epigenetic information from cell generation to cell generation remains to be elucidated. 1.2.2.7 Histone Deimination In addition to demethylation by the dioxygenase JMJD6, the methyl group from arginine can also be removed in a reaction called demethylimination or deimination that generates citrulline, which can’t be methylated (Cuthbert et al. 2004). Deimination is carried out by peptidylarginine deiminase 4 (PADI 4) and results in an irreversible alteration of histone H3 and H4 structure. Its role in the establishment of epigenetic memory is unknown at present. 1.2.2.8 Histone Proline Isomerization: A Noncovalent Histone Modification In histone H3 of Saccharomyces cereviseae the peptidyl-prolyl isomerase (PPIase) Fpr4 interconverts the cis and trans isomers of the alanine 15-proline 16 peptide bond, and it was observed that acetylation at the neighbouring lysine 14 (K14) residue promotes the trans conformation (Howe et al. 2014). Proline isomerization at proline 38 of histone H3 affects the ability of Set2 to methylate lysine 36 (K36) that suggests a role for a noncovalent histone modification in epigenetic regulation (Nelson et al. 2006). 1.2.2.9 Histone O-GlcNAcylation N-acetylglucosamine, briefly GlcNAc, may form a covalent linkage with serine or threonine residues of proteins when transferred from UDPGlcNAc to them by OGT (O-GlcNAc transferase). This modification occurs on cytosolic, nuclear and mitochondrial proteins and marks also the core histones. O-GlcNAcylation of histone tails 14 J. Minarovits et al. may activate or repress promoter activity, depending on the residue modified. OGT belongs to the polycomb group of proteins, and it is a component of Polycomb Repressive Complex 1 (PRC1). In fact, OGT was found to be identical with the polyhomeotic (PH) protein that interacts with EZH2 (Enhancer of Zeste Homolog 2), the lysine methyltransferase of the other PcG complex, PRC2 (reviewed by Dehennaut et al. 2014). In addition, OGT is also a partner of the TET proteins of dioxygenases that demethylate 5mC (see Sect. 1.2.1). TET2 and TET3 may recruit OGT to the chromatin (Chen et al. 2013). The O-GlcNAc mark is removed by OGA (O-GlcNAcase). Because UDP-GlcNAc is a product of the hexosamine biosynthetic pathway, it was suggested that the nutritional state of the organism may affect the epigenotype and result in metabolic diseases and cancer due to epigenetic reprogramming (Dehennaut et al. 2014). However, the exact role of O-GlcNAcylation in epigenetic inheritance remains to be clarified. 1.2.2.10 Histone Crotonylation Histone lysine crotonylation (Kcr) is a newly identified chromatin mark enriched at active promoters and potential enhancers (Tan et al. 2011). In male germinal cells Kcr marks sex chromosome-linked genes activated following meiosis. 1.2.3 Polycomb and Trithorax Complexes Polycomb group (PcG) and Trithorax group (TrxG) protein complexes are capable to establish heritable chromatin states and transmit cell type specific gene expression patterns from cell generation to cell generation. PcG and TrxG complexes were discovered in Drosophila melanogaster as regulators of Hox (homeotic) genes (reviewed by Schuettengruber et al. 2007). TrxG complexes were characterized as activators of Hox genes both in Drosophila and in mammals, a phenomenon attributed to the histone lysine methyltransferase members of the TrxG complex that deposit the euchromatic mark H3K4me3 to the chromatin. In mammals, the enzymes involved in histone H3K4 methylation are the SET1A, SET1B, and mixed lineage leukemia (MLL) proteins 1–4 (Shilatifard 2012). These proteins require additional subunits for activity, similarly to the yeast methylase Set1, and the complex of proteins associated with the histone H3K4 methylases is termed COMPASS (Miller et al. 2001). TrxG proteins in humans also form COMPASS-like multisubunit complexes with SET1A/B and MLL1–4 (Shilatifard 2012). PcG proteins were identified as silencers of Hox genes in Drosophila (reviewed by Schuettengruber et al. 2007; Simon and Kingston 2009). Similarly to TrxG proteins, PcG proteins also form multisubunit complexes called Polycomb-repressive complex 1 and 2 (PRC1 and 2) (Margueron and Reinberg 2011; Simon and Kingston 2013; Voigt et al. 2013; Scelfo et al. 2015). Gene silencing by PRC2 was attributed to the deposition of H3K27me3, a heterochromatic mark, to the chromatin by the histone methyltransferases EZH2 and EZH1 (enhancer of zeste homologs). Subsequently, H3K27me3 is bound by Polycomb (PC), a member of PRC1 in Drosophila, or its mammalian and human homologs Cbx2, −4, −6, −7, and −8 (chromobox protein homolog 4, 6, 7, and 8), although the latter proteins bind with lower affinity to H3K27me3 peptides and they can’t distinguish between H3K27me3 and H3K9me3 marks (Kaustov et al. 2011). There are at least five distinct PRC1 subcomplexes that contain, in addition to the RING1A/B ubiquitin ligase that monoubiquitinates histone H2A, different PCGF (Polycomb group RING finger) proteins, in addition to the core components EED (Embryonic Ectoderm Development), SUZ12 (Suppressor of Zeste 12), and Retinoblastoma binding proteins 46 and 48 (also designated as RBBP7 and RBBP4) (Gao et al. 2012; Tavares et al. 2012; Simon and Kingston 2013). Histone H2AK119 monoubiquitination plays an important role in Polycomb mediated silencing of the Ubx gene in Drosophila (Wang et al. 2004). An alternative model suggests that noncanonical PRC1 complexes can be recruited to the targeted chromatin regions first, followed by 1 Epigenetic Regulation ubiquitination of histone H2A. Such an H3K27me3-independent pathway would rely on an unknown signal recognized by the RYBP protein. RYBP is a partner of RING1B, the catalytic subunit of PRC1. Subsequently, H2Aubq deposited by RING1B would induce PRC2 activity, resulting in H3K27 trimethylation (Tavares et al. 2012; Scelfo et al. 2015). In addition to creating repressive histone marks, either directly by monoubiquitination or indirectly by recruiting histone lysine methyltransferases, PRC1 can also affect chromatin structure and silence gene expression by direct compaction of chromatin (Francis et al. 2004; Eskeland et al. 2010). The Ring1B-mediated compaction of nucleosomal arrays is independent of Ring1B enzymatic activity and occurs even on nucleosomes assembled from tail-less histones (Francis et al. 2004). In the absence of PRC1, i.e. in Ring1B null cells, there is a large-scale decompaction of chromatin that can be detected at the level of chromosomes using FISH (Eskeland et al. 2010). PcG proteins were originally identified as gene silencers. Recent data suggest, however, that they may play a role in promoter activation, too. Although the heterochromatic histone mark H3K27me3 is deposited by PRC2 to CpG-rich inactive promoters, it was observed that in embryonic stem cells the monomethylated H3K27me1 – that is also deposited by PRC2 – accumulates within transcribed genes (Ferrari et al. 2014). In addition, EZH1, a PRC2 member, was associated with transcriptionally competent euchromatic regions in mouse skeletal muscle myoblasts, and it was recruited to transcriptionally active promoters in differentiating myocytes (Mousavi et al. 2012). It is worthy to note, however, that in another cell type, i.e. in differentiated osteoblasts, EZH1 is recruited to silent promoters marked with a dual histone modification, H3K9me3/S10ph (histone H3 lysine 9 trimethylated and serine 10 phosphorylated, Sabbattini et al. 2014). In resting B lymphocytes, the PRC1 subunit Ring1B is deposited to active promoters marked by Aurora kinase B that phosphorylates histone H3S28 (Frangini et al. 2013). Ring1B apparently 15 functions as a coactivator of transcription at these promoters. One may speculate that the function of the PRC1 subunit Ring1B is context dependent: it may activate promoters when associated with Aurora kinase B that blocks its E3 ubiquitin ligase activity, but acts as a transcriptional repressor in other chromatin environments (Frangini et al. 2013). Aurora kinase B also enhances binding and activity of the USP16 deubiquitinase at transcribed genes, further decreasing the level of monoubiquitinated histone H2A (Frangini et al. 2013). It is remarkable that the histone specific lysine methyltransferases (KMTs) of TrxG and PcG complexes are active only when incorporated into large protein complexes and their activity and specificity is defined by the subunits of the respective complexes (Del Rizzo and Trievel 2011). Whereas TrxG and PcG proteins may exert antagonistic effects during interphase, it was observed that in living transgenic lines of Drosophila a small fraction of both TrxG and PcG proteins, fused to enhanced green fluorescent protein (EGFP), interact in a cooperative manner and remain bound to mitotic chromatin (Fonseca et al. 2012; Steffen et al. 2013). In Drosophila embryos, both PcG and TrxG proteins remain associated with newly replicated chromatin as well (Petruk et al. 2012). These data suggest that similarly to variant histones and pioneer transcription factors (see Sects. 1.2.4 and 1.2.5), PcG and TrxG proteins bound to newly replicated DNA molecules and to mitotic chromatin may bookmark certain promoters for repression or activation in the daughter cells, establishing thereby heritable chromatin states (epigenetic memory). 1.2.4 Variant Histones In addition to covalently modified tails of core histones, certain histone variants that replace invariant histones in histone octamers may also convey epigenetic information (Volle and Dalal 2014). In contrast to core histones which associate with newly replicated DNA, variant histones can be incorporated into the chromatin through- 16 out the interphase in a replication-independent manner. Thus, it was observed that the histone variant H2A.Z marked transcriptional start sites of active genes in interphase cells and retained its position even in highly condensed mitotic chromosomes (Kelly et al. 2010). Its nucleosome occupancy changed in mitotic chromatin. H2A.Z acted like a bookmarking protein that permitted promoter activation in the daughter cells after chromosome decondensation. Based on these data, Kelly and Jones suggested that altered nucleosome occupancy may form a novel epigenetic mechanism (Kelly et al. 2010; Kelly and Jones 2011). In Caenorhabditis elegans, the histone variant H2A.Z, also called HTZ-1, acts in concert with BET-1, a member of the bromodomain and extra terminal (BET) family of acetylated histone binding proteins to maintain the expression status of selector genes that govern the fates of cell groups (Shibata and Nishiwaki 2014). Although the level of histone acetylation decreases during the mitotic phase and most of the acetylated histone-binding proteins do not bind to mitotic chromatin, BET-1 remains associated with selector gene loci enriched in acetylated histones where H2A.Z is deposited. In this case H2A.Z represses transcription, but maintains the poised state of RNA polymerase II at the promoters (Shibata and Nishiwaki 2014). Shibata and Nishiwaki (2014) suggested that, upon receiving a proper differentiation signal, H2A.Z is released permitting the transcription of selector genes that specify cell, tissue or regional identity. Variant histones function not only as epigenetic marks, but also as transcriptional regulators, although their deposition may facilitate chromation access of both activating and repressive regulatory complexes (Weber and Henikoff 2014). In addition, the variants H2A.X and H2A.Z are involved in the repair of double-strand DNA breaks (Volle and Dalal 2014). A subset of active promoters, enhancers and insulator regions is enriched in two distinct variant histones, H3.3 and H2A.Z (Jin et al. 2009). Nucleosome core particles containing H3.3/H2A.Z double variant histones are instable, a feature facilitating the access of transcription factors to such areas J. Minarovits et al. (“nucleosome-free regions”) (Jin et al. 2009). It is worthy to note that histone chaperones play an important role in nucleosome assembly (reviewed by Burgess and Zhang 2013), as well as in the regulation of histone modifications (Begum et al. 2012; Stevenson and Liu 2013; Wang et al. 2013). 1.2.5 Pioneer Transcription Factors It was observed that in living cells transcription factors are regularly unable to access the majority of their consensus binding sites in the genome due to the nucleosomal structure of the chromatin and the folding of nucleosomes into higher order structures (reviewed by Zaret and Carroll 2011). Cooperative binding with other transcription factors may circumvent this problem and a unique category of nuclear regulatory proteins, called pioneer transcription factors can access their target sites even on nucleosomes located to heterochromatic regions. Their binding to their recognition sites is usually stable and preceeds the binding of other transcription factors to promoter regulatory sequences. The association of pioneer transcription factors with enhancer regions may speed up inductive responses or alter the local chromatin structure that enables the binding of other transcription factors (reviewed by Zaret and Carroll 2011). Heterochromatin binding, the remarkable capacity of the pioneer transcription factors belonging to the FoxA (forkhead box protein A) family is due to their structural similarity to the linker histone: there is a winged helix motif present in both FoxA proteins and histone H1 that facilitates nucleosome binding. In addition, the C-terminal domain of FoxA proteins contributes to chromatin opening, whereas the N-terminal transactivating domain may recruit coregulator proteins. In addition to variant histones, direct binding of distinct non-histone proteins to regulatory regions of the genome may also constitute epigenetic marks that can be inherited to daughter cells. It was demonstrated that “pioneer” transcription factors or “bookmarking” proteins remain bound to chromatin even in mitotic chro- 1 Epigenetic Regulation mosomes, and accelerate transcriptional reactivation following mitosis (Zaret et al. 2008; Caravaca et al. 2013). Not only can pioneer factors bind to highly methylated DNA sequences and occupy the position of histone H1 at nucleosomes, but they can establish euchromatic regions as well by inducing local cytosine demethylation. As described above, their binding to tissue-specific enhancers preceeds transcriptional activation of the genes associated by such pre-marked enhancers (Zaret et al. 2008). 1.2.6 Long Noncoding RNAs Long noncoding RNA (lncRNA) molecules are potential carriers of epigenetic information. They may directly interact with PRC2 and target the activity of the histone lysine methyltransferase EZH2 to selected genomic loci, resulting in trimethylation of histone H3 at lysine 27 (H3K27me3), chromatin condensation, and promoter silencing. LncRNAs interact with other chromatin remodeling complexes as well (reviewed by Nie et al. 2012). In principle, lncRNAs may affect chromatin structure by acting as tethers, scaffolds, allosteric regulators or decoys, and may directly interfere with transcription as well (reviewed by Keller and Buhler 2013). Tethering or recruitment of chromatin-modifying complexes by lncRNAs may either silence or activate gene expression. Approximately 3,300 large intergenic noncoding RNAs (lincRNAs) – encoded in genomic regions located between genes – were detected in various human cell types (Khalil et al. 2009). In addition to the well-characterized lincRNA HOTAIR (HOX transcript antisense RNA) that binds both the PRC2 (Rinn et al. 2007) and the lysinespecific histone demethylase LSD1 (Tsai et al. 2010), a series of other lincRNAs also associated with PRC2 or other chromatin modifying complexes. Thus, it was suggested that certain lincRNAs may guide chromatin-modifying complexes to specific genomic loci and control promoter activity (Khalil et al. 2009). The role of lncRNAs in the transmission of epigenetic memory remains to be established. 17 1.2.7 Long-Distance Chromatin Interactions DNA methylation and histone modifications may spread from their primary sites of deposition to neighbouring chromatin areas resulting in the establishment of extended heterochromatic or euchromatic regions, i.e. nuclear subcompartments repressing or facilitating transcription, respectively (reviewed by Gyory and Minarovits 2005; Doerfler 2012). Long-distance chromatin interactions mediated by CCCTC binding factor (CTCF) and cohesin proteins may insulate chromatin domains and allow coregulation of promoters within the loops by preventing the spread of chromatin modifications from adjacent areas. Burke et al. observed that CTCF was bound to mitotic chromosomes and found that a chromatin loop at the Igf2/H19 locus could also be detected in mitosis (Burke et al. 2005). They also observed the loss of a neighbouring chromatin loop in mitotic chromatin. This finding indicated, however, that certain chromatin loops may be preserved in mitotic chromosomes and could possibly contribute to epigenetic memory (Burke et al. 2005). In contrast, the insulator upstream of the c-MYC gene changed its structure in mitotic HeLa cells in parallel with the disappearance of the sequence-specific direct binding of CTCF (Komura et al. 2007). This observation suggested that the nucleoprotein complex involving this particular insulator element must be reassembled de novo after cell division (Komura et al. 2007). The potential contribution of long distance chromatin interactions to epigenetic memory needs further studies. 1.3 Chromatin Types 1.3.1 Euchromatin (Active Chromatin) Euchromatic regions are characterized by a relaxed, decondensed structure that permits the access of transcription factors, RNA polymerases and recombinases. Transcribed housekeeping genes and cell type or tissue specific genes are 18 J. Minarovits et al. preferentially located to such active chromatin domains. Promoters and regulatory sequences located to euchromatic domains are typically unmethylated or hypomethylated and they are frequently marked by euchromatic, activating histone modifications including acetylated histone H3 and histone H4 or histone H3K4me2/ me3. Active chromatin is organized in three dimensions, forming topologically associating domains (TADs) that are flanked by insulator elements and characterized mainly by internal chromatin interactions (Ciabrelli and Cavalli 2015). The 3D folding of the chromatin, i.e. the formation of DNA loops is mediated by insulator binding proteins including CTCF and cohesin complexes that may interact with coactivator proteins to connect enhancer and promoter sequences; such interactions may generate cell type-specific or developmental stage-specific looping, and corresponding gene expression patterns (Rubio et al. 2008; Kagey et al. 2010). It was observed that transcriptionally active chromatin regions and euchromatic domains involved in recombination are preferentially located in the nuclear interior and translocation of inactive, heterochromatic domains from the nuclear periphery to more central regions facilitates the switching-on of silent promoters and creates a favourable environment for the gene recombination in cells of the adaptive immune system (reviewed by Gyory and Minarovits 2005). 1.3.2 Hererochromatin (Repressive Chromatin) Heterochromatic regions are characterized by a condensed structure that usually hinders the access of transcription factors, RNA polymerase complexes and recombinases to their recognition sequences. Silent genes are frequently located to such inactive chromatin domains. The promoters and regulatory sequences of silent genes are typically hypermethylated and are frequently marked by heterochromatic, repressive histone modifications including deacetylated histone H3 and histone H4 or histone H3K9me3 and H3K27me3. Depending on the epigenetic marks enriched in heterochromatic domains, one can distinguish between various subtypes of heterochromatin. 1.3.2.1 Polycomb-Repressed Chromatin Polycomb-repressed chromatin corresponds to genomic regions silenced by PRC1 and PRC2 repressive complexes. These compacted chromatin domains are typically enriched in histone H3K27me3. Long-range interactions between TADs may displace Polycomb-repressed chromatin from active chromatin regions or laminaassociated nuclear areas (reviewed by Ciabrelli and Cavalli 2015). 1.3.2.2 Null Chromatin, LaminAssociated Domains The so called null chromatin apparently lacked typical histone marks in genome-wide studies. It was highly enriched, however, in lamin proteins that form a meshwork just below the nuclear membrane and frequently silence the lamin associated domains (LADs) of chromatin at the nuclear periphery. LADs are insulated by CTCF from the neighbouring chromatin areas that frequently contain transcribed housekeeping genes (reviewed by Ciabrelli and Cavalli 2015). It is worthy to note that in addition to its location at the nuclear periphery, lamin B1 is also present in the nucleoplasm as an internal lattice, and it is associated with matrix attachment regions (MARs), i.e. DNA sequences acting as epigenetic insulators (Luderus et al. 1992; Arope et al. 2013). MARs correspond to LADs and typically contain AT-rich elements (AT core) that may unwind when exposed to superhelical strain. The AT core is relatively histone-poor but enriched in RNA polymerase II, CTCF and the euchromatic mark histone H3K4me3 (Arope et al. 2013). MARs may augment gene expression, whereas depletion of lamin B1 may severely inhibit both RNA polymerase II and RNA polymerase I mediated transcription (Tang et al. 2008; Arope et al. 2013). 1.3.2.3 Constitutive Heterochromatin Constitutive heterochromatin is located to centromeric and telomeric regions that are usually 1 Epigenetic Regulation marked by H4K20me3 and H3K9me2/me3. The latter mark is typically bound by HP1a (heterochromatin protein 1a). Constitutive heterochromatin is highly compacted and it is devoid of euchromatic histone modifications. The repressive mark H3K27me3 is also absent (reviewed by Ciabrelli and Cavalli 2015). 1.4 Coregulation of Gene Batteries Coordinated expression of tissue- or cell-typespecific genes could be achieved by repetitive regulatory sequences situated at the control regions of coregulated genes. Alternatively, the organization of chromatin into higher order structures, e.g. chromatin loops may ensure the coordinated activation or silencing of gene batteries (reviewed by Gyory and Minarovits 2005). Clustering of genes may arrange them into domains enriched in transcription factors, and such co-transcribed genes located to “transcription factories” may be located quite far from each other, even on different chromosomes in interphase nuclei (reviewed by Osborne 2014, see also Sect. 1.4.2). Although RNA polymerase II may reside in transcription factories only transiently, proteomics studies support the existence of such stable, lamin-associated compartments for active transcription (Melnik et al. 2011). Active tranacription may affect the three-dimensional organization of the genome, in concert with CTCF and cohesins that establish and stabilize chromatin loops (Choi and Feeney 2014; Osborne 2014). 1.4.1 Locus Control Regions, Chromatin Loops, Topologically Associated Domains Locus control regions (LCRs) typically contain enhancer sequences and insulator elements that separate the genes located within a chromatin loop from the surrounding chromatin domains and regulatory elements. A locus control region may bind tissue-specific transcription factors that 19 may contribute to regional DNA hypomethylation and demethylation of methylated histones, too (for review see Gyory and Minarovits 2005). The boundaries of TADs are determined by the insulator binding protein CTCF that prevents spreading of heterochromatin and blocks enhancer activity. CTCF binding is frequently associated with the attachment of cohesin subunits that stabilize the chromatin loops formed by CTCF-CTCF interactions (Choi and Feeney 2014). The architecture of chromatin loops at the major histocompatibility comlex (MHC) is affected by the matrix attachment region (MAR)-binding protein SATB1 (special AT-rich sequence binding protein 1). SATB1 interacts with PML nuclear bodies, a subnuclear structure first described in promyelocytic leukemia cells (Kumar et al. 2007). 1.4.2 Nuclear Subcompartments Switching of the nuclear environment may facilitate or suppress both transcription and genetic recombination. Relocation of V, D, and J gene segments of the active IgH allele occurs during B lymphocyte development: in pro-B cells they move away from the nuclear periphery to an euchromatic domain before recombination and subsequent transcription (reviewed by Gyory and Minarovits 2005). It is interesting to note, however, that active genes may also associate with the nuclear periphery: Recently, using threedimensional imaging and chromatin immunoprecipitation (ChIP)-chromosome conformation capture (3C) techniques, Park et al. observed that in murine plasma cells active immunoglobulin genes were located in the vicinity of the nuclear periphery (Park et al. 2014). In addition, active IgH, Igκ and IgJ genes, coding for immunoglogulin heavy and light chains and for J (joining) chain, a component of IgM and IgA molecules secreted to the mucosa, were preferably colocalized in transcription factories (see below, Sect. 1.4.2). Because IgH, Igκ and IgJ are located on three different chromosomes, such an arrangement may facilitate trans-chromosomal emhancer interactions and the utilization of shared transcription factors (Park et al. 2014). 20 J. Minarovits et al. In parallel with the nuclear subcompartment switch, the B cell and T cell antigen receptor loci undergo large-scale structural changes (locus contraction). The rosette-like chromatin loops formed by CTCF and cohesins collapse into a single globule that permits recombination even between variable (V), diversity (D) and joining (J) gene segments located at a long distance from each other (Choi and Feeney 2014). These 3D changes are facilitated by transcription of noncoding RNAs (ncRNAs) encoded in euchromatic regions that are brought into juxtaposition by a long-range enhancer to the same transcription factory (Choi and Feeney 2014). 1.4.3 Transcription Factories, Active Chromatin Hubs Transcription factories are nuclear foci that contain two or more DNA-dependent RNA polymerase enzymes active on at least two different templates (Osborne et al. 2004; Papantonis and Cook 2013). 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