REVIEW Getting down to the core of histone modifications Antonia P. M. Jack & Sandra B. Hake Received: 17 January 2014 /Revised: 8 April 2014 /Accepted: 9 April 2014 # Springer-Verlag Berlin Heidelberg 2014 Abstract The identification of an increasing number of posttranslationally modified residues within histone core do- mains is furthering our understanding of how nucleosome dynamics are regulated. In this review, we first discuss how the targeting of specific histone H3 core residues can directly influence the nucleosome structure and then apply this knowl- edge to provide functional reasoning for their localization to distinct genomic regions. While we focus mainly on transcrip- tional implications, the principles discussed in this review can also be applied to their roles in other cellular processes. Finally, we highlight some examples of how aberrant modifi- cations of core histone residues can facilitate the pathogenesis of some diseases. Keywords Chromatin . H3K56 . H3K64 . H3K79 . H3T118 . H3K122 . Nucleosome . Transcription . Enhancer Introduction The nucleosome, the most basic chromatin particle, both solves and poses cellular problems. Consisting of a core of basic histone proteins, around which approximately 1.65 turns of negatively charged DNA is wrapped, the nucleosome pro- vides an elegant means of partially alleviating the repulsive forces that would otherwise prevent a naked DNA strand from folding up (Luger et al. 1997). In doing so, it provides the first level of compaction that is required to store vast amounts of genetic information within the confines of a eukaryotic cell nucleus. While this may seem advantageous, it also has some underlying problems. By associating tightly with the histone octamer (consisting of two of each of the histones H3, H4, H2A, and H2B), the substrate for all DNA-based processes becomes largely inaccessible, and the presence of nucleo- somes obstructs the path of essential protein machineries. In addition, formation of inter-nucleosomal contacts and associ- ation of chromatin-related proteins lead to the assembly of higher-order structures, augmenting the accessibility barriers generated by nucleosomes alone (Tremethick 2007). However, chromatin and the nucleosome structures within it are highly dynamic, and by local manipulation of the com- ponents that comprise them, functionally distinct genomic regions, based on DNA accessibility, can be established. Im- portantly, it is this malleability that confers the central role of chromatin as a mediator of all DNA-based processes. The timing and locality of chromatin changes are determined by the coordinated action of several processes including DNA methylation, ATP-dependent remodeling, incorporation of histone variants, and posttranslational modifications (PTMs) of the histone proteins, the sum of which determines the chromatin environment (Bonisch et al. 2008). Until technological advancements made in the last decade, our knowledge of the latter process was limited to modifica- tions of the intrinsically disordered and flexible histone N- terminal tail domains, which protrude out of the nucleosome structure (Luger et al. 1997). Such extension away from the nucleosome core makes the N-terminal tails not only amena- ble to experimental detection but also more accessible to enzymes that set and erase modifications, known respectively as writers and erasers. In addition, the exposure of residues in these regions promotes the association of chromatin-related proteins, termed readers, that possess conserved domains ca- pable of recognizing and binding specific modifications and A. P. M. Jack (*) : S. B. Hake (*) Adolf-Butenandt Institute, Department of Molecular Biology, LMU Munich, Schillerstrasse 44, 80366 Munich, Germany e-mail: [email protected]e-mail: [email protected]S. B. Hake Center for Integrated Protein Science Munich (CIPSM), Munich, Germany Chromosoma DOI 10.1007/s00412-014-0465-x
Transcript
REVIEW
Getting down to the core of histone modifications
Antonia P. M. Jack & Sandra B. Hake
Received: 17 January 2014 /Revised: 8 April 2014 /Accepted: 9 April 2014# Springer-Verlag Berlin Heidelberg 2014
Abstract The identification of an increasing number ofposttranslationally modified residues within histone core do-mains is furthering our understanding of how nucleosomedynamics are regulated. In this review, we first discuss howthe targeting of specific histone H3 core residues can directlyinfluence the nucleosome structure and then apply this knowl-edge to provide functional reasoning for their localization todistinct genomic regions.While we focus mainly on transcrip-tional implications, the principles discussed in this review canalso be applied to their roles in other cellular processes.Finally, we highlight some examples of how aberrant modifi-cations of core histone residues can facilitate the pathogenesisof some diseases.
The nucleosome, the most basic chromatin particle, bothsolves and poses cellular problems. Consisting of a core ofbasic histone proteins, around which approximately 1.65 turnsof negatively charged DNA is wrapped, the nucleosome pro-vides an elegant means of partially alleviating the repulsiveforces that would otherwise prevent a naked DNA strand fromfolding up (Luger et al. 1997). In doing so, it provides the first
level of compaction that is required to store vast amounts ofgenetic information within the confines of a eukaryotic cellnucleus. While this may seem advantageous, it also has someunderlying problems. By associating tightly with the histoneoctamer (consisting of two of each of the histones H3, H4,H2A, and H2B), the substrate for all DNA-based processesbecomes largely inaccessible, and the presence of nucleo-somes obstructs the path of essential protein machineries. Inaddition, formation of inter-nucleosomal contacts and associ-ation of chromatin-related proteins lead to the assembly ofhigher-order structures, augmenting the accessibility barriersgenerated by nucleosomes alone (Tremethick 2007).
However, chromatin and the nucleosome structures withinit are highly dynamic, and by local manipulation of the com-ponents that comprise them, functionally distinct genomicregions, based on DNA accessibility, can be established. Im-portantly, it is this malleability that confers the central role ofchromatin as a mediator of all DNA-based processes. Thetiming and locality of chromatin changes are determined bythe coordinated action of several processes including DNAmethylation, ATP-dependent remodeling, incorporation ofhistone variants, and posttranslational modifications (PTMs)of the histone proteins, the sum of which determines thechromatin environment (Bonisch et al. 2008).
Until technological advancements made in the last decade,our knowledge of the latter process was limited to modifica-tions of the intrinsically disordered and flexible histone N-terminal tail domains, which protrude out of the nucleosomestructure (Luger et al. 1997). Such extension away from thenucleosome core makes the N-terminal tails not only amena-ble to experimental detection but also more accessible toenzymes that set and erase modifications, known respectivelyas writers and erasers. In addition, the exposure of residues inthese regions promotes the association of chromatin-relatedproteins, termed readers, that possess conserved domains ca-pable of recognizing and binding specific modifications and
A. P. M. Jack (*) : S. B. Hake (*)Adolf-Butenandt Institute, Department of Molecular Biology, LMUMunich, Schillerstrasse 44, 80366 Munich, Germanye-mail: [email protected]: [email protected]
S. B. HakeCenter for Integrated Protein Science Munich (CIPSM), Munich,Germany
ChromosomaDOI 10.1007/s00412-014-0465-x
which are responsible for the recruitment and assembly ofmulti-protein complexes (Jenuwein and Allis 2001; Tavernaet al. 2007). It is the overall balance of these complexes thateventually defines the function of specific genomic regions(Filion et al. 2010).
PTMs not only function as recruitment platforms but canalso act directly bymodulating the strength of histone-DNA orhistone-histone contacts, which are not only important fornucleosome assembly but also higher-order chromatin folding(Hansen 2002). Charge alteration by modifications such asacetylation and phosphorylation can result in the interferenceof electrostatic interactions, whereas methylation may inhibitthe formation of hydrogen bonds. Although the histone tailshave been shown to be important for higher-order chromatinassembly, affecting inter-nucleosomal interactions (Hansen2002), their protrusion away from the nucleosome limits theirparticipation in intra-nucleosomal interactions that occur at theheart of the nucleosome and are likely to be directly affectedby PTMs.
The conserved histone globular regions make up the coreof the nucleosome. Although once thought to be inaccessibleto histone modifying enzymes, technological advances haveled to the discovery that several residues within these regionscan in fact be modified and that these residues occupy func-tionally significant positions within the nucleosome (Freitaset al. 2004). These studies open up exciting new possibilitiesin chromatin regulatory processes and are reviewed below.
Localization of modified core residueswithin the nucleosome
H3K56 at the DNA entry/exit site
Although part of the core globular histone domain, lysine 56of histone H3 (H3K56) lies in the amino-terminal alpha helix(H3αN) (Fig. 1a), which is significantly less structured thanthe histone fold. Nevertheless, this region has been shown tobe important for stabilization of the nucleosome and is posi-tioned by the H2A docking domain to interact with the pen-ultimate 10 bp of DNA, at the DNA entry/exit site (Fig. 1b,top left) (Luger et al. 1997). In addition, in Saccharomycescerevisiae (referred to as yeast for the rest of the review), it hasbeen shown that ∼30 % of transcription factor binding sitesreside within this region (North et al. 2012). H3K56 is welldocumented as a site of acetylation (ac), occurring on ∼30 %of yeast histone H3 (Xu et al. 2005) and is predominantlyassociated with the incorporation of H3 during DNA replica-tion and repair (Chen et al. 2008; Li et al. 2008). In highereukaryotes, H3K56ac is far less abundant appearing on lessthan 1% of mammalian H3 (Xie et al. 2009). However, unlikein yeast, H3K56 can also be methylated (me) in these organ-isms, with monomethylation (me1) playing a key role in DNA
replication (Yu et al. 2012) and trimethylation (me3) likelyhaving a role in heterochromatin formation (Jack et al. 2013).Acetylation of H3K56 disrupts the normal water-mediatedinteraction between it and the DNA entry gyre (Davey et al.2002) and has been shown, by single-molecule fluorescenceresonance energy transfer (FRET), to increase the rate of DNAunwrapping by 7-fold as compared to the unmodified residue(Neumann et al. 2009). Although X-ray crystallography sug-gests that there is no change in the nucleosome structure orDNA conformation (Watanabe et al. 2010), this is compatiblewith the FRET data given that crystallization stabilizes theDNA in a “closed” conformation (Suto et al. 2003). It hasbeen shown that although there is extensive histone-DNAinteraction at the entry/exit site, these contacts are relativelyweak compared to those at the nucleosome dyad (Hall et al.2009). Loosening of the DNA, therefore, facilitates the bind-ing of proteins to the chromatin fiber by promoting a moreaccessible nucleosomal environment (Shimko et al. 2011). Itis conceivable that these proteins include chromatinremodelers (Xu et al. 2005), and it was previously reportedthat a H3K56Q mutation, chemically mimicking acetylationof this residue, thereby contributes modestly to nucleosomerepositioning (Neumann et al. 2009). Additionally, it had beenproposed that given the pivotal location of H3K56, its acety-lation might lead to destabilization of the nucleosome. Re-cently, however, these findings have been disputed (Shimkoet al. 2011; Simon et al. 2011).
There is also some controversy over the exact effectH3K56ac has on chromatin compaction in vitro. While it isgenerally accepted that H3K56ac has no effect on cis actinginteractions within individual nucleosomal arrays, discrepan-cies lie in the effects it has on trans array-array interactions(Neumann et al. 2009; Watanabe et al. 2010). One postulatedreason for this is that H3K56 acetylation may only disruptoligomerization of subsaturated arrays, whichmay better reflectthe in vivo local chromatin environment in which H3K56ac isfound, but such arrays were not tested in all studies. The use ofH3K56ac to decrease interactions between multiple arrays maybe a means of maintaining nucleosome-depleted regions(NDRs) of chromatin permissible for such functions as DNAreplication and repair (Watanabe et al. 2010).
Recently, using pulsed electron-electron double resonancespectroscopy coupled with site-directed spin labeling, thestructure of the (H3-H4)2 histone tetramer was investigatedand revealed that, while the H3-H3 interface retains a similarstructure as observed in a nucleosomal context, the H3αNextension is more heterogeneous, so that in this conformation,additional flexibility may enhance the likelihood of posttrans-lational modifications and further interactions with chromatin-associated proteins (Bowman et al. 2010). In yeast, H3K56 isprimarily acetylated by the lysine acetyltransferase (KAT)Rtt109 (Schneider et al. 2006), which requires the presenceof the histone chaperone Asf1 and therefore occurs on (H3-
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H4)2 tetramers before incorporation into chromatin. In mam-mals and flies, H3K56ac is mediated by the KATs, CBP/p300(Das et al. 2009), and GCN5 (Tjeertes et al. 2009).
In higher eukaryotes, H3K56 has also been identified, bymass spectrometry (MS), as a site of methylation (Garcia et al.2007; Zhang et al. 2003) and further in vivo data demonstrated
Fig. 1 a Sequence (NCBI Reference Sequence NP_002098.1) and sec-ondary structure of human histone H3.3 with core modified residues, forwhich there is more than just mass spectrometry data, highlighted inpurple (methylated and acetylated), blue (acetylated), and red (methylat-ed). Writers and erasers are displayed above and below the sequence,respectively, and the coloring of the surrounding box matches the mod-ification color-coding used for the residues. y and m denote yeast and
mammalian, respectively. b The crystal structure of the nucleosome (PDBID: 1AoI) (Luger et al. 1997). H3 is shown in blue, H4 in green, H2A inyellow, H2B in red, and DNA in gray. Discussed H3 core modifications(H3K56, H3K64, H3K79, and H3K122) are highlighted in cyan. Zoomedimages show H3K56 and the H2A docking domain (top left), H3K122(top right), H3K64 (bottom left), and H3K79 and the H4 basic patch(bottom right)
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the presence of me1 (Yu et al. 2012) and me3 (Jack et al.2013). Recent crystal structures, reconstituted with methylat-ed histones, show that although this type of modification isthought to induce less severe structural changes compared tocharge-altering modifications, it can still alter the capacity of aresidue to hydrogen bond and can change the conformation ofthe side chain (Lu et al. 2008). In this respect, methylation ofthis residue may alter the water-mediated hydrogen bondbetween H3K56 and the DNA backbone, although to date,there is no available experimental data to confirm this.
Adjacent to H3K56 is serine 57, which has been identifiedby MS as a site of phosphorylation (ph) (Aslam and Logie2010). Although there is currently no further evidence for thismark in vivo, it is imaginable that the presence of this markcould affect the binding of proteins at H3K56, acting as aphospho-methyl binary switch (Fischle et al. 2003).
H3 PTMs at the dyad
H3K122 acetylation is the most recent core modification to becharacterized and is found at the nucleosome dyad axis(Fig. 1b, top right), where the contact between histone andDNA is at its strongest (Hall et al. 2009). So far, this modifi-cation has only been characterized in higher eukaryoteswhere, like H3K56ac, it is deposited by CBP/p300 (Fig. 1a)(Tropberger et al. 2013). Similar to H3K56, addition of anacetyl group at H3K122 is likely to disrupt a water-mediatedcontact between it and the nucleosomal DNA (Iwasaki et al.2011). Interestingly, recent in vitro studies showed that unlikePTMs at the DNA entry/exit site, those at the dyad do notaffect DNA unwrapping but rather function by facilitatingnucleosome disassembly (Manohar et al. 2009; Simon et al.2011) and sliding (Flaus and Owen-Hughes 2003;Muthurajanet al. 2003). Given that salt-dependent nucleosome disassem-bly has recently been shown to begin with the “loosening” ofthe (H3-H4)2 tetramer and the (H2A-H2B) dimer interface,before (H2A-H2B) dissociation from the DNA (Bohm et al.2011), one could speculate that H3K122ac and other modifi-cations within the dyad axis facilitate this process. H3T118can be phosphorylated and sits between, although not directlyadjacent to, H3K122 and H3K115, the latter of which has alsobeen identified, by MS, as a site of acetylation. Mutations ofH3T118 fall into the class of SWI/SNF (switching and sucrosenon-fermentation) independence (SIN) histone mutations,which in yeast, can functionally compensate for the loss ofthe ATP-dependent chromatin remodeller SWI/SNF and areeither found near the dyad-DNA contact points or thetetramer-dimer packing interface (Kruger et al. 1995). Likethe other two dyad modifications, H3T118ph facilitates nu-cleosome disassembly and does not appear to affect DNAunwrapping in vitro (Simon et al. 2011). Interestingly, a recentstudy, in which the binding of nucleosome assembly proteins(NAPs) was assessed in the presence of different
modifications, showed that H3T118ph enhanced NAP-peptide interactions, while H3K122ac diminished them(Kumar et al. 2012). Engineered histone H3 containing site-specific genetically incorporated acetyl lysine (Neumann et al.2009) was, however, successfully assembled into chromatin,in vitro, using a combination of NAP1 and ATP-dependentchromatin assembly factor (ACF) (Tropberger et al. 2013).This suggests that further studies will be needed to delineatethe in vivo functions and relationship of these modifications.
H3K79 on the solvent-exposed nucleosome surface
H3K79 is found on the first loop of the histone H3 globulardomain (Fig. 1a) and is exposed on the solvent accessiblesurface of the nucleosome (Fig. 1b, bottom right). Unlike theDNA entry/exit point, this region does not contact the DNA;however, mutational analysis has shown that residues sur-rounding H3K79 are important for heterochromatic silencingin yeast (Park et al. 2002; Thompson et al. 2003). H3K79 canbe mono-, di-, and tri-methylated (me1, me2, me3) by theenzyme disruptor of telomeric silencing (Dot1) (Fig. 1a)(Lacoste et al. 2002; Singer et al. 1998; van Leeuwen et al.2002). This enzyme is conserved and in most organismsanalyzed to date and with the exception of trypanosomes(Janzen et al. 2006), the only enzyme identified as able tomediate this modification. In the case of H3K79, addition ofmethyl groups has been shown to disrupt a weak hydrogenbond between it and the L2 loop of histone H4 and it has beendemonstrated that the induced side chain rearrangementcauses the partial uncovering of a hydrophobic pocket linedby H3L82 and H4V70 (Lu et al. 2008). While such changesare small, alterations of the electrostatic potential and nucleo-somal surface may lead to a more cumulative effect. In addi-tion, the nucleosomal position of H3K79, in close proximityto the histone H4 N-terminal tail (Fig. 1b, bottom right), islikely to facilitate its methylation by Dot1, given that this H4region contains a stretch of basic residues necessary for Dot1methyltransferase activity (Altaf et al. 2007; Fingerman et al.2007). Furthermore, higher levels of H3K79 methylation aredependent on another trans-mediated interaction, yH2BK123/hH2BK120 ubiquitination (H2Bub). This modification is aprerequisite for Dot1-mediated H3K79me2 and me3 as wellas H3K4 methylation by COMPASS (Dover et al. 2002; Leeet al. 2007; Shilatifard 2006). Indeed, in vitro studies showedthat H2Bub could actually stimulate H3K79me (McGintyet al. 2008). The position of H2BK123, on the same solvent-exposed surface and in close proximity to H3K79, is likely tofacilitate such cross talk between the two modifications(Nguyen and Zhang 2011).
H3K79 has also been shown, by MS, to be acetylated inboth humans (Garcia et al. 2007) and yeast (Bheda et al.2012). In both organisms, H3K79ac occurs in low abundance
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and the only regulatory data that exists is in yeast, where Sir2has been shown to catalyze its removal (Fig. 1a).
H3K64 on the lateral surface
H3K64 is located at the beginning of the α1-helix (Fig. 1a)and tri-methylation of this residue is indirectly mediated, bythe known H3K9 methyltransferases, suppressor of variega-tion 39 homologue 1 (SUV39h1) and 2 (SUV39h2) (Daujatet al. 2009). This residue is located on the lateral surface of thenucleosome (Fig. 1b, bottom left), and the crystal structure ofthe nucleosome core particle (NCP) shows that it contacts thenucleosomal DNA via hydrogen bonds between the main-chain amide nitrogen of H3K64 and the phosphates of theDNA backbone (Iwasaki et al. 2011). In addition, the aminogroup of H3K64 may participate in stabilizing the H3 α1 andα2 helices, via water-mediated hydrogen bonds. Consideringthe addition of methyl groups can alter hydrogen bonding, theaforementioned interactions may be disrupted, althoughmodeling shows steric complementarity between the methylgroup and the nucleosomal DNA (Daujat et al. 2009). BothMS data (Garcia et al. 2007) and antibody detection (Di Cerboet al. 2014) show that this residue can be acetylated in mam-mals. Like H3K56ac and H3K122ac, H3K64ac is catalyzedby CBP/p300 (Fig. 1a) (Di Cerbo et al. 2014). Crystal struc-tures of the NCP containing H3K64Q show that while thepresence of acetylation is not likely to affect hydrogen
bonding to the DNA backbone, it may alter the interactionswith the H3 α1 and α2 helices as a result of a significantchange in side-chain orientation (Iwasaki et al. 2011). Thesefindings are somewhat contradicted by recent FRET studies,which were used to analyze the stability of nucleosomal DNAinteractions in nucleosomes containing chemically incorpo-rated acetyl lysine at K64 (Di Cerbo et al. 2014). Using thissecond technique, it appears that H3K64ac does in fact neg-atively impact the stability of DNA-histone interactions,reflecting possible limitations of using K to Q substitutionsin some assays. Interestingly, the latter study also showed thatthis modification facilitates ATP-dependent chromatin remod-eling in an enzyme-specific manner, behaving, in this respect,differently from H3K56ac.
Genome-wide localization of H3 core modificationsand their functional implications
As well as occupying important positions within a nucleo-some and thereby possibly influencing its structural integrity,the discussed core histone modifications are distributed in anonrandom manner throughout the genome (Table 1).
In this next section, we integrate the in vitro data on H3core modifications with information on their genomic distri-bution. Although we focus primarily on the impact of coremodification localization on transcription, the principles of
Table 1 Summary of histone H3 core modification distribution at genomic elements
PTM Chromatintype
Promoter/TSS region
Genebody
Enhancer Telomere(inc. subtelomere)
Centromere(inc. pericentric)
References
H3K56ac E, FH ++ + ? (Xie et al. 2009; Xu et al. 2005)
H3K56me1 RFa (Yu et al. 2012)
H3K56me3 CHa +a (Jack et al. 2013)
H3K64ac Ea ++a +a (Di Cerbo et al. 2014)
H3K64me3 CHa +a ++a (Daujat et al. 2009; Lange et al. 2013)
H3K79ac ? (Bheda et al. 2012)
H3K79me1 E, FH + + + (Barski et al. 2007; Liu et al. 2011;Steger et al. 2008; Wang et al. 2008)
H3K79me2 E, FH, CHa + ++ +a +a (Barski et al. 2007; Im et al. 2003; Joneset al. 2008; Liu et al. 2011; Ng et al.2003; Schubeler et al. 2004; Schulzeet al. 2009; Steger et al. 2008;Wang et al. 2008)
H3K79me3 E, FH + ++ (Barski et al. 2007; Liu et al. 2011; Pokholoket al. 2005; Schulze et al. 2009; Steger et al.2008; Vakoc et al. 2006; Wang et al. 2008)
H3K122ac Ea ++a +a (Tropberger et al. 2013)
Presence of a modification at an element is represented by (+) and when present in more than one element (++) reflects the location with the mostsupporting data
E euchromatin, FH facultative heterochromatin, CH constitutive heterochromatin, RF replication focia Indicates that the presence of the modification at these regions has only been detected in mammals
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how they mediate this process can be extended to and arecompatible with postulated roles in other important cellularfunctions.
Core modifications at promoter and enhancer regions
The chromatin organization within distinct regions associatedwith any particular gene is a key in defining its transcriptionalplasticity and output. At gene promoters, for example, thedensity and positioning of nucleosomes determines the acces-sibility of essential cis-regulatory elements, which can eitherbe exposed or occluded, facilitating or hindering, respectively,events such as transcription factor binding. Likewise, nucleo-some arrangement around the transcriptional start site (TSS)and in the body of genes influences processes such as RNApolymerase II (RNAPII) recruitment and transcriptional initi-ation, elongation, and termination (Li et al. 2007; Pokholoket al. 2005).
Promoters of actively transcribed genes are associated witha high nucleosome turnover and are typically enriched inhistone acetylation. Correlating this knowledge with thein vitro properties of H3K56ac, H3K122ac, and H3K64ac, itis not surprising that these three core modifications are mostabundant within these regions (Fig. 2 and Table 1) (Di Cerbo
et al. 2014; Tropberger et al. 2013; Xie et al. 2009; Xu et al.2005). One could speculate that the increased DNA breathingH3K56ac confers at the DNA entry/exit site facilitates thepreferred binding of transcription factors close to these regionsor helps the invasion by RNAPs (Luger 2006). Profiling ofH3K122ac-containing nucleosomes showed they are enrichedfor H3K56ac as well as other hallmarks of active transcription,but not H3K36me3 (Tropberger et al. 2013), which is typical-ly found at the 3′end of actively transcribed genes and isassociated with elongation (Pokholok et al. 2005). Thesefindings substantiate genome-wide profiling data ofH3K122ac, which showed that its distribution is limited tothe flanking regions of the TSS (Fig. 2), similar to H3K27acand H2A.Zac. Likewise, H3K64ac is also enriched around theTSS (Fig. 2) of active genes and is anticorrelated with repres-sive marks (Di Cerbo et al. 2014). H3K56ac has also beenshown to overlap with H2A.Z at vertebrate promoters. Inter-estingly, recent findings show that H3K56Q-containing nu-cleosomes enhance the replacement of H2A.Z with H2A(Watanabe et al. 2013), indicating that H3K56ac may prepareH2A.Z nucleosomes for exchange. H3K122ac levels are pro-portional to the amount of messenger RNA (mRNA) expres-sion (Tropberger et al. 2013), suggesting a role in transcrip-tional activation. The correlation to transcriptional activation
Fig. 2 Schematic diagram depicting the major chromosomal distributionpatterns of histone H3 core modifications. The distribution patterns ofH3K122ac and H3K64ac (blue), H3K56ac (cyan), H3K79me (purple),H3K64me3 (bright red), and H3K56me3 (dark red) at common chromo-somal features including the enhancer (light gray), promoter (mid-gray),and gene body (dark gray) of an active gene, telomeres (short, off-whitearrows) and centromeres (long, off-white arrows). Distributions are
generalized, based on genome-wide, single locus and immunofluores-cence studies from different organisms and give a broad overview ofhistone H3 core modification genomic localization. Not depicted aremore minor or species-specific distribution patterns, which are discussedin the main text. The general regions of a chromatid at which thechromosomal features are found are shown on the right
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is interesting, given the promotion of nucleosome disassemblywhen H3K122 is acetylated (Simon et al. 2011). One couldspeculate that H3K56ac allows limited access to a smallregion of nucleosome-obscured DNA, requiring relativelylittle energy, given the weaker DNA-histone contacts at thesesites, andmaintaining it in a poised state. Indeed, experimentalevidence implicates H3K56ac in nucleosome disassemblyduring transcription (Williams et al. 2008). When the condi-tions favor transcriptional activation, acetylation at H3K122could act as a switch to reinforce the signal and facilitate themore energy-demanding nucleosome disassembly or nucleo-some sliding required for promoter clearance. The latter pointis substantiated, by the confirmation of a direct function forH3K122ac in transcriptional activation, by the group of R.Schneider (Tropberger et al. 2013). Using an in vitro tran-scription assay, they showed that unlike the tail modificationH3K18ac, H3K122ac alone could stimulate transcriptionfrom a chromatin template. Furthermore, histone evictionexperiments demonstrated that nucleosomes displayingH3K122ac were more susceptible to displacement, reiteratingthe likely mechanism by which it functions. Given the nega-tive effects, H3K64ac has on nucleosome stability and itscorrelation with transcriptional activation (Di Cerbo et al.2014), it will be interesting, in future studies, to assess anycross talk that may occur between these modifications. Therole of H3K56ac in the proposed hypothesis, as a mechanismof opening up chromatin but not activating transcription, issupported experimentally. In both yeast and humans,H3K56ac is found at some repressed genes and regions ofDNA repair and therefore does not necessarily correlate withmRNA expression levels (Chen et al. 2008; Xie et al. 2009).
Genome-wide profiling in human embryonic stem (hES)cell lines showed that genes associated with the highest levelsof H3K56 acetylation include almost all canonical histonegenes, similar to a published study in yeast (Xu et al. 2005).Furthermore, in hES cells, H3K56ac was also found to asso-ciate with many pluripotency regulators, such as Nanog,Sox2, and Oct4 (NSO) (Xie et al. 2009). The recent findingthat H3K56ac and Oct4 interact directly, both in vitro andin vivo (Tan et al. 2013), suggests a direct mechanism bywhich these factors could be recruited to specific regions andalso highlights the accessibility of H3K56 to binding proteins.The latter point is further substantiated by the finding thatduring G1 phase of the cell cycle, H3K56me1 acts as achromatin docking site for PCNA, thereby facilitating DNAreplication (Yu et al. 2012). Interestingly, upon differentiation,H3K56ac is redistributed to the promoters of genes involvedin development, such as theHOX genes, and those involved insomatic cell maintenance (Xie et al. 2009). In support of thesefindings, a study in mature adipocytes also found H3K56acadjacent, but not overlapping, to some transcription factorbinding sites as well as hyperacetylation of this residue atdevelopmental genes (Lo et al. 2011). Again, this supports a
role for H3K56ac in maintaining transcriptional plasticityrather than mRNA levels per se.
Findings by the group of B. Ren suggest that cell type-specific modification patterns at enhancer regions play a ma-jor role in driving differences in gene expression profilesassociated with cell fate decisions (Heintzman et al. 2009). Ithas been postulated that the modification pattern of specifi-cally placed nucleosomes may act to display transcriptionfactor binding sites (He et al. 2010). H3K4me1/me2 enrich-ment is associated with enhancers and is often coupled withH3K27me3 if the gene that it is regulating is repressed or withH3K27ac when the gene is activated (Creyghton et al. 2010;Heintzman et al. 2007). In keeping with a similar distributionto H3K27ac, genome-wide analyses of H3K122ac andH3K64ac showed that both are also present at active enhancerregions (Di Cerbo et al. 2014; Tropberger et al. 2013). This iscompatible with a role in transcriptional activation, wherebythe presence of H3K64ac at enhancers likely decreases nucle-osome stability and H3K122ac facilitates nucleosome disas-sembly, thereby permitting the binding of transcriptional acti-vators to cis-regulatory sites.
Previous work in murine ES cells (mES) has shown thatNSO co-occupancy at specific genomic regions is indicativeof enhancer activity and that these factors are able to recruitp300, an acetyltransferase associated with H3K56ac,H3K122ac, and H3K64ac (Chen et al. 2012). More recently,super-enhancers have been discovered, which regulate thetranscription of master regulator genes that control cell iden-tity (Whyte et al. 2013). In mES cells, these super-enhancersconsist of clusters of enhancers, which are densely occupiedby NSO and have high levels of the mediator co-activatorcomplex (Whyte et al. 2013). Given previous data showingco-localization of H3K56ac with NSO, the fact that p300 cancatalyze addition of this modification and the established roleof H3K56ac in ES cell identity, one might speculate that thismodification could also be a mark of super-enhancers and thatits misregulation could have deleterious effects on cellspecification.
Taken together, the presence of acetylation within the his-tone H3 core at promoters and enhancers seems to function bypromoting an “open” and binding-permissive chromatin con-formation. While the nucleosomal changes induced byH3K56ac do not seem to be strong enough to direct transcrip-tion alone, it appears that this modification may mark specificregions that respond to regulatory cues, for example, duringdifferentiation. In addition, it is directly involved in proteinrecruitment. The stronger effects of H3K64ac and H3K122ac,on the other hand, likely serve as a switch to commit tonucleosome disassembly and transcriptional activation. Giventhe long-range chromatin interactions between promoters andenhancers and the presence of these modifications on bothelements (Tolhuis et al. 2002), it will be interesting to see ifthese three core marks have additional roles in facilitating
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chromatin looping and the selective association of specificregulatory regions.
Core modifications in the gene body
Unlike H3K122ac, H3K56ac is not only enriched at thepromoters but also extends into the gene body of highlytranscribed genes (Fig. 2) (Schneider et al. 2006), suggestingthat while H3K122ac plays a role in transcriptional initiation,H3K56ac may also function in proximal promoter pauserelease. Although nucleosome occupancy in gene bodies ishigh compared to promoter regions, they must retain a level ofdynamicity in order to allow chromatin disassembly at thetranscriptional machinery and reassembly following its pas-sage along the gene (Schwabish and Struhl 2004). WhereasH3K122ac appears to function solely in nucleosome disas-sembly, there is evidence that H3K56ac also functions innucleosome assembly (Chen et al. 2008; Li et al. 2008),providing a possible reason for the presence of this modifica-tion within the gene body of highly transcribed genes(Rufiange et al. 2007). Interestingly, neither recruitment ofpre-initiation complex components to promoters of activelytranscribed genes nor the presence of RNAPII within thecoding sequences was affected in Asf1-mutant yeast strains,in which H3K56ac levels were diminished (Schneider et al.2006). Transcriptional repression of genes within heterochro-matic loci has been suggested to be regulated at the level ofelongation rather than initiation considering the successfulrecruitment and binding of transcriptional activators, compo-nents of the pre-initiation complex and RNAPII to promoterswithin these regions (Sekinger and Gross 1999, 2001). Rtt109or Asf1 yeast mutant strains as well as H3K56R substitutioninhibit transcription at a heterochromatinized locus. In con-trast, the H3K56Q substitution is able to restore transcriptionin the Rtt109 mutant, reiterating that the presence of this markis important in allowing RNAPII progression (Varv et al.2010).
Although H3K56ac and RNAPII overlap (Schneider et al.2006), the finding that RNAPII is still present at codingsequences in Asf1 mutants (Schneider et al. 2006) suggeststhat H3K56ac is not directly involved in its recruitment totranscribed regions. At these sites, it could therefore functionby promoting the progression of RNAPII by loosening thenucleosomal DNA and/or by recruiting a factor that has notyet been identified.
In vitro data suggesting that H3K56ac can destabilize the(H3-H4)2 tetramer but not the nucleosome indicate that(H3K56ac-H4)2 tetramers favor assembly with (H2A-H2B)dimers in order to form a more stable complex or they areotherwise disassembled (Bowman et al. 2010). Consideringboth processes occur at transcriptionally active loci, it is hardto decipher the exact role of H3K56ac within these regions.One could postulate that the presence of other histone
modifications and different histone variants could favor theone or the other outcome.
H3K79 methylation is the core PTM most well known forits enrichment in the gene body (Fig. 2 and Table 1). At thesesites, it is associated with the presence of RNAPII, suggestinga role in transcriptional elongation (Bitoun et al. 2007;Mueller et al. 2007; Wang et al. 2008). In addition, withinthese regions, H3K79me2 and me3 overlap with the presenceof Dot1 (Steger et al. 2008). This is not surprising given therequirement of several trans-histone interactions for Dot1-mediated H3K79 methylation and therefore the targeting ofthis enzyme to chromatin-incorporated rather than soluble H3.Dot1 has been found as part of several elongating complexes(summarized in Nguyen and Zhang 2011), consistent with theoverlap of H3K79me and RNAPII and a role in gene regula-tion. A recent study showed that Dot1-like methyltransferase(Dot1L), the mammalian homologue of Dot1, can directlybind the phosphorylated C-terminal domain (CTD) ofRNAPII. Interestingly, this interaction occurs through a regionon Dot1L that is unique to the multicellular eukaryotes (Kimet al. 2012), reflecting the possible evolution of a morestreamlined process. In addition to its histone methylationroles, Dot1 has recently been proposed to function in otherways (Ooga et al. 2013; Stulemeijer et al. 2011) and thereforecorrelations with this methyltransferase may not always serveas a means of analyzing H3K79me actions.
Given that all three H3K79me states share a common,nonprocessive methyltransferase, there has been some debateover the individuality of their functions. On the one hand, ithas been shown that Dot1 works in a distributive manner andthat all states are functionally redundant (Frederiks et al.2008); however, the observation that H3K79me2 and me3are differentially distributed in yeast (Schulze et al. 2009)suggests otherwise. The latter finding is not only intriguingin terms of functional implications but also in terms of how thetwo modifications are independently regulated. Firstly, fromthe functional perspective, one could imagine that modifiedH3K79 behaves as a binding platform, given that in vitro datasuggests methylation of this residue induces a more flexibleside-chain conformation (Lu et al. 2008), likely facilitatingassociation of this solvent-exposed residue with potentialbinding partners. However, the accessibility of H3K79 withinpolynucleosomes is still unclear. Although, so far, there islittle data on H3K79me binding partners, it is conceivable thatsuch interactions are dependent on the extent of methylation,with H3K79me2 recruiting a different repertoire of proteinscompared to H3K79me3, thereby establishing different func-tional domains. Secondly, from the regulation perspective, thefinding that Dot1 is present in multiple complexes makes itplausible that the differential distribution of H3K79me2 andme3 is established by the presence, or lack, of other complexcomponents that may be involved in H3K79me. Furthermore,H3K79me3 but not H3K79me2 has been found to overlap
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with yH2BK123ub, suggesting the presence of the latter markmay influence localization. Interestingly, in Caenorhabditiselegans, Dot1 forms a negative feedback loop by opposingH2Bub, thereby reducing RNAPII transcription through po-lymerase pausing (Cecere et al. 2013), indicating the need tobetter understand the molecular aspects of correlative studies.
Both yH2BK123ub and H3K79me3, but not H3K79me2,often associate with longer genes (Schulze et al. 2011). Likethe deposition of H3K36me3 at the 3′end of genes, it could bethat the presence of H3K79me3 and yH2BK123ub preventaberrant transcription in the wake of RNAPII. Indeed, there isalready some evidence that H2Bub functions in this respect bypromoting the assembly of nucleosomes (Cecere et al. 2013;Feng et al. 2002). What is compelling is how these modifica-tions could function in both transcriptional elongation, wherenucleosome turnover must take place, as well as cryptic tran-scription, where it must be prevented.
In yeast, over 90 % of total H3 is methylated at K79 withme3 making up the majority (van Leeuwen et al. 2002). InmES cells, this modification is far less abundant, and the vastmajority of the 11% of H3K79 that is methylated harbors me3and to a lesser extent me2 (Garcia et al. 2007; Jones et al.2008). This is interesting in light of the recent ChIP-seq datafrom mouse adipocytes, which provides evidence that inmammals, the conversion of H3K79me1 to H3K79me2/3correlates with a transition from low to high level transcription(Steger et al. 2008). Considering, under normal growth con-ditions, most of the yeast genome is transcribed (Harrisonet al. 2002), differences in gene density and transcriptionalactivity could provide an explanation for the organismal dif-ferences with regards not only to the total levels of H3K79mebut also the abundance of the individual states.
Most of the data linking H3K79 methylation to transcrip-tional elongation come from correlative studies, and it is hardto say whether it is a cause or consequence. There is continualdebate over whether this modification facilitates transcription-al activation or whether it plays a role in repression. Whilesome of the earlier discrepancies have been accounted for bytechnical differences in ChIP protocols, there remains consid-erable controversy (Barski et al. 2007; Buttner et al. 2010;Pokholok et al. 2005; Wang et al. 2008; Zhang et al. 2006).Although strong correlations between H3K79me and tran-scriptional activation fit with the finding that Dot1 is moreabundant at the transcribed regions of active genes comparedto inactive (Schubeler et al. 2004; Steger et al. 2008; Vakocet al. 2006), H3K79me has also been found at some repressedregions (Liu et al. 2011; Shahbazian et al. 2005; Zhang et al.2006). In addition, disruption of Dot1L in mice does not affectall transcriptional pathways (Ho et al. 2013), suggesting that ifit does play a role in mediating gene expression, this occurs ina targeted manner. Unlike H3K79me2/me3, which are foundin proximity to the TSS, H3K79me1 covers a broader gene-associated area extending both upstream and downstream of
regions enriched for Dot1 (Steger et al. 2008) in mouseadipocytes. This suggests that while H3K79me2/3 plays arole in the early steps of elongation, H3K79me1 has a differ-ent function. Although there is some overlap with H3K4me1,which has previously been shown to associate with enhancers,the presence of H3K79me1 does not appear to be a generaldemarcation of these regulatory elements (Wang et al. 2008).However, its co-localization with some transcriptional activa-tors at their binding sites has been shown, indicating that itstargeted deposition within certain intergenic regions may playa gene-specific role (Steger et al. 2008; Wang et al. 2008).
H3K79me1 has also been found at some poised genes(Steger et al. 2008), suggesting that it may function to setthe stage for gene activation in response to specific cues.Consistent with the presence of H3K79 methylation at theseregions, RNAPII is also known to accumulate at some tightlyregulated genes, such as human c-myc (Bentley and Groudine1986) and Drosophila hsp70 (Gilmour and Lis 1986), underrepressive conditions. During the process of differentiation,dynamic changes in H3K79me2/3 within transcribed regionshave been shown, which parallel changes in mRNA levels(Steger et al. 2008;Wang et al. 2008), suggesting that togetherthe three modification states may also serve specific functionsin cell specification.
Core modifications at repeat elements: telomeresand centromeres
The formation and confinement of genomic silencing at con-stitutive heterochromatin is crucial for the maintenance ofgenomic integrity and has been shown to be partly dependenton histone H3 core modifications.
Despite the high abundance of repeat sequences and lowlevel of transcription within these regions, they are not func-tionally inert. Telomeres, for example, cap eukaryotic chro-mosome ends, preventing their recognition as sites of DNAdamage and play an essential role in limiting the loss ofprotein-coding regions, which would otherwise occur as aresult of the end replication problem (Blackburn 1991). Al-though similar to telomeres in their dense nucleosomal pack-ing and epigenetic features, centromeres serve a very differentfunction and are responsible for assembly of the kinetochore,which serves as the attachment point for the mitotic spindleand is required for proper sister chromatid segregation duringmitosis (Westhorpe and Straight 2013). In both mentionedstructures, as well as other silenced genomic regions, therepressive, heterochromatic architecture is essential for themaintenance of genomic stability, preventing rearrangements,which might otherwise occur between the highly similar se-quences (Grewal and Jia 2007). In higher eukaryotes, thisarchitecture is promoted by several epigenetic features includ-ing DNA methylation, which induces tighter wrapping of theDNA around the nucleosome (Lee and Lee 2012) and
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H3K9me3, which recruits the chromo-domain protein, hetero-chromatic protein 1 (HP1) (Grewal and Jia 2007). As a resultof its compact conformation, any genes within constitutiveheterochromatin will be poorly expressed, a trait which canspread into adjacent, non-repetitive regions through a phe-nomenon known as position effect variegation (PEV)(Henikoff 1990).
H3K79 methylation has known functions in telomere si-lencing, and in the yeast genome, it is specifically excludedfrom these and other silenced regions (Stulemeijer et al. 2011;van Leeuwen et al. 2002). Indeed, mutation of Dot1 methyl-transferase activity or substitution of H3K79 both lead tosevere silencing defects (Ng et al. 2002; Singer et al. 1998;van Leeuwen et al. 2002). In several of the studies cited, thesedefects were measured by activation of a URA3 gene inte-grated in a region that is normally silenced, for example closeto the telomere. This technique, however, has recently re-ceived some criticism since it was discovered that the silenc-ing defects resulting from mutation of some proteins, includ-ing Dot1 and PCNA, are actually determined by unevennucleotide metabolism at the URA3 promoter rather than theeffects of the tested proteins themselves (Rossmann et al.2011). Despite these novel insights, a recent study monitoringchanges at natural telomeric genes in H3K79-methylationdefective mutants showed that this modification is still impor-tant for the regulation of some coding sequences (Takahashiet al. 2011), although overall effects seem to be milder thanformerly predicted. In addition, when other pathways arecompromised, the role of Dot1 in natural silencing may be-come more apparent (van Welsem et al. 2008), highlightingthe need for further investigation.
In yeast, silencing is mediated by the assembly of thesilencing information regulator (SIR) complex, consisting ofthe H4K16 deacetylase Sir2 together with Sir3 and Sir4 (Luoet al. 2002; Moretti et al. 1994). Mutation of any of theseproteins leads to a complete loss of silencing (Aparicio et al.1991). Several lines of evidence suggest H3K79me and Sir3share an antagonistic relationship. Firstly, Sir3 and Dot1 bothbind the same short basic patch of the histone H4 tail (Altafet al. 2007; Fingerman et al. 2007) and therefore compete forthis site. Secondly, methylation at H3K79 disrupts contactsbetween the bromo-adjacent homology (BAH) and AAA+domains of Sir3 and the lateral surface of the nucleosome(Armache et al. 2011; Ehrentraut et al. 2011). In this regard,H3K79me appears to function by altering the binding affinityof certain proteins rather than causing direct effects on thenucleosome structure. It was postulated that H3K79me distri-bution throughout most of the yeast genome prevents Sirbinding, causing its localization to discrete, silenced regions(Ng et al. 2003; van Leeuwen et al. 2002).
In higher eukaryotes, there is very little mechanistic infor-mation on how H3K79me functions. However, H3K79me2marks a distinct set of replication origins in the human (Fu
et al. 2013) and trypanosome genome (H3K76me2), and it hasbeen shown that in Trypanosoma brucei overexpression ofDot1A, one of two DOT1 homologs causes continuous repli-cation of nuclear DNA (Gassen et al. 2012). In humans, thesedata favor the view that H3K79me2’s association with theseorigins and replicated chromatin during S-phase may play arole in preventing re-replication, thereby preserving genomestability. In trypanosomes, however, H3K76me1/2 occurs ex-clusively after replication and most likely initiates licensing,raising interesting questions regarding functional conserva-tion. Several mammalian proteins contain a BAH domainsimilar to Sir3, raising the possibility that methylation ofH3K79 may alter the binding of these proteins. In highereukaryotes, several of the proteins harboring the BAH mod-ule, including ORC1, are important for replicative events(Callebaut et al. 1999). In addition, the BAH domain is partof the mouse DNA methyltransferase 1 (DNMT1) enzymeand may be involved in its recruitment to the replicationorigins, suggesting a functional link between DNA methyla-tion and replication (Callebaut et al. 1999). Finally, MTA1, asubunit of the repressive, HDAC-containing NuRD complex,also contains a BAH domain and one could speculate thatmethylation of H3K79 could disrupt a nucleosomal interac-tion and hinder targeted chromatin remodeling and histonedeacetylation within specific regions.
In mammals, H3K79me2 is present throughout heterochro-matic regions (pericentric, centromeric, telomeric, and sub-telomeric) (Table 1) (Jones et al. 2008); however, its absencein Dot1L mutant cells leads to a general reduction in hetero-chromatic marks including H4K20me3 and H3K9me2 (butnot H3K9me3) and an increase in H3K9ac. As a functionalconsequence, loss of Dol1L activity resulted in aberrant telo-mere elongation through activation of the alternative length-ening of telomere (ALT) pathway (reviewed in Cesare andReddel 2010), a telomerase-independent mechanism ofcounteracting the end replication problem. The effects ofdysfunctional Dot1L on telomere length appear to be con-served as similar alterations have been observed in yeast Dot1mutant strains (Singer et al. 1998). As the authors point out,this finding is intriguing given the presence of H3K79me-containing nucleosomes at mammalian telomeres and com-plete lack of nucleosomes at yeast telomeres. Moreover, theresults in this study point to Dot1L playing a promotional rolein heterochromatin maintenance, which seems counterintui-tive to its link with transcriptional activation and the enrich-ment of H3K79me in euchromatin. Possible functions forDot1L in heterochromatin maintenance could be mediatingthe expression and/or distribution of heterochromatin-associated factors, or effects might result from changes to asyet unknown nonhistone Dot1L targets.
In addition to alterations in H3K79me levels, H3K56 sub-stitutions in yeast, also lead to silencing defects, especially atthe telomeric regions (Hyland et al. 2005; Xu et al. 2007). The
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silencing effects are, however, neither due to altered Sir pro-tein recruitment or spreading nor due to changes in otheracetylated residues, such as H4K16 (Xu et al. 2007), althoughit may facilitate slight loosening of Sir binding (Oppikoferet al. 2011). In yeast, H3K56 deacetylation is globally medi-ated by the HDACs Hst3 and Hst4 (Celic et al. 2006; Maaset al. 2006), mutation of which leads to defects in telomericsilencing as a result of hyperacetylation. This occurs despiterecruitment of Sir2 (Yang et al. 2008). Although Sir2 itself haspreviously been implicated in H3K56 deacetylation, it ispossible that alone it is not able to compensate for the lossof Hst3 and Hst4. In addition, its role in H3K56 deacetylationis controversial and there are several reports presenting con-flicting data (Oppikofer et al. 2011; Xu et al. 2007; Yang et al.2008). It has recently been shown that HDACs mediate thestability of heterochromatin through the suppression of his-tone turnover (Aygun et al. 2013) and given that H3K56ac isconducive to DNA unwrapping at the entry/exit site of thenucleosome, removal of this modification may facilitate thisprocess by inducing a more closed conformation at these sites.A similar mechanism is likely to be used in higher eukaryotes,given that the HDACs SIRT1 and SIRT6, which can bothdeacetylate H3K56, have been shown to be associated withmammalian telomeres and to be important for their integrity(Das et al. 2009; Gil et al. 2013; Michishita et al. 2008;Palacios et al. 2010).
Not only does it appear that regulation of H3K56 modifi-cation is important for maintaining a compact conformation atthe telomeres but there is also evidence that it is involved inthe localization of the telomeres to the nuclear peripheryduring yeast DNA replication (Hiraga et al. 2008; Tanakaet al. 2012). It is thought that tethering of heterochromaticloci to the nuclear lamina contributes to their transcriptionalstate (reviewed in Kind and van Steensel 2010) and may beimportant for DNA damage repair (Therizols et al. 2006), twoprocesses with which H3K56ac is linked. Interestingly, K56Ror K56Q mutants showed similar localization defects, indicat-ing that cycling between the acetylated and non-acetylatedform is likely to be important for this function. It has recentlybeen shown that replication triggers the release of telomeresfrom the nuclear periphery (Ebrahimi and Donaldson 2008)and this must presumably occur for nucleosome disassemblyto take place. This process is mediated by suppression of Ku-mediated anchoring (Laroche et al. 1998). Ku is a heterodimercomplex (Ku70/Ku80) that is involved in nonhomologousend joining (NHEJ) (reviewed in Daley et al. 2005) and hasrecently been implicated in the clustering of Tfretrotransposon elements at Schizosaccharomyces pombe cen-tromeres, a process which appears to be disrupted byH3K56ac due to its interference with Ku localization at theseregions (Tanaka et al. 2012). It is feasible, therefore, that at thetelomeres, lack of H3K56ac is permissive to Ku-mediatedtethering, while the presence of this mark facilitates the
disruption of this interaction, which is necessary for the rep-lication to take place. Considering Ku and H3K56ac are bothinvolved in other events such as DNA damage repair, it islikely that these factors form an intimate relationship andtogether ensure correct timing of nucleosome assembly anddisassembly. Moreover, it is likely that this relationship isevolutionary conserved and extends to humans (Michishitaet al. 2008; Myung et al. 2004). Further investigation isrequired to decipher if the effects of H3K56ac on Ku associ-ation are direct or if other factors are involved.
While H3K56ac is largely excluded from constitutive het-erochromatic regions, HeLa cell metaphase chromosomespreads show a specific localization of H3K56me3 to thecentromeres, although surprisingly not the telomeres (Fig. 2and Table 1) (Jack et al. 2013). In mammals, the DNA-PKcomplex, containing the Ku and the catalytic subunit of DNAdependent protein kinase (DNA-PKcs), has recently beenimplicated in normal cell cycle progression through mitosis(Lee et al. 2011), where it is thought to modulate chromosomealignment and the meta-to-anaphase transition. It would beinteresting to see if, in mammalian systems, H3K56ac is alsorefractory to Ku binding. If this is the case, then it may be thatconcentration of mammalian H3K56me3 at the mitotic cen-tromeres serves to prevent acetylation within these regions,allowing recruitment of DNA-PK and successful alignment ofchromosomes before segregation, although this is purely spec-ulative. The fact that little H3K56me3 is found at telomeressuggests that it plays a specialized role at centromeres; how-ever, considering it appears to be regulated by the sameenzymes as H3K9me3, it will be important to find out howthese differential patterns are established. One hypothesis isthat if H3S57ph exists in vivo; this could occur in a telomere-specific manner and function by, for example, recruitingH3K56me3 erasers. Another explanation is that since allstudies use antibodies, it is possible that a nearby phosphory-lation mark occludes the H3K56me3 antibody epitope andtherefore some regions appear devoid of the methyl mark. Inaddition, H3K56me3 may be regulated by other enzymes thathave not yet been identified and which may act independentlyof H3K9me3, leading to differential patterning of the twomodifications at specific loci. Indeed, although G9a has beenshown to regulate H3K56me1, this modification is still de-tectable in G9a−/−cells, supporting the idea that other KMTscan regulate this modification in vivo (Yu et al. 2012). To date,there is unfortunately no available genome-wide data for thismodification, possibly due to its low abundance and technicaldifficulties with analyzing repeat sequences.
H3K64me3 is found at constitutive heterochromatin, andChIP-qPCR showed that it is enriched on repetitive DNA,including pericentromeric heterochromatin (Fig. 2 and Table 1)(Daujat et al. 2009). Interestingly, this localization does notrequire DNA methylation, consistent with other tail modifica-tions in these regions, nor is it affected by HP1 localization
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(Lange et al. 2013). Instead, there appears to be cross talkbetween H3K64me3 and H3K9me3. Suv39h1/h2 catalyzeshigher degrees of H3K9me, and knockout of both enzymescauses a severe reduction in not only this modification but alsoH3K64me3. An elegant experiment by Lange et. al., in whichH3K9me3 was artificially recruited to pericentromeric hetero-chromatin in Suv39h1/h2 double knockout cells, showed res-toration of H3K64me3 at these regions. This indicates a de-pendency of H3K64me3 on H3K9me3 rather than on theSuv39hs (Lange et al. 2013). Interestingly, in mouse NIH3T3cells, overexpression of H3 in which K64 was substituted toarginine (R) resulted in a reduction of PTMs and factorsrepresenting hallmarks of constitutive heterochromatin, includ-ing H3K9me3, H4K20me3, and HP1, indicating an importantrole for H3K64me3 in heterochromatin maintenance.
Mislocalization of core modifications and disease
Given the importance and implications of histone core mod-ification genomic localization, it is not surprising that theirmislocalization is implicated in several pathological process-es. The occurrence of abnormal fusion proteins through geno-mic rearrangement is a common feature of many cancers. Inover 70 % of infant leukemias, for example, the 5′-region ofthe mixed lineage leukemia (MLL) gene is fused to varioustranslocation partners, many of which are involved in tran-scriptional initiation and elongation (reviewed in de Boer et al.2013). In addition to other fundamental cellular processes,MLL is involved in transcriptional control of specific geneswithin the developmentally regulatedHox cluster (Milne et al.2002). Among the MLL fusion partners are CBP/p300 andseveral elongating complex members which are capable ofinteracting with Dot1. Fusion-facilitated mistargeting of thesehistone-modifying activities alters the pattern of PTMs atspecific regions, such as the Hox genes, resulting in aberrantgene expression. In this regard, altered H3K79me patterns cancontribute to the disruption of normal hematopoiesis and theprogression of leukemia (Bernt et al. 2011; Okada et al.2005)—a finding that has been substantiated using an induc-ible MLL fusion protein expression system. Indeed, Dot1inhibitors are effective at reducing the growth of MLLs(Daigle et al. 2013) and are currently in phase 1 clinicaltrials (ClinicalTrials.gov Identifier: NCT01684150, Drug:EPZ-5676).
A common fusion protein linked to prostate cancer isformed through the joining of the 5′-UTR region of theandrogen-regulated TMPRSS2 gene to the oncogenic tran-scription factor, ERG (Kumar-Sinha et al. 2008; Tomlinset al. 2005). It is thought that this fusion is brought aboutthrough the interaction of the androgen receptor (AR) atspecific binding sites, which mediates chromatin looping,thereby inducing abnormal spatial proximity between the
two gene partners (Mani and Chinnaiyan 2010). Recent stud-ies have shown that AR-binding sites at TMPRSS2 and ERGbreakpoints are enriched in H3K79me and H4K16ac, raisingthe possibility that histone modifications may play a novelrole in chromatin looping (Lin et al. 2009; Wu et al. 2011) andpromote the formation of fusion proteins. Finally, it has alsobeen demonstrated that DNA tumor viruses encodeoncoproteins, which among other regulatory proteins targetCBP/p300 (Avantaggiati et al. 1996; Eckner et al. 1996; Lillet al. 1997). A recent publication showed that in cells express-ing the simian virus 40 T antigen, higher levels of CBP/p300resulted in an increase in H3K56ac and H4K12ac (SaenzRobles et al. 2013). In the case of adenovirus early region1A, CBP/p300 is re-localized from the promoters of genesinvolved in differentiation and antiviral defense to those in-volved in cell proliferation, altering their histone acetylationpatterns, including H3K56ac distribution (Ferrari et al. 2008).
Furthering our understanding of the effects of H3 coremodifications on transcription and long-range interactionswill be crucial in delineating their role in disease pathogenesis.In addition, identifying the enzymes that regulate them couldenhance our array of potential drug targets.
Concluding remarks
The identification of histone core modifications and theirgenome-wide mapping gives us further insight into the regu-lation of DNA accessibility across the genome. While manyfunctional conclusions have already been drawn about the roleof these modifications at specific genomic loci, most of thedata is based on correlative studies. In addition, the interpre-tation of mutational studies can be problematic. For example,the commonly used K to Q substitution to mimic acetylationbehaves differently, in multiple assays, compared to acetyla-tion itself. Suchmutations also lead to 100% of histones being“acetylated” all the time, which does not reflect the in vivosituation. The finding that several lysine residues can be bothmethylated and acetylated also complicates such studies asreplacement with a Q not only mimics acetylation but alsoprevents methylation. Deciphering whether histone PTMstake place as a cause or consequence of the processes withwhich they are linked also remains problematic (discussed inHenikoff and Shilatifard 2011). H3K56ac and H3K79me haveboth been implicated in cellular functions outside of transcrip-tion, for example, mitotic and meiotic regulation and the DNAdamage response. Such roles are consistent with their predict-ed mode of action in transcription.
With the development of more sensitive MS machines, therepertoire of core histone PTMs is increasing (Arnaudo andGarcia 2013; Garcia-Gimenez et al. 2013). In addition, theexplosion of data implicating core modifications in DNAdamage repair, cell cycle regulation, cell fate determination,
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and disease pathogenesis highlights the need to better under-stand the mechanisms underlying their functions and regula-tion. Finally, with the implementation of chromatin capturetechniques, our understanding of the importance of chromatinorganization in the nucleus is growing and it is likely that, asin the case of H3K56ac and telomere localization, new func-tions for histone modifications in mediating processes such aslong-range interactions will become apparent. Given the piv-otal role of nonrandom nuclear organization in maintaininggenomic stability, it will be intriguing to see how we canintegrate this new knowledge with what we already know tofurther our understanding of cellular homeostasis and diseasepathogenesis.
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