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Primer compacted metaphase domains, although more and more structures. This examples of modifications within organization of DNA into the central domains of the fibers hinders its have been identified Histones and accessibility to that must (Figure 1). Given the number of ‘read’ and/or copy the nucleotide new modification sites that are base sequence, and consequently identified each year, it seems modifications such structures must be dynamic likely that nearly every histone and capable of regulated residue that is accessible to unfolding–folding transitions. solvent may be a target for post- Craig L. Peterson and Each of the core histones has a translational modification. Marc-André Laniel related globular domain that Why should histones be the mediates histone–histone target for so much enzymatic Imagine trying to stuff about interactions within the octamer, activity? Given that chromatin is 10,000 miles of spaghetti inside a and that organizes the two wraps the physiological template for all basketball. Then, if that was not of nucleosomal DNA. Each DNA-mediated processes, histone difficult enough, attempt to find a histone also harbors an amino- modifications are likely to control unique one inch segment of pasta terminal 20–35 residue segment the structure and/or function of from the middle of this mess, or that is rich in basic amino acids the chromatin fiber, with different try to duplicate, untangle and and extends from the surface of modifications yielding distinct separate individual strings to the ; is functional consequences. Indeed, opposite ends. This simple unique in having an additional ~37 recent studies have shown that analogy illustrates some of the carboxy-terminal site-specific combinations of daunting tasks associated with domain that protrudes from the histone modifications correlate the , repair and nucleosome. These histone ‘tails’ well with particular biological replication of the nearly 2 meters do not contribute significantly to functions (see Table 1). For of DNA that is packaged into the the structure of individual instance, the combination of H4 confines of a tiny eukaryotic nor to their stability, K8 , H3 K14 nucleus. The solution to each of but they do play an essential role acetylation, and H3 S10 these problems lies in the in controlling the folding of is often assembly of the eukaryotic nucleosomal arrays into higher- associated with transcription. into chromatin, a order structures. Indeed, in vitro Conversely, tri- of H3 structural polymer that not only removal of the histone tails results K9 and the lack of H3 and H4 solves the basic packaging in nucleosomal arrays that cannot acetylation correlates with problem, but also provides a condense past the beads-on-a- transcriptional repression in dynamic platform that controls all string 10 nm fiber. Although the higher . Particular DNA-mediated processes within highly basic histone tails are patterns of histone modifications the nucleus. generally viewed as DNA-binding also correlate with global The basic unit of chromatin is modules, their essential roles in chromatin dynamics, as the nucleosome core particle, tail-mediated chromatin folding diacetylation of at K4 which contains 147 bp of DNA also involve inter-nucleosomal and K12 is associated with wrapped nearly twice around an histone–histone interactions. histone deposition at , octamer of the core histones. The and phosphorylation of histone is composed of a Post-translational modifications H2A (at S1 and T119) and H3 (at central heterotetramer of histones of histones: encoding or T3, S10 and S28) appear to be H3 and H4, flanked by two patterning? hallmarks of condensed mitotic heterodimers of histones H2A and Histones are subject to an chromatin. H2B. Each nucleosome is enormous number of post- These and other observations separated by 10–60 bp of ‘linker’ translational modifications, have led to the idea of a histone DNA, and the resulting including acetylation and modification ‘code’ which might nucleosomal array constitutes a methylation of (K) and be read by various cellular chromatin fiber of ~10 nm in (R), phosphorylation of machineries. The term ‘code’ may diameter. This simple ‘beads-on- (S) and threonines (T), be a misnomer, however, as it a-string’ arrangement is folded ubiquitylation and sumoylation of implies that a particular into more condensed, ~30 nm lysines, as well as ribosylation combination of histone marks will thick fibers that are stabilized by (Figure 1; Table 1). Adding to the always dictate the same biological binding of a linker histone to each complexity is the fact that each function. By analogy, the genetic nucleosome core (note that linker residue can accept one, two code is always the same no histones are not related in or even three methyl groups, and matter which is analyzed, in sequence to the core histones). an can be either mono- or any type or tissue: TAG Such 30 nm fibers are then further di-methylated. The majority of always means STOP. In the case condensed in vivo to form these post-translational marks of histone modifications, however, 100–400 nm thick occur on the amino-terminal and there are clear exceptions — a fibers or the more highly carboxy-terminal histone tail particular mark or set of marks Magazine R547 can have different or even opposite biological P Ac Ub consequences. For instance, the H2A Ac- S G R G K Q G G K A R A ... A V L L P K K T E S H H K A K G K -COOH generally inhibitory H3 K9 1 5 119 methylation can in some cases be associated with actively Ac AcP Ac Ac Ub transcribed , and NH2- P E P V K S A P V P K K G S K K A I N K ... V K Y T S S K -COOH acetylation can be inhibitory 5 12 14 15 20 120 (123 in yeast) rather than stimulatory for Me Me Me transcription. Thus, rather than a Me P Ac Ac P P Ac MeAc Ac Me Ac P Me Me there are instead clear patterns of histone marks H3 NH2- A R T K Q T A R K S T G G K A P R K Q L A S K A A R K S A ... G V K K ... E F K T D ... 2 3 4 9 10 11 14 17 18 23 262728 36 79 that can be differentially interpreted by cellular factors, depending on the gene being P MeAc Ac Ac Ac Me studied and the cellular context. H4 Ac- S G R G K G G K G L G K G G A K R H R K V L R D N I Q G I T ... 1 3 5 8 12 16 20 Current Patterning chromatin: targeting the Figure 1. Post-translational modifications of the core histones. Although histone modifications The colored shapes represent known post-translational modifications of the core his- have been studied for over 30 tones. The histone tails can be methylated at lysines and arginines (green pentagons), years, the identification of the phosphorylated at serines or threonines (yellow circles), ubiquitylated (blue stars) and acetylated (red triangles) at lysines. histone modifying enzymes themselves remained elusive until the first nuclear histone SAGA interacts with the subunits that recognize DNA acetyltransferase (HAT), a transcriptional activation backbone-distorting base homolog of yeast domains of a variety of yeast adducts, targeting Gcn5, was identified in 1996. In gene-specific activator proteins, acetylation activity to sites of vivo studies in yeast had and these interactions target HAT nucleotide excision repair. previously characterized Gcn5 as activity to specific A quite different strategy uses a transcriptional co-activator regions in vivo. Likewise, small noncoding to target , and thus its identification unliganded nuclear hormone histone H3 K9 methylation to as a HAT solidified the view that receptors interact with HDAC chromatin surrounding histone modifications directly complexes, such as NCoR and mammalian and fission yeast regulate transcription. SMRT, which direct histone . These centromeric Subsequently, a variety of other deacetylase activity to target regions are characterized by transcriptional co-activators, genes and contribute to repetitive DNA sequences that such as CBP/p300 were found to subsequent gene repression. In are transcribed at low levels. The have intrinsic HAT activity, and addition to targeting via gene- resulting double-stranded RNAs many co-, such as specific regulators, the yeast provide substrates for processing Rpd3, were found to have histone Set1 and Set2 HMTs are found by the RNA interference (RNAi) deacetylation (HDAC) activity. associated with RNA polymerase machinery which produces small, Histone modification enzymes II holoenzymes, directing histone 21–23 nucleotide RNAs. Recent are now organized into large H3 K4 or K36 methylation, studies have shown that an intact HAT, HDAC, histone respectively, during RNAi pathway is essential for (HMT) and transcriptional elongation. targeting H3 K9 methylation to histone kinase families Targeting histone modifications centromeric chromatin, and (see Table 1). is not unique to transcriptional furthermore that these small The precise combination of control, as DNA repair and RNAs actually associate with locus-specific histone centromeric use several chromatin components. modifications is due to the distinct mechanisms to generate The resulting novel combined effects of targeting novel patterns of histone marks. ribonucleoprotein complex histone modifying enzymes to In the case of DNA repair, the ultimately targets the Clr4p HMT specific loci, as well as to the DNA lesion itself seems to play a to centromeric repeats, via either inherent substrate specificity of central role in targeting histone RNA–RNA (nascent centromeric the enzymes themselves. In the modifications. For instance, the transcripts) or RNA–DNA case of transcription, it is clear DNA damage checkpoint kinase homologous pairing. Subsequent that targeting of histone ATM (Mec1p in yeast) is recruited leads to modifications is achieved by to a DNA double strand break recruitment of proteins such as direct interactions between where it phosphorylates histone Heterochromatin Protein 1 (HP1), histone modifying enzymes and H2A (in yeast) or the histone H2A which directs formation of highly DNA sequence-specific variant, H2AX (in mammals). condensed, heterochromatin transcriptional regulators. For Likewise, the human STAGA HAT structures required for instance, the yeast HAT complex complex contains DNA binding function. Current Biology Vol 14 No 14 R548

Table 1. A current view of histone modifications.

Modification Histone Site Possible function

Acetylation H2A K4 (S. cerevisiae) Esa1 Transcriptional activation K5 (mammals) Tip60 Transcriptional activation p300/CBP Transcriptional activation K7 (S. cerevisiae) Hat1 ? Esa1 Transcriptional activation H2B K5 ATF2 Transcriptional activation K11 (S. cerevisiae) Gcn5 Transcriptional activation K12 (mammals) p300/CBP Transcriptional activation ATF2 Transcriptional activation K16 (S. cerevisiae) Gcn5 Transcriptional activation Esa1 K15 (mammals) p300/CBP ATF2 Transcriptional activation K20 p300 Transcriptional activation H3 K4 Esa1 Transcriptional activation Hpa2 ? K9 ? Histone deposition Gcn5 Transcriptional activation SRC-1 Transcriptional activation K14 Gcn5, PCAF Transcriptional activation Esa1, Tip60 Transcriptional activation DNA repair SRC-1 Transcriptional activation Elp3 Transcription elongation Hpa2 ? hTFIIIC90 RNA polymerase III transcription TAF1 RNA polymerase II transcription Sas2 ? Sas3 Transcriptional activation/elongation? p300 Transcriptional activation K18 Gcn5 (SAGA/STAGA complex) Transcriptional activation DNA repair p300, CBP DNA replication Transcriptional activation K23 Gcn5 (SAGA/STAGA complex) Transcriptional activation Sas3 DNA repair p300, CBP Transcriptional activation/elongation? Transcriptional activation K27 Gcn5 Transcriptional activation H4 K5 Hat1 Histone deposition Esa1, Tip60 Transcriptional activation DNA repair ATF2 Transcriptional activation Hpa2 ? p300 Transcriptional activation K8 Gcn5, PCAF Transcriptional activation Esa1, Tip60 Transcriptional activation DNA repair ATF2 Transcriptional activation Elp3 Transcription elongation p300 Transcriptional activation K12 Hat1 Histone deposition Telomeric silencing Esa1, Tip60 Transcriptional activation DNA repair Magazine R549

Table 1. A current view of histone modifications (continued).

Modification Histone Site Enzyme Possible Function

K12 Hpa2 ? K16 Gcn5 Transcriptional activation MOF (D. melanogaster) Transcriptional activation Transcriptional activation Esa1 (yeast), Tip60 (mammals) DNA repair ATF2 Transcriptional activation Sas2 Euchromatin Methylation H3 K4 Set1 (yeast) Permissive euchromatin (di-Me) Set9 (vertebrates) Active euchromatin (tri-Me) Transcriptional elongation/ (tri-Me) Transcriptional activation MLL, Trx Transcriptional activation Ash1 (D. melanogaster) Transcriptional activation K9 Suv39h, Clr4 Transcriptional silencing (tri-Me) DNA methylation (tri-Me) G9a Transcriptional repression Imprinting SETDB1 Transcriptional repression (tri-Me) Dim-5, Kryptonite DNA methylation (tri-Me) Ash1 (D. melanogaster) Transcriptional activation R17 CARM1 Transcriptional activation K27 Ezh2 Transcriptional silencing X inactivation (tri-Me) K36 Set2 Transcriptional elongation Transcriptional repression? K79 Dot1p Euchromatin Transcriptional elongation / memory H4 R3 PRMT1 Transcriptional activation K20 PR-Set7 Transcriptional silencing (mono-Me) Suv4-20h Heterochromatin (tri-Me) Ash1 (D. melanogaster) Transcriptional activation K59 ? Transcriptional silencing? Phosphorylation H2A S1 ? ? Chromatin assembly? MSK1 Transcriptional repression T119 NHK-1 Mitosis S129 (S. cerevisiae) Mec1 DNA repair S139 (mammalian H2AX) ATR, ATM, DNA-PK DNA repair H2B S14 (vertebrates) Mst1 Apoptosis S33 (D. melanogaster) TAF1 Transcriptional activation H3 T3 ? Mitosis S10 Aurora-B kinase Mitosis, MSK1, MSK2 Immediate-early activation Snf1 Transcriptional activation T11 (mammals) Dlk/ZIP Mitosis S28 (mammals) Aurora-B kinase? Mitosis MSK1, MSK2 Immediate-early activation H4 S1 ? Mitosis Ubiquitylation H2A K119 (mammals) HR6A,B? H2B K120 (mammals) HR6A,B? Meiosis K123 (S. cerevisiae) Rad6 Transcriptional activation Euchromatin H3 ? ? Spermatogenesis Sumoylation H4 ? Ubc9 Transcriptional repression Current Biology Vol 14 No 14 R550

Overlaid on top of these locus- distinct combinations of histone unlikely to perturb ionic specific marks is the genome- modifications. interactions with DNA. wide, bulk chromatin Cross-talk among different Consistent with this view, in modifications that may control the histone marks can also have a vivo laser crosslinking studies day-to-day folding dynamics of profound effect on enzyme have shown that histone . For instance, activity. For instance, hyperacetylation does not release newly synthesized histones that ubiquitylation of H2B K123 by the tails from DNA, and nucleosomes are deposited after passage of E2 conjugating enzyme that harbor >12 acetates per replication forks in S phase are Rad6 is required for subsequent octamer wrap DNA normally in enriched in acetylated isoforms of di-methylation of H3 K4 by Set1p vitro and have hydrodynamic histones H3 and H4, and the or H3 K79 by Dot1p. Prior histone properties that are nearly identical formation of condensed marks can also inhibit subsequent to unmodified nucleosomes. chromosomes in mitosis is modifications. For instance, H3 Although it is true that histone associated with phosphorylation S10 phosphorylation inhibits hyperacetylation does disrupt the of histones H3 and H2A. subsequent H3 K9 methylation, folding dynamics of nucleosomal In addition to these marks and of course H3 K9 methylation arrays in vitro, even in this case linked to the , there can also block acetylation of this 6–12 acetates per nucleosome are appears to be a constant battle same residue. An excellent required. Although most site- among HATs and HDACs on a example of even more complex specific patterns of histone global, nontargeted level that cross-talk is exemplified during modifications have yet to be maintains a baseline, equilibrium p53-dependent transcriptional generated and tested in vitro, the level of histone acetylation activation in vitro. In this case prevailing view is that these throughout the genome. Histone methylation of H4 R3 by protein histone marks may not alter deacetylase inhibitors, such as arginine methyltransferase 1 nucleosomal dynamics by trichostatin or sodium butyrate, (PRMT1) stimulates CBP-p300 themselves. disrupt this equilibrium, leading to acetylation of H4 K5, K8, K12 and In contrast to the lack of a general increase in bulk histone K16, which in turn promotes the evidence pointing to direct acetylation. Such genome-wide methylation of H3 R2, R17 and changes in chromatin structure, activities of histone modifying R26 by another PRMT family there is now a wealth of examples enzymes likely act in concert with member, CARM1. Thus, positive where specific histone the cell-cycle-linked changes in and negative crosstalk ultimately modifications control the binding bulk chromatin to enhance the generates the complex patterns of of nonhistone proteins to the general dynamic nature of gene or locus-specific histone chromatin fiber. These nonhistone eukaryotic chromosomes. marks associated with distinct proteins then elicit the function chromatin states. that is associated with a particular Patterning chromatin: controlling histone mark. A hallmark of many enzyme substrate specificity Patterns of histone proteins that bind to histone tails Recruitment of a histone modifications: what happens is the presence of small histone modifying enzyme to the right next? binding modules. For example, place at the right time is only the Once a pattern of histone some bind to first step in establishing a modifications is established at a methylated lysines, whereas combination of histone marks that target locus, what do they do? specify binding to may direct a biological outcome. Many older models proposed that acetylated lysines. Furthermore, The second step in this process histone modifications might these modules often bind to only revolves around the specificity of directly influence either the a particular modified histone the enzyme for individual histone structure of individual residue. For example, the tails and for specific histone nucleosomes or the folding within HP1 residues (Table 1). For example, dynamics of nucleosomal arrays. interacts specifically with a yeast and human Gcn5 and Indeed one common dimethylated K9 of histone H3, human PCAF preferentially misconception is that histone whereas the chromodomain of the acetylate lysine residues within modifications that alter the charge Polycomb protein binds to a the histone H3 amino-terminal tail, of a residue, such as lysine dimethylated K27 of histone H3. at K9 and K14. In contrast, the acetylation or In contrast, the binding of yeast and human NuA4 HAT phosphorylation, will disrupt bromodomains to different complexes preferentially acetylate histone–DNA interactions leading acetylated lysines does not show K4, K8, K12 and K16 of histone to ‘open’ or ‘active’ chromatin as much specificity. For instance, H4. Even more extreme specificity structures. There is not actually acetylation of K8 within histone is seen with HMTs. For instance, much evidence for such models. H4 can promote the recruitment the HMT Set7/9 is restricted to For example, the histone H3 tail of the ATP-dependent chromatin mono-methylation of histone H3 contains 13 positively charged remodeling enzyme, human at K4, whereas the Dim-5 HMT is amino acids, and thus acetylation SWI/SNF — via a a tri-methylase specific for H3 K9. of one to four residues will only within the Brg1 subunit — but a Thus, recruitment of different yield a 10–30% decrease in similar bromodomain within the HATs or HMTs can result in positive charge, levels that are Swi2 subunit of the yeast Magazine R551

SWI/SNF complex interacts with a structure or change the folding functions. Some of these domains broader range of acetylated H3 properties of nucleosomal arrays can be propagated through DNA and H4 tails. is not known. It is also not clear replication and mitosis, The interactions of how many of these variant guaranteeing the inheritance of bromodomains and nucleosomes are localized to chromatin states to progeny. chromodomains with modified specific DNA sequences; for Histone lysine methylation may tails is also subject to example, why are CENP-A- play a central role in the stability modification crosstalk — the containing nucleosomes found of these chromatin states, as to modification of adjacent residues only at centromeres? Notable date no enzymes are known that can positively or negatively exceptions include the deposition catalyze lysine demethylation. regulate binding. Thus, in many of H3.3 to chromatin of RNA Furthermore, several nonhistone ways histone tails can be viewed polymerase II transcribed genes proteins, such as HP1 or the as complex protein–protein via a novel replication- PRC1 polycomb complex, not interaction surfaces that are independent assembly complex, only bind to methylated histone regulated by numerous post- and the exchange of H2AZ for lysines, but also recruit the translational modifications. canonical H2A via the ATP- methylase itself, thus providing a Furthermore, it is clear that the dependent SWR1 complex. means for templating new histone overall constellation of proteins Once a histone variant is methylation events — for bound to each tail plays a primary targeted to a specific locus, there example, following replication role in dictating the biological is the potential for creation of fork passage — or for spreading functions of that chromatin novel chromatin domains that the domain to adjacent domain. have distinct regulatory nucleosomes. properties. For instance, the How ‘open’ states are Chromatin fiber heterogeneity: amino-terminal tail of CENP-A propagated through cell divisions lacks the phosphorylation and is not clear, especially as histone Throughout this primer we have acetylation sites that are normally lysine acetylation or serine described chromatin (in its modified in histone H3 at phosphorylation can be rapidly simplest form) as a linear array of transcriptionally active regions. reversed by HDACs or histone canonical nucleosomes. An in vivo Thus, CENP-A might produce phosphatases. Future studies will chromatin fiber, however, is islands of unmodified histone H3 no doubt continue to identify the actually an extremely that help to maintain centromeric functional and biochemical heterogeneous chromatin in its condensed, properties of new chromatin filament, even at the nucleosome inactive state. In contrast, the domains as well as to elucidate level. First and foremost, in histone H3.3 variant contains an the principles that govern their addition to canonical amino-terminal tail that is virtually maintenance and propagation. nucleosomes, in vivo chromatin identical to that of histone H3, and arrays also contain novel types of thus it seems likely that many of Further reading nucleosome that harbor one or the transcription-associated Hansen, J.C. (2002). Conformational more variant isoforms of the core marks that have been attributed to dynamics of the chromatin fiber in solution: determinants, histones. For instance, histone H3 are likely also mechanisms, and functions. Annu. nucleosomes assembled at yeast occurring on the histone H3.3 Rev. Biophys. Biomol. Struct. 31, and mammalian centromeres variant. 361–392. contain a histone H3 variant, In the case of H2AZ, Elgin, S.C.R., and Grewal, S.I.S. (2003). Heterochromatin: silence is golden. Cse4/CENP-A, which is essential biochemical studies suggest that Curr. Biol. 13, R895–R898. for centromere function or nucleosomal arrays containing Fischle, W., Wang, Y., and Allis, C.D. assembly. Another histone H3 H2AZ may only partially compact, (2003). Binary switches and variant, H3.3, replaces canonical resisting formation of large modification cassettes in histone biology and beyond. Nature 425, histone H3 during transcription, 100–400 nm fibers and thereby 475–479. generating a mark of the facilitating transcription. Thus, Fischle, W., Wang, Y., and Allis, C.D. transcription event. Several incorporation of histone variants (2003). Histone and chromatin variants of histone H2A have also into chromatin fibers might cross-talk. Curr. Opin. Cell Biol. 15, been identified. The macro-H2A enhance chromosome dynamics 172–183. Korber, P., and Horz, W. (2004). variant is restricted to metazoans by creating domains of chromatin SWRred not shaken; mixing the and functions in with novel properties. histones. Cell 117, 5–7. inactivation, while H2AZ (also Tagami, H., Ray-Gallet, D., Almouzni, known as H2A.F/Z or H2AvD) is Concluding remarks G., and Nakatani, Y. (2004). H3.1 and H3.3 complexes mediate found in all eukaryotes. Histone variants, distinct patterns nucleosome assembly pathways Surprisingly, H2AZ is required for of posttranslational modifications dependent or independent of DNA one or more essential roles in of histones, and histone tail synthesis. Cell 116, 51–61. chromatin structure that cannot binding proteins all contribute to be replaced by bona fide histone establishment of various ‘open’ or Program in Molecular Medicine, University of Massachusetts Medical H2A. ‘closed’ chromatin domains that School, Worcester, Massachusetts In most cases, how histone have specialized folding 01605, USA. variants alter nucleosome properties and biological E-mail: [email protected]