Histone H3 variants and their potentialrole in indexing mammalian genomes:The ‘‘H3 barcode hypothesis’’Sandra B. Hake and C. David Allis*

Laboratory of Chromatin Biology, The Rockefeller University, Box 78, 1230 York Avenue, New York, NY 10021

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on May 3, 2005.

Contributed by C. David Allis, January 31, 2006

In the history of science, provocative but, at times, controversialideas have been put forward to explain basic problems thatconfront and intrigue the scientific community. These hypotheses,although often not correct in every detail, lead to increaseddiscussion that ultimately guides experimental tests of the princi-pal concepts and produce valuable insights into long-standingquestions. Here, we present a hypothesis, the ‘‘H3 barcode hy-pothesis.’’ Hopefully, our ideas will evoke critical discussion andnew experimental approaches that bear on general topics, such asnuclear architecture, epigenetic memory, and cell-fate choice. Ourhypothesis rests on the central concept that mammalian histone H3variants (H3.1, H3.2, and H3.3), although remarkably similar inamino acid sequence, exhibit distinct posttranslational ‘‘signa-tures’’ that create different chromosomal domains or territories,which, in turn, influence epigenetic states during cellular differ-entiation and development. Although we restrict our comments toH3 variants in mammals, we expect that the more general conceptspresented here will apply to other histone variant families inorganisms that employ them.

histone H3 variants H3.1, H3.2, H3.3 � ‘‘barcode hypothesis’’ � epigeneticmemory � cell differentiation

Chromatin and Its Role in Cellular Processes

Every eukaryotic cell contains genetic information in the formof DNA that is compacted to varying degrees in a confined

nuclear space. However, DNA is packaged in such a way thatenables its readout, replication, and repair in response to cellularneeds and external stimuli. This condensation is achieved by anintimate interaction between DNA and histone proteins to formchromatin. The fundamental unit of chromatin is the nucleo-some particle, consisting of core histone proteins (H2A, H2B,H3, and H4) around which the DNA is wrapped. Chromatin isoften broadly divided into two cytologically distinct fractions:euchromatin, which is generally permissive for transcription, andheterochromatin, which is largely repressive. Two basic varietiesof heterochromatin exist, constitutive and facultative; DNAwithin constitutive heterochromatin is obligately silenced; fac-ultative heterochromatin is silenced only in certain contexts.

Relevant to our proposed ‘‘H3 barcode hypothesis’’ is theextent to which the chromatin fiber is constant or variable.Constancy is provided by the nearly universal nucleosomalpackaging theme of histones and DNA in all eukaryotes. Vari-ation is provided by subtle changes in this packaging theme thatprovide ‘‘instructions’’ as to how the DNA template is to be‘‘read’’ when needed. Histone proteins are, for example, wellknown to be extensively modified by a vast array of covalentmodifications on ‘‘external’’ (N- and C-terminal tails) as well as‘‘internal’’ (histone-fold) domains, often leading to complexmodification patterns that correlate closely with various states ofgene expression or other DNA-templated processes. This stag-gering number of posttranslational modifications (PTMs) hasprompted theories as to how these chemical marks might be

translated into meaningful biological responses (1, 2). The‘‘histone code’’ hypothesis states that a specific histone modifi-cation, or combinations thereof, can affect distinct downstreamcellular events by altering the structure of chromatin (cis mech-anisms) or by generating a binding platform for effector proteins(trans mechanisms). Such effectors specifically recognize par-ticular PTM(s) and initiate events that ultimately lead to down-stream events, such as gene activation or silencing. Tests of thishypothesis, as well as extensions of it (3), are gaining experi-mental support (e.g., refs. 4 and 5), although alternative viewshave been expressed (6, 7). Despite these uncertainties, emerg-ing evidence underscores elaborate mechanisms for introducingvariation, covalent and noncovalent, into the chromatin polymer(reviewed in ref. 8). The challenge remains as to how thisvariation is converted into meaningful biological readout.

Histone H3 Variants and Their EvolutionWith the exception of H4, all core histone proteins have variantcounterparts, which often differ in surprisingly few amino acids(reviewed in ref. 9). Histone genes encoding these variants canbe classified into three main subtypes on the basis of theirexpression pattern and genomic organization (10, 11): replica-tion-dependent (RD), replication- and cell cycle phase-independent (RI), and tissue-specific (TS) histones. RI expres-sion of histone genes reinforces the general view that histoneproteins evolved to participate actively in DNA-templated pro-cesses rather than to serve simply a passive DNA-packaging role(see below). Nowhere is the concept of histone variants betterillustrated than with the family of H3 histones.

Most eukaryotes express a centromere-specific H3 variant(Saccharomyces cerevisiae, Cse4; Drosophila, CID; and Homosapiens, CENP-A) that is evolutionarily well conserved in itsglobular core region but not in its N-terminal tail (reviewed inref. 12) and is essential for cell survival because of its funda-mental role in centromeric function during mitosis (13). Inter-estingly, during evolution, additional genes encoding H3 variantshave emerged (Fig. 1A). For example, outside of the centromericH3 variant, the unicellular yeast S. cerevisiae possesses only H3.3,a H3 variant that is expressed and incorporated into chromatinin a RI fashion and associated in higher eukaryotes withtranscriptional activation (see below). Although budding yeastcontains well defined ‘‘silent’’ chromatin, several hallmark fea-tures of constitutive heterochromatin in higher eukaryotes (e.g.,H3 K9, and K27 methylation) have yet to be observed in S.cerevisiae (14). This observation correlates well with the presence

Conflict of interest statement: No conflicts declared.

Abbreviations: LBR, lamin B receptor; PTM, posttranslational modification; RD, replication-dependent; RI, replication-independent.

See accompanying Profile on page 6425.

*To whom correspondence should be addressed. E-mail: alliscd@rockefeller.edu.

© 2006 by The National Academy of Sciences of the USA

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of only a H3.3 variant and the streamlined gene-rich compositionof the yeast genome. Interestingly, the fission yeast Saccharo-myces pombe contains one H3, with characteristics from bothH3.3 and H3.2, a finding consistent with this yeast havingconstitutive heterochromatin more typical of higher organisms(15). Organisms such as plants, f lies, frogs, and birds contain, inaddition to H3.3, another H3 variant H3.2 that differs in onlyfour amino acids from H3.3 (Fig. 1B); H3.2 is expressed andincorporated into chromatin in a RD fashion. Only in mammalshave two additional H3 variants evolved: H3.1 and a testis-specific H3.1 variant (H3.1t) (Fig. 1 A). H3.1 differs from H3.2in only one amino acid (amino acid 96: cysteine�serine, respec-tively) and is also expressed in RD fashion, whereas H3.1t isexpressed only in testis and has four additional amino acidsubstitutions when compared with H3.1 (see Fig. 1B). Are these

modest changes in primary sequence among H3 variants unim-portant, a likely consequence of evolutionary ‘‘drift?’’ Alterna-tively, the small number of amino acid changes in these H3variants lead to unique protein structures and, in turn, to uniquenucleosomal architecture and chromosomal domains that mightgovern H3 variant-specific biological functions (as is the case forcentromere-associated H3s) (16). Future studies aimed at de-termining the x-ray structures of nucleosomes containing dif-ferent histone variants may provide structural insights into theireffects on nucleosome stability and organization.

The literature on H3 variants does not contain a universalnomenclature for these variants, and, therefore, we propose toadopt the following convention: histone H3 protein containingS31, A87, I89, and G90 will be called H3.3; H3 with A31, S87,V89, M90, and S96 will be called H3.2; and H3.1 has thesequence of H3.2, with the exception of position 96, where itcontains a cysteine. Amino acids 87–90 in H3.3 have been shownto be important for RI incorporation into chromatin (17), andthese data suggest that this region might act as a ‘‘chaperonerecognition domain’’ where HIRA binds to H3.3 and CAF-1 toH3.1 (see below and ref. 18). It is as yet unknown whether H3.2binds to a different chaperone and whether amino acid position96 plays any role in this potential chaperone recognition domain(Fig. 1B).

Elegant experiments have shown that H3.3 is associated withtranscriptionally active gene loci and is enriched in covalentmodifications associated with gene activation in flies, plants, andhumans (17, 19–21). In contrast, in Drosophila and Arabidopsis,H3.2 has been shown to be enriched in marks that are associatedwith gene silencing (19, 20). These observations suggest that,during evolution, organisms draw on different profiles of phys-iologically relevant PTMs but also selective employment (re-cruitment and replacement) of different histone H3 variants, aconcept well articulated by Henikoff and colleagues (22). Be-cause H3.1 and H3.2 differ by only a single amino acid, moststudies tend to group these variants as one. However, recentresults provide evidence that human H3.1, H3.2, and H3.3 differin both their expression and PTM patterns as follows: H3.3 isenriched in PTMs associated with gene activation (hyperacety-lation and dimethylation of K36 and K79), H3.2 is enriched inPTMs associated with gene silencing (K27 di- and trimethyla-tion), and H3.1 is enriched in PTMs associated with geneactivation (K14 acetylation) and gene silencing (K9 dimethyla-tion), suggesting that these mammalian H3 variants may, indeed,have separate biological functions (23). These studies under-score a general conclusion: Remarkably similar histone proteinsmay vary considerably in their expression and PTM profiles.Determining how these differences translate into different bio-logical functions and, notably, whether different functions, in-deed, exist for the closely related H3.1 and H3.2 remains achallenge for future research.

The mechanism(s) by which histone variants and their PTMsare transmitted through the cell cycle also remains unsolved.Depending on the precise mechanism of nucleosome assembly atthe time of DNA replication, histone variants may provide abridge for the transmission of epigenetic information from onecell or one sexual generation to the next (18). If, for example, theincorporation of histone variants into replicating chromatin isnonrandom, we envision that the variants may provide potential‘‘backup’’ for the more labile histone PTMs by playing a role inthe establishment of ‘‘epigenetic memory.’’ Central to thisconcept is the general view that H3 variants can impart structuraldifferences to individual nucleosomes, nucleosomal arrays, orhigher-order chromatin domains that contain them before PTMsare added (or removed) (24). Below, we present several ideas forhow such differences might occur, even though only a smallnumber of amino acid differences exist between H3 variants.

Fig. 1. H3 variants in different organisms. (A) Schematic of evolutionaryappearance of histone H3 variants. All organisms express a centromere-specific H3 variant (CENP-A, filled blue circle). In addition to the centromericH3 variant, the following H3 variants are expressed in these organisms: S.cerevisiae contains only H3.3 (blue gradient circle); S. pombe expresses ahybrid H3 protein that contains amino acids characteristic for H3.3 and H3.2;Arabidopsis thaliana, Xenopus laevis, and Drosophila melanogaster (for ex-ample) express H3.3 and H3.2 (blue circle with white dots); mammals such asMus musculus and H. sapiens express H3.3, H3.2, H3.1 (white circle with bluedots), and a testis-specific H3.1t (white circle with blue stripes) variant ofunknown function. H3.3 has been associated with euchromatin and transcrip-tional activation. H3.2 and H3.1 might localize to heterochromatin and areinvolved in transcriptional silencing. (B) Alignment of human noncentromerichistone H3 variants. Differences in amino acid sequence among human H3.3,H3.2, H3.1, and H3.1t are shown in white boxes. Cysteine residues are high-lighted in red (Cys 96 in dark red and Cys 110 in pink). Identical amino acids areshown in gray. TS, tissue-specific. The region where most amino acid differ-ences between the variants are found is underlined as a potential chaperonerecognition domain (see text for details), and the chaperones binding to H3variants are depicted below.

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Cysteines of H3 Variants and Their Potential Rolein Nuclear ArchitectureWell established in the literature, but relatively underappreci-ated, is the fact that most members of the histone H3 familycontain one or more cysteine(s) in their protein core and thatthis feature is a hallmark property of histone H3; all otherhistone proteins lack cysteine (Fig. 1B). Cysteine is one of themost rarely used amino acids in nature (1.9% occurrence inproteins) (25), suggesting that it plays a specialized role in thefunction of proteins that contain it. Equally well established isthe fact that cysteines can form disulfide bonds under oxidativeconditions and are involved in the homotypic or heterotypicdimerization and oligomerization of proteins. As shown in Fig.1B, the histone H3 variants (except H3 in S. cerevisiae) containone cysteine at position 110 that is located in their a2 helix, theregion where both H3 proteins are closely apposed in thenucleosome core particle (26). The region immediately sur-rounding amino acid 110 is important to hold together twohistone H2A–H2B–H3–H4 tetramers, because C110E muta-tions, for example, destabilized the H3–H3 hydrophobic four-helix bundle tetramer interface in vitro (27). It is not yet clearwhat role, if any, cysteine 110 plays in this process in vivo. Wepropose that Cys-110, common in essentially all H3 variants,forms an ‘‘intra’’-disulfide bond with H3 in the same nucleosomeunder oxidative conditions, adding stability to the H3–H4 tet-ramer (Fig. 2A). In support, a crosslinked H3-H3 octamer canstill form a nucleosome in vitro (28). No cysteine exists in S.cerevisiae H3, and giving yeast an artificial cysteine in place of itsserine at 110 does not appear to have clear phenotypic conse-quences (29). We note that budding yeast lacks many of thebetter known heterochromatin marks and related machinery(i.e., K9 methylation in H3, HP1-like homologues, etc.). Thus,the tentative conclusion that cysteine utilization is unimportantbased solely on experiments conducted in budding yeast may notbe warranted. We look forward to the generation of H3 cysteinemutants in organisms that use more classical heterochromatin.

Although the extent to which the nucleus contains an oxidizingor reducing environment is not well established, redox-sensingmechanisms appear to play important roles in the nucleus.Certain transcription factors, for example, NF-�B, contain acysteine that has been shown to participate in intermoleculardisulfide formation (30) and must be in a reduced state in orderfor NF-�B to bind to DNA. Reduction is achieved by the actionof molecules that are unique to the nucleus (31). In contrast,other transcription factors have an increased DNA-bindingaffinity under oxidative conditions (32), lending support to thegeneral notion that physiologically relevant, redox-sensitivemechanisms may occur inside the nucleus.

It is intriguing to revisit earlier literature (33, 34) aimed atdetermining whether the cysteines in histone H3 variants‘‘sense’’ changes in the redox state of the nucleus. If so, does theproximity of the two cysteines at the interface between homo-typic H3 dimers within each nucleosome play a stabilizing rolein the architecture of the chromatin polymer that, in turn,impacts on the regulation of gene expression? Roughly 20 yearsago, Allfrey and colleagues (35) hypothesized a meaningfuldifference between euchromatin and heterochromatin, as as-sayed by accessibility to sulfhydryl reagents, which can formdisulfide bonds with exposed cysteines under oxidative condi-tions. Transcriptionally active regions were labeled preferentiallywith sulfhydryl-specific reagents, whereas nucleosomes in het-erochromatin and nontranscribed regions were not. Moreover,these reagents preferentially bound to the cysteines in chromatinfractions enriched for hyperacetylated H3, suggesting that tran-scriptional activity ‘‘opens’’ the otherwise more tightly com-pacted chromatin, exposing the H3 cysteine so that it can bebound by sulfhydryl-reactive molecules (36). These observations

correlate well with results showing that exposure of fibroblaststo mercury leads to the accumulation of this metal into euchro-matin but not into heterochromatin (37, 38). Enrichment of‘‘active,’’ hyperacetylated chromatin, obtained by virtue of itsability to bind to mercury-containing columns, formed the basisof several intriguing experiments, including fractionation of

Fig. 2. Potential usage of H3 variant-specific cysteines 110 and H3.1-specificcysteine 96. (A) H3 cysteine 110 forms a potential intramolecular disulfidebond (light red box) with H3� cysteine 110 in the same nucleosome (for details,see text). For simplicity, only the H3–H4 tetramer is shown as top view (Left).All mammalian H3 variants contain cysteine 110 and can potentially partici-pate in disulfide bonding. (Right) H3–H4 dimers. (B) H3.1 cysteine 96 poten-tially forms intermolecular disulfide bonds (dark red box) with H3.1� cysteines96 in different nucleosomes, leading to chromatin condensation and hetero-chromatin generation (for details, see text). (C) H3.1 cysteine 96 is envisionedto potentially form disulfide bonds (dark red box) with cysteine in LBR on thenuclear envelope or with a cysteine in an as yet unknown protein (X?) in thenucleus (for details, see text). We speculate that chromatin containing H3.1nucleosomes is preferentially located near the nuclear membrane and irre-versibly rendered for transcription regardless of PTMs.

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yeast chromatin with an artificial cysteine at position 110 in placeof its natural serine (29).

These data suggest that cysteine 110 in H3 is more accessibleto sulfhydryl-reactive reagents in euchromatin and may be moreburied in heterochromatin, providing a potential molecularmarker, underscoring a physical change in the nature of higher-order chromatin structure that may reflect different physiolog-ical states. It remains unclear whether the inaccessibility ofcysteine 110 in transcriptionally silent regions is an indirectconsequence of chromatin compaction. Alternatively, a moredirect effect is possibly due to a disulfide bonding between bothcysteines 110 in the two H3s in the same nucleosome that, in turn,compacts nucleosomal and higher-order structures (Fig. 2 A).Determining the extent to which H3 ‘‘oxidation�reduction’’occurs in vivo, if at all, remains a worthwhile challenge for futurestudies.

Interestingly, two mammalian histone H3 variants, H3.1 andH3.1t, contain an additional cysteine 96 in their protein-coreregion besides the more highly conserved cysteine 110 discussedabove (Figs. 1B and 2 A). Because cysteine 96 is likely located onthe protein’s surface (26), we speculate that it may play anunappreciated role in chromatin compaction and gene silencingby its ability to form disulfide bonds with other H3s in differentnucleosomes or with other cysteine-containing proteins underoxidative conditions or in the presence of an as yet undeterminedoxidizing molecule(s). Several scenarios for cysteine 96 can beenvisioned:

(i) The H3.1-specific histone chaperone CAF-1 (18) mayspecifically recognize the region containing cysteine 96 in H3.1as part of a chaperone-specific replacement mechanism thatserves to direct H3.1 to target genomic loci (see below and Fig.1B). In Drosophila, the region of H3.3 that differs most from thatof H3.2 (amino acids 87, 89, and 90) is important for RIincorporation (17). These findings suggest that this region of H3is important for the binding of specialized histone chaperones,and we speculate that cysteine 96 plays a role in this process,thereby distinguishing H3.1 from H3.2 and H3.3. To our knowl-edge, little is known as to how histone-specific chaperones (e.g.,CAF-1 versus HIRA; see ref. 18) recognize the appropriatetarget histones, nor is it known whether H3.2 is escorted tochromatin by its own unique chaperone (see Fig. 1B).

(ii) Nucleosomes that contain H3.1 might bind to otherH3.1-containing nucleosomes through internucleosomal disul-fide bonds between cysteines 96. We envision that this eventwould serve to provide additional stability to higher-ordernucleosomal contacts and may provide an explanation for H3.1-mediated condensation of heterochromatin (Fig. 2B). The con-cept of cysteine 96-mediated disulfide ‘‘bridging’’ suggests thatH3.1 might play a unique role in the formation of constitutiveheterochromatin that stably represses transcription through thegeneration of H3.1-containing oligonucleosomes. Here, we notethat the formation of H3 dimers (H3.1, H3.2, and H3.3) and H3oligomers (H3.1 only) occurs, at least in vitro, under oxidativeconditions and is inhibited in a reducing environment (S.B.H.and C.D.A, unpublished observations). The in vivo significanceof these findings, if any, remains unclear.

(iii) H3.1 might form disulfide bonds with other nuclearcysteine-containing nonhistone proteins (Fig. 2C). One attrac-tive candidate is the nuclear membrane-associated protein laminB receptor (LBR); other disulfide ‘‘partners’’ (‘‘X’’) are alsopossible (Fig. 2C). LBR has been shown to bind distinct hetero-chromatin-enriched fractions (39). Moreover, Makatsori andcoworkers (23) found that LBR-associated purified fractionscontain histone H3 enriched in PTMs associated with transcrip-tional silencing similar to those that we have observed on H3.1.Additionally, LBR binds heterochromatin as a higher oligomer.Interestingly, another study reports the formation of a higher-order complex including H3, H4, LBR, and heterochromatin

protein 1 (HP1) (40) that has been found to interact with histoneH3 methylated at lysine 9 (41). We have shown that H3.1 isenriched in K9 dimethylation, suggesting that H3.1, but not H3.2or H3.3, might be the H3 variant that selectively binds HP1 andinteracts with LBR at the nuclear envelope. It remains to bedetermined whether LBR-bound heterochromatin contains onlyH3.1 and, if so, whether cysteine 96 is important for theestablishment of nuclear membrane-associated heterochroma-tin. In conclusion, we speculate that the unique cysteines in H3variants might be important for nucleosomal and chromatinhigher-order structures in ways that remain to be determined. Inturn, we speculate that these structures determine, directly orindirectly, transcriptional regulatory states and distinct nucleardomains or compartments (see below).

Histone H3 Variants and Epigenetic MemoryDuring the development of multicellular organisms, cells differ-entiate by changing their gene expression profiles in response tostimuli or environmental cues. Long after these external stimuliare gone, ‘‘cellular memory’’ mechanisms enable cells to remem-ber their chosen fate over many cell divisions (reviewed in ref.42). Chromatin has long been suspected to play a major role inthese mechanisms, but how an epigenetic memory, defined bynetworks of inherited sets of expressed and silenced genes, isfaithfully transmitted to daughter cells during each S-phaseremains unresolved. We favor the general view that histonevariants, especially H3.1, H3.2, and H3.3, contribute to not onlygene expression and silencing events, but also to the mainte-nance of epigenetic inheritance. In this view, histone PTMsalone cannot explain the establishment of epigenetic memoryduring several cell divisions. We propose that histone H3variants contribute to ‘‘indexing’’ the genome into functionallyseparate domains (e.g., euchromatin, facultative heterochroma-tin, and constitutive heterochromatin) that, in turn, establish andmaintain epigenetic memory for each individual cell type. Ifcorrect, one requirement for H3 variants to play a major role inepigenetic inheritance is that nucleosomes contain ‘‘homo’’-dimers of the same H3 variant, which are deposited by differentchaperones (see Fig. 3). In support, Nakatani and colleagues(18) provided evidence that mammalian histone H3 variantsH3.1 and H3.3 are incorporated into chromatin by separatechaperones (CAF-1 and HIRA, respectively). Once properlydeposited into chromatin, H3 variants must be read by mecha-nisms that remain unclear but are likely to involve PTMs (seebelow).

Different models have been proposed to explain how epige-netic memory can be achieved (reviewed in ref. 43). Henikoffand coworkers (44) recently proposed that histone states are notactively duplicated but are reestablished each cell cycle by activetranscription and new deposition of histone variants, in partic-ular H3.3 (Fig. 3, see nonreplicating DNA). Although transcrip-tion-coupled histone-variant deposition may function to estab-lish and reestablish active euchromatin, it is unlikely to be thesole means of epigenetic inheritance, because it does not accountfor the inheritance of silenced chromatin. The timing of thereplication of different chromatin states during S-phase mightalso play an important role in establishing epigenetic memory. Itis well known that transcriptionally active chromatin is replicatedin early S-phase, whereas heterochromatin is replicated in lateS-phase (reviewed in ref. 45). It will also be interesting todetermine whether facultative and constitutive heterochromatinreplicate at different times during late S-phase, which mightcoincide with the expression of H3.2 and H3.1 and�or theirspecific chaperones, therefore providing one regulatory step inachieving epigenetic memory.

Much experimental evidence points toward another model ofinheritance, the conservative model. This model suggests thatintact parental nucleosomal cores are most likely dispersively

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segregated to daughter strands (46, 47) (Fig. 3 Left). In thismodel, the maintenance of epigenetic inheritance is difficult toenvision but might be achieved by the replication timing differ-ences between chromatin stages or H3 variant-specific chaper-ones that sense adjacent H3–H4 tetramers on the same daughterstrand. Another possibility is that the topology of DNA differsamong different H3 variant-H4 tetramers, and, therefore, onlyspecific H3 variant-containing nucleosomal cores are depositedonto the DNA during replication. Although not impossible,little, if any, evidence exists to support these scenarios.

In contrast, the semiconservative inheritance model proposesthat nucleosomes are ‘‘split’’ into H3–H4 dimers that are dis-tributed to each daughter strand (Fig. 3 Right). Although con-troversial (see ref. 43), these findings suggest that H3 and H4may be deposited into chromatin as a dimeric unit rather thanas a tetrameric unit, as has been proposed (48). The semicon-servative model suggests that parental (H3–H4)2 tetramers aredissociated into two H3–H4 dimers during DNA replication andsegregated evenly onto daughter DNA strands (18). One con-sequence of this model is that each daughter strand obtains one

posttranslationally modified parental H4–H3 variant dimer,thereby acquiring epigenetic memory. New H3.1–H4 dimers aredeposited in chromatin through CAF-1 chaperone (RD-expressed), resulting in (H3.1–H4)2 tetramers. In contrast,HIRA is believed to deposit newly synthesized H3.3–H4 dimersinto chromatin, forming (H3.3–H4)2 tetramers on the daughterstrands.

We speculate that there may be an additional, as yet uniden-tified, H3.2-specific histone chaperone that deposits onlyH3.2–H4 dimers (or tetramers). As discussed above, whetherserine 96 in H3.2 (as compared with cysteine 96 in H3.1; see Fig.1B) is an important recognition site for this hypothetical H3.2chaperone is not known. Regardless of which model is morecorrect, CAF-1 may recognizes both H3.1 and H3.2. Here, thesite-specific incorporation of each variant into chromatin woulddepend on the template variant in the parental H3–H4 dimer,the time when H3.1 and H3.2 are expressed during late S-phase,etc. ‘‘Daughter’’ mononucleosomes would then be completedwith the addition of H2A–H2B dimers donated by other chap-erones or exchange machinery (reviewed in refs. 49 and 50).

Fig. 3. Epigenetic memory and H3 variants: graphic of different models of epigenetic inheritance (for details, see text). Nucleosomes contain two of H3.3 (bluegradient circle), H3.2 (blue circle with white dots) or H3.1 (white circle with blue dots), and H4 (yellow circle). N-terminal tails of H3 variants are posttranslationallymodified: H3.3, active PTMs (green flag); H3.2, silencing PTMs (red flag); H3.1, silencing PTMs that differ from those observed on H3.2 (orange flag). Outside ofS-phase, H3.3 can be deposited into chromatin in a RI manner [as either H3.3–H4 tetramers (Left) or H3.3-H4 dimers (Right)] to activate gene transcriptionimmediately, as proposed by Henikoff and colleagues (44). The conservative inheritance model proposes that, during replication, H3–H4 tetramers are distributedon daughter strands in a random fashion. (Left) H3 variant-specific chaperones deposit H3–H4 tetramers onto daughter strands to fill in the gaps, distributingH3 variants by potentially sensing adjacent H3 variants on the same daughter strand. (Right) The semiconservative model of replication, as proposed by Tagamiet al. (18), is shown. During replication, nucleosomes are separated into two H3–H4 dimers that are distributed equally onto daughter strands. H3 variant-specificchaperones deposit H3.3–H4 dimers (HIRA), H3.1–H4 dimers (CAF-1), and H3.2–H4 dimers (unknown, ?) to histone dimers on the daughter strands forminghomogenic nucleosomes.

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Several chromatin-remodeling complexes, including the SWI�SNF, RSC, and ISWIb complexes, can catalyze the exchange ofH2A–H2B dimers between chromatin fragments in an ATP-dependent reaction (51). On the other hand, the H2A.Z varianthas recently been shown to be incorporated into chromatin by aspecialized ATP-dependent nucleosome remodeling complex;the SWR1 complex, which consists of 13 subunits, including theSwi2�Snf2-related ATPase Swr1 (52). It remains to be seenwhether other H2A variants, such as H2A.X, macroH2A, andH2A.Bbd are also incorporated into chromatin by other spe-cialized chaperones and whether all these H2A variants mightthen be pairing specifically with different H3 variants in onenucleosome. After completion of the newly assembled chroma-tin, we envision that appropriate histone-modifying enzymes willadd or remove PTMs, maintaining a specific histone code foreach particular chromosomal region.

We suggest, then, that H3.1, H3.2, and H3.3 have differentbiological functions, based on differences in cell and tissue-specific expression patterns and PTMs (23). We favor thegeneral view that histone variants index select chromosomalregions by using selective chromatin-assembly mechanisms ofthe type described above, regardless of which model of inheri-tance is actually happening in the cell. Once in place, we envisionthat variant nucleosomes, marked by different PTMs, influencegene expression and nuclear architecture and, therefore, achievepersistent epigenetic memory over multiple cell generations.

Histone H3 Variants and Cell Lineage Restriction:The H3 Barcode HypothesisAdult mammals contain hundreds of cell types distributedamong specialized tissues and organs, each with an identicalDNA content. Yet, each of these cell types has a unique patternof gene expression. In simple terms, genes behave in three waysduring development: Some genes are subject to lineage-dependent activation events, such as PAX-5, PU-1, E2A, andEBF, leading to the generation of cell-type-specific precursors,in this case, B cell precursors, in the hematopoietic system (53),whereas others undergo lineage-dependent silencing events,such as X-chromosome inactivation and the silencing of embry-onic genes such as Oct 4 (54). Lastly, the expression of house-keeping genes is maintained constitutively.

Stem cell and animal cloning (nuclear transfer) experimentshint that much of the molecular basis of tissue-specific geneexpression and developmental potential is deeply rooted in thedetails of chromatin structure and epigenetic mechanisms (55).In addition, the intranuclear ‘‘architecture’’ of chromatin likelyhas a bearing on its regulation. Transcriptionally inactive genes,for example, reside in a position near the nuclear periphery (56),or interphase centromeres (57), whereas active genes are main-tained near the center of nuclei. The nuclear location of genesmay therefore affect their transcriptional status, and someevidence suggests that this is a dynamic process involved in celldifferentiation (58).

The extent to which H3 variants factor into these events, if atall, is largely unexplored. We propose that histone H3 variantsplay a major role in cell differentiation and cell lineage restric-tion, and we put forward a speculative hypothesis, the H3barcode hypothesis, to explain how this may occur. Our modelsuggests that mammals have evolved an additional way ofregulating their genetic information over many cell generations.We propose that the mammalian genome is indexed by histoneH3 variants (Fig. 4A) in a nonrandom fashion that reflects theassembly mechanisms and ‘‘personalized’’ chaperones and ex-change factors described above (Fig. 3). We envision that H3.3is incorporated into transcriptionally active regions, whereas, incontrast, H3.2 is deposited in transcriptionally silent areas thatcan be reversibly activated, depending on cellular needs (facul-tative heterochromatin). In our model, H3.1 might then be

localized to genes that are constitutively silent or to genomic locicontaining little or no apparent protein-coding information,whereas CENP-A is localized to highly specialized centromeres(Fig. 4A). If correct, we envision that this barcoding of genomicDNA with histone H3 variants would be subjected to changeduring stem cell differentiation, when chromatin-remodelingevents take place. We further speculate that these remodelingpathways impart a memory to cell lineage-dependent geneexpression in light of the epigenetic inheritance models pre-sented above.

In considering the H3 barcode hypothesis, we propose thatpatterning of histone PTMs would serve to regulate the imme-diate responses of genes to external stimuli and maintain net-works of gene expression or silencing over short developmentaltime periods (Fig. 4B). Our hypothesis suggests that genes areswitched ‘‘on’’ or ‘‘off’’ according to their PTM pattern duringone cell cycle. For long-term memory (many cell generations) ofa cell’s particular ‘‘epigenetic state;’’ however, we propose thatthe selective incorporation of histone H3 variants into variouschromosomal domains plays a role in establishing gene-expression profiles exhibited by a particular cell type at aparticular point in time. In support of this hypothesis, Loppinand coworkers (59) have recently suggested that H3.3 is incor-porated by HIRA chaperone into the chromatin of the malepronucleus in Drosophila, thereby replacing protamines andleading to sperm nucleus decondensation. In the mouse, H3.1 isabsent in the male pronucleus, which is largely decondensed(60), a finding also consistent with our hypothesis. In addition,a recent study from Felsenfeld and colleagues showed that, inchicken erythroid cells, exogenous H3.3 expression resulted inincreased expression of folate receptor and VEGF-D genes,whereas H3 (H3.2) caused decreased expression of these genes,therefore implying a difference in function for H3 (H3.2) andH3.3. All of the above studies support the general notion thatH3.3 is associated with decondensed open chromatin, whereasmammalian H3.1 and chicken H3.2 mark heterochromatin thatis in a ‘‘closed’’ state. An important feature of our hypothesis isthat chromatin structures change during cell differentiationthrough the selective incorporation of different histone H3variants.

By combining all of the above ideas and models, we proposethat it should be possible to distinguish cell types by the genomiclocalization of H3.1, H3.2, and H3.3, producing a pattern orbarcode of staining along chromosomal regions much likecharacteristic band�interband regions of Drosophila polytenechromosomes (Fig. 4A). In this speculative model, chromosomesfrom cell type A contain H3 variants in different genomic locithan chromosomes from cell type B, because different sets ofgenes are activated and�or silenced by selective deposition orexchange of appropriate H3 variants. Additionally, we proposethat each chromosome in any given cell type should have adifferent distribution of the H3 variants along their chromosomearms, outside of more constant chromosomal landmarks such ascentromeres and telomeres that are also likely marked with theirown H3 variant signatures (e.g., CENP-A at centromeric re-gions). One test of the H3 barcode hypothesis would be to displaythe different H3 variants with differentially marked or coloredtags, asking whether a barcode pattern is revealed that differsfrom chromosome to chromosome and cell type to cell type.Ultimately, chromatin immunoprecipitation (ChIP) assays, com-bined with whole-genome microarray and tiling analyses (ChIPby chip; for one example, see ref. 62) will provide a powerful testof these ideas, when the appropriate immunological reagentsbecome available for these highly conserved proteins. As men-tioned above, histone genetics in mammalian models presents achallenge for those histone genes that are present in high copynumbers, such as H3.1 and H3.2. However, because H3.3 isencoded by only two genes in mouse and human (H3.3A and

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H3.3B, each encoding identical H3.3 proteins), histone geneticswith this H3 variant may be possible.

In conclusion, we speculate that at least three differentbiological codes, the genetic code, a PTM histone code, and aH3 barcode (and potentially other histone variant barcodes),may act together to ensure proper gene activation and silenc-ing (Fig. 4B). We favor the view that, at least in mammaliancells, histone H3 variants index the genome as follows: H3.3 islargely, if not exclusively, associated with euchromatin, H3.2with facultative heterochromatin, and H3.1 with constitutiveheterochromatin (although we note that there might be ex-ceptions to this rule). We propose that this barcoding ofgenomic regions with appropriate H3 variants ensures long-term cellular memory of the transcriptional status of genesthat, in addition, can be inherited over many cell generations.On the other hand, we propose that PTMs play an importantrole in the maintenance of these transcriptional stages and arealso involved in the regulation of short-term gene expression.In this view, certain PTMs enable a cell to immediately turnspecific genes on or off after the cell receives appropriate

stimuli, and this switch in gene expression is formally accom-plished without the exchange of one H3 variant with another.Outside of gene regulation, PTMs are likely to contribute toat least two other biological processes, deposition-relatedPTMs (e.g., acetylation of K5�K8�K12 on H4) (63) and,possibly, the active exchange of histone variants, a mechanismabout which not much is known. Taken together, we envisionthat the selective employment of histone H3 variants, togetherwith their PTM signatures, regulate gene expression by bar-coding the genome according to specific functions: H3.3,euchromatin; H3.2, facultative heterochromatin; H3.1, consti-tutive heterochromatin. However, many questions remain tobe answered.

One specific question is how the H3 barcode and the histonecode are connected or how different H3 variants becomeassociated with distinct PTMs in the first place. One possibilityis that the distinct H3 variants, through their ability to differ-entially regulate nucleosome stability, control the precise higher-order folding of chromatin that then makes these fibers suitablesubstrates for the appropriate modifying enzymes. For example,

Fig. 4. The H3 barcode model to index genomic information and ensure epigenetic memory. (A) Theoretical visualization of H3 variants in two chromosomes(1, 2) of A and B cell types show different banding patterns (white with blue dots, H3.1; blue with white dots, H3.2; blue, H3.3). This H3 variant barcode differsfrom chromosome to chromosome and cell type to cell type. In this model, H3.1 localizes to constitutive heterochromatin, H3.2 to facultative heterochromatin,and H3.3 to euchromatin. (B) Graphical combination of the three biological codes: the genetic code, the H3 barcode, and the histone code. DNA contains geneticinformation in the form of genes (white boxes) that have to be activated or silenced at appropriate times and noncoding regions, such as centromeres, telomeres,and satellites (dotted line). Actively transcribed genes contain H3.3 (blue gradient circle) in their chromatin, whereas silenced genes have H3.2 (blue circle withwhite dots) incorporated. A majority of DNA does not contain any meaningful genetic information and also genes, which are constitutively silent. These genomicregions are indexed by the presence of H3.1 (white circles with blue dots) in the chromatin. The next regulatory step to ensure proper gene expression is theregulation of genes with posttranslational histone modifications (green flag, activation PMTs; red and orange flags, different silencing PMTs). We propose thatshort-term alterations in gene expression is achieved by the employment of specialized PMTs (e.g., acetylation), but long-term establishment (epigeneticmemory) of gene expression involves more stable histone modifications as well as the incorporation of the appropriate histone H3 variants.

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H3.3-containing nucleosomes may be less stable, thereby keep-ing chromatin fibers in a somewhat, but precise, unfolded state.These more open fibers may be the preferred substrates foractivating enzymes (such as MLL�Set1, the H3 K4 HMTase). Incontrast, H3.1 and H3.2 may result in generating more stablenucleosomes (in particular H3.1 through disulfide bonds with itscysteine 96) that lead to more compacted or folded chromatinfibers that are the preferred substrates for repressing enzymes(such as Ezh2, the H3 K27 HMTase). Thus, the precise chro-matin structure (or fibers) these variants create and also theirlocalization in the nuclear architecture may be, in part, thereason why they are modified in different ways with PTMs.

Consistent with the H3 barcode hypothesis, the first layer ofchromatin organization (and epigenetic memory) would bedictated by the particular histone variant, whereas the potentialactions of a specific modifying enzyme(s) depends, in part, on theunique structure of that chromatin fiber that the variant gener-ates. In addition, DNA-binding transcriptional activators orrepressors that recognize unique chromatin structures might

recruit the appropriate enzyme(s) and, thereby, prevent inap-propriate marks and create the final biological effect. In support,a subpopulation of H3.3 is phosphorylated during mitosis at itsunique S31 (64). Also, nucleosomes containing H2A.Z are poorsubstrates for certain histone-modifying enzymes (65).

Finally, for the H3 barcode to be functional, it must have acellular reader that interprets or scans the proposed patterns ofH3 variant stripes in their entirety (66). Although such areader(s) has yet to be identified, we suspect that PTMs, carriedby the H3 variants, will hold some clues, if indeed such readersexist. We look forward to experimental tests of this hypothesisand extensions of it in the years to come

We thank all members of the Allis laboratory for insightful discussions.We especially thank E. Bernstein, A. Goldberg, C. Janzen, T. Milne, andJ. Wysocka for critical review of the manuscript. Valuable input was alsoprovided by A. Annunziato, B. Strahl, and M. Smith before the submis-sion of this article. This work was supported by National Institutes ofHealth MERIT Award GM 53512 (to C.D.A.) and The RockefellerUniversity’s Women and Science Fellowship Program (S.B.H.).

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