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how assembly of H3H4 into nucleosomes is regulated is extremely these two processes.

Biochimica et Biophysica Acta 1819 (2012) 238246

Contents lists available at ScienceDirect

Biochimica et Bi

j ourna l homepage: www.e lsimportant and will be the focus of this review.In general, nucleosome assembly is classied into replication-

coupled (RC) nucleosome assembly and replication-independent (RI)nucleosome assembly (Fig. 1A and Table 1). Both RC and RInucleosome assembly processes occur in both yeast and mammaliancells despite the fact that yeast cells have only one form of histone H3,which is most similar to the mammalian H3 variant H3.3. Inmammalian cells, H3.3 is mainly assembled into nucleosomes in a RI

2. Replication coupled (RC) nucleosome assembly

Nucleosomes are barriers for DNA replication and therefore,nucleosomes ahead of the replication fork must be temporarilydisassembled or remodeled in order for the DNA replication machineryto gain access to the DNA. Immediately following DNA replication,replicated DNA is assembled into nucleosomes using both parental and This article is part of a Special Issue entitled: Histoassembly.

Corresponding author. Tel.: +1 507 538 6074; fax:E-mail address: Zhang.Zhiguo@mayo.edu (Z. Zhang)

1 These two authors contributed equally to this work

1874-9399/$ see front matter 2011 Published by Edoi:10.1016/j.bbagrm.2011.06.013nheritance of epigeneticntegrity. Understanding

of RC and RI nucleosome assembly, highlighting the roles of histonechaperones and modications on newly synthesized H3 and H4 intranscription is likely to be a key step in the iinformation and maintenance of genome iChromatin, a complex of DNA andiverse processes including gene tranDNA repair [1]. The fundamental unit oconsisting of 147 bp of DNA wound aphistone octamer of one (H3H4)2 tetraIn order to form nucleosomes, (H3H4followed by the rapid deposition of H2that the assembly of (H3H4)2 tetramelimiting step of nucleosome assemblyinto nucleosomes following DNA repciated proteins, governson, DNA replication andatin is the nucleosome,ately 1.6 turns around ad two H2AH2B dimers.mers are deposited rst,. Therefore, it is believednucleosomes is the rate-ver, assembly of H3H4, DNA repair and gene

into nucleosomes in a RC manner. H3.1 and H3.2 differ by only oneamino acid and therefore, throughout the review, wewill refer to H3.1as the canonical histone H3 in mammalian cells. While H3 moleculesare different between yeast and mammalian cells, most of the histonechaperones, a group of proteins that bind histones and promotenucleosome assembly and/or exchange without being nal products,are conserved from yeast to human cells [2]. Histone chaperones arekey factors in regulating nucleosome assembly. Further regulationcomes from post-translational modications of the histone proteins.Therefore, we will separate our discussion below into the regulationnewly synthesizeassembly of repliDNA replicationassembly to DNAstructure, propagand maintenancmutations in gen

ne chaperones and Chromatin

+1 507 284 9759...

lsevier B.V.1. Introduction manner while the canonical histone H3 (H3.1 and H3.2), whoseexpression peaks during S phase in mammalian cells, is assembledReview

All roads lead to chromatin: Multiple pat

Qing Li 1, Rebecca Burgess 1, Zhiguo Zhang Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, 200

a b s t r a c ta r t i c l e i n f o

Article history:Received 9 May 2011Received in revised form 28 June 2011Accepted 29 June 2011Available online 7 July 2011

Keywords:Nucleosome assemblyHistone modicationHistone chaperoneHIRACAF-1Rtt106

Chromatin, a complex of DNDNA replication and DNA reDNAwound about 1.6 turnsorder to form nucleosomes,H2B. It is believed that thenucleosome assembly. Moreand gene transcription is likof genome integrity. In this ractions of histone chaperonSpecial Issue entitled: Histoays for histone deposition

Street SW, Rochester, MN 55905, USA

d associated proteins, governs diverse processes including gene transcription,. The fundamental unit of chromatin is the nucleosome, consisting of 147 bp ofund a histone octamer of one (H3H4)2 tetramer and two H2AH2B dimers. InH4)2 tetramers are deposited rst, followed by the rapid deposition of H2Aembly of (H3H4)2 tetramers into nucleosomes is the rate-limiting step ofr, assembly of H3H4 into nucleosomes following DNA replication, DNA repairto be a key step in the inheritance of epigenetic information and maintenanceew, we discuss how nucleosome assembly of H3H4 is regulated by concertedand modications on newly synthesized H3 and H4. This article is part of achaperones and Chromatin assembly.

2011 Published by Elsevier B.V.

ophysica Acta

ev ie r.com/ locate /bbagrmd histone proteins. Numerous studies indicate that thecatedDNA into nucleosomes is coupled to the on-going[3,4]. It is hypothesized that coupling nucleosomereplication ensures proper inheritance of chromatination of epigeneticmarks on histones to daughter cellse of genome integrity. Supporting this hypothesis,es involved in DNA RC nucleosome assembly result in

239Q. Li et al. / Biochimica et Biophysica Acta 1819 (2012) 238246increased sensitivity to DNA damaging agents and compromisedmaintenance and inheritance of heterochromatin states in yeast andmammalian cells [59].

At the molecular level, two separate pathways are likely involvedin RC nucleosome assembly. First, parental histones in front of thereplication fork are transferred onto the replicated DNA. While howthis process is coupled to ongoing DNA replication remains elusive,recent evidence indicates that (H3H4)2 tetramers are transferred asa single unit for nucleosome formation. While it has been known for awhile that parental histones do not mix with newly synthesized

Fig. 1. Nucleosome assembly of new H3H4. (A) There are two major nucleosome assembly(RI) nucleosome assembly. Histone chaperone Asf1 binds a H3H4 dimer, which will be t(B) Models of formation of histone (H3H4)2 tetramers, the rst building block of a nucleoso(H3H4)2 tetramer on a monomeric or dimeric form of the histone chaperon. Alternatively, awill deposit two H3H4 dimers sequentially onto DNA for formation of a (H3H4)2 tetram

Table 1A summary of factors involved in RC and RI nucleosome assembly.

Nucleosome assemblypathway

Replication-coupled(RC)

Replication-independent(RI)

Biological process DNA replication Transcription/othersFunction Epigenetic inheritance Histone turnover

Gene expressionHistone chaperone Asf1 Asf1

CAF-1 HIRA(HIR1)Rtt106 (Yeast) Daxx

Rtt106 (Yeast)Histone H3 H3.1 H3.3Histone H3 modicationsa H3K56Ac H3K56Ac

H3 N-terminal tail AcHistone H4 modications H4K5,12Ac H4S47ph

a We mentioned solely those modications that have been shown to have a role inregulating the indicated nucleosome assembly pathways.histones to form nucleosomes during S phase of the cell cycle [1012],several studies proposed that parental (H3H4)2 tetramers may splitinto two dimers for nucleosome formation [13,14]. Recently, usingstable isotope labeling of amino acids in cell culture (SILAC) combinedwith quantitative mass spectrometry (MS), it has been shown thatparental (H3.1H4)2 tetramers do not mix with newly synthesized(H3.1H4)2 tetramers, whereas parental H2AH2B and newly-synthesized H2AH2B can be found within one nucleosome followingDNA replication [15]. These results not only clarify a major question inthe eld, but also reinforce the idea that the assembly of (H3H4)2tetramers, both parental and newly synthesized, is likely to be a keystep in the inheritance of chromatin states and high order chromatinstructure. Since only one daughter cell receives the parental (H3H4)2tetramer at any given DNA position, epigeneticmarks on H3H4 in theindividual nucleosome cannot be maintained. Instead, epigeneticmarks on H3H4 can only be maintained in a group of nucleosomeswithin a functional domain.

Second, newly synthesized H3H4 can be deposited onto replicatingDNA to form nucleosomes in a histone chaperone dependent manner.Below,we introduce the histone chaperones involved in RCnucleosomeassembly and then discuss how newly synthesized (H3H4)2 tetramersare deposited onto replicating DNA during S phase of the cell cycle.

2.1. Histone chaperones involved in de novo nucleosome assembly of H3H4

The classic histone chaperone regulating RC nucleosome assembly ischromatin assembly factor 1 (CAF-1), identied in human cells using a

pathways: replication coupled (RC) nucleosome assembly and replication independentransferred to histone chaperones that are involved in RC or RI nucleosome assembly.me. Two H3H4 dimers from Asf1 can be transferred to a histone chaperone to form oneH3H4 heterodimer will be transferred from Asf1 to another histone chaperone, whicher.

240 Q. Li et al. / Biochimica et Biophysica Acta 1819 (2012) 238246SV40 replication-coupled nucleosome assembly assay [3,16,17]. CAF-1is a three subunit (p150, p60 and RbAp48 in humans) protein complexknown to bind histones H3H4 containing modications indicative ofnewly synthesized H3H4 [18,19]. Thus, CAF-1 is likely involved inpromoting de novo nucleosome assembly of newly synthesized H3H4.Whether CAF-1 has any role in the transfer of parental H3H4 during Sphase of the cell cycle is not known. In human cells, CAF-1 preferentiallyco-puries with the canonical histone, H3.1, in comparison to H3.3,indicative of CAF-1's role in the deposition of H3.1H4during S phase ofthe cell cycle [13]. However, how CAF-1 recognizes H3.1H4 preferen-tially over H3.3H4 remains to be determined.

Anti-silencing factor 1 (Asf1)was discovered as a histone chaperonethat promotes CAF-1 dependent nucleosome assembly [20,21]. Asf1puried from yeast to human cells cannot promote RC nucleosomeassembly by itself using an in vitro SV40 DNA RC nucleosome assemblyassay [22,23], consistent with the idea that Asf1's role in RC nucleosomeassembly is not direct deposition of H3H4 onto replicating DNA.Several studies indicate that Asf1 is likely involved in regulating themodication and/or nuclear import of newly synthesized H3H4. Forexample, in budding yeast, Asf1 is essential for acetylation of histoneH3lysine 56 (H3K56Ac) [2427], a modication on newly synthesized H3[28]. Moreover, genetic studies indicate that Asf1's role in nucleosomeassembly and other processes in budding yeast are likely due to itsability to regulate H3K56Ac [26]. In human cells, there are two sequencehomologs of Asf1,Asf1aandAsf1b. BothAsf1a andAsf1b are required forH3K56Ac in human cells, with Asf1a being the predominant form inregulating H3K56Ac [29,30]. In addition, it is known that Asf1a andAsf1b associate with H3H4 prior to the import of newly synthesizedH3H4 into the nucleus, suggesting that Asf1 in human cells mayregulate nuclear import of new H3H4 [31].

In addition to binding newly synthesized H3H4 and promotingRC nucleosome assembly of newly synthesized H3H4, Asf1 has otherfunctions. First, yeast cells lacking Asf1 exhibit increased supercoilingof an endogenous 2 plasmid, suggesting a role for Asf1 innucleosome disassembly [32]; however, Asf1's role in nucleosomedisassembly is unclear. It is possible that Asf1 alone disassemblesnucleosomes by removing H3H4. Alternatively, Asf1 may collaboratewith ATP dependent chromatin remodeling complexes to disassemblenucleosomes. Second, in Schizosaccharomyces pombe, Asf1 binds thehistone deacetylase Clr6 and is required for deacetylation of histoneH3 lysine 9 [33]. In Saccharomyces cerevisiae, however, Asf1 isrequired for efcient acetylation of H3 lysine 9 [34]. These distinctfunctions of Asf1 in budding yeast and ssion yeast are furtherhighlighted in the fact that Asf1 is essential for cell growth of S. pombe,whereas S. cerevisiae cells lacking Asf1 are viable. Third, human Asf1binds histones with marks representing parental histones, suggestingthat Asf1 may have a role in parental H3H4 transfer [35]. How Asf1 isinvolved in parental histone transfer remains to be determined.

In S. cerevisiae, Rtt106 is another H3H4 histone chaperone found tobe important for mediating de novo histone deposition. Rtt106'sfunction as a histone chaperone was originally identied in a yeastgenetic screen for enhancers of silencing defects of mutant PCNA cellsdefective for CAF-1 binding [36]. Genetic and biochemical evidencesuggest that Rtt106 functions in parallel with CAF-1 to promote RCnucleosome assembly [37,38]. However, it remains to be resolvedexactly how Rtt106 functions in parallel with CAF-1 in nucleosomeassembly. It is possible that CAF-1 and Rtt106 deposit H3H4 at distinctchromatin regions. Alternatively, CAF-1 and Rtt106 may functiondistinctly as histone chaperones for either the leading or lagging strand.Further supporting distinct roles for CAF-1 and Rtt106, Rtt106 bindsdsDNA with no sequence specicity, and this, along with the histonebinding ability, appears to be important for Rtt106's function intranscriptional silencing [39]. Therefore, Rtt106 may have roles outsideof RC nucleosome assembly.

As the assembly of newly-synthesizedH3H4 is tightly coupledwith

DNA replication, it is not surprising that several of the histonechaperones involved in RC nucleosome assembly exhibit interactionswith DNA replication proteins, suggesting that the replication forkmachinery recruit histone chaperones to the fork for histone deposition.CAF-1 is recruited to the replication fork through a direct interactionbetween the large subunit of CAF-1, p150, and PCNA [40,41], theprocessivity factor for the DNA polymerases, an interaction conservedfrom yeast to humans [42]. In yeast, Asf1 directly binds RFC, a proteincomplex that loads PCNAontoDNA [43]. Inmammalian cells, bothAsf1aand Asf1b are found to complex with the replicative helicaseMCM, andthis association is required for fork progression [35]. It is still unclear asto how Rtt106 is recruited to the replication fork.

Furthermore, there appears to be signicant coordination among theH3H4 histone chaperones in order to effectively deposit newly-synthesized H3H4 at the replication fork. Various studies support amodel in which newly synthesized H3H4 molecules bind Asf1. Asf1binds H3 through the same H3 interface involved in formation of(H3H4)2 tetramers, and in vitro, Asf1 disrupts (H3H4)2 tetramers[44]. This raises the following question, how are (H3H4)2 tetramers,the rst building block of a nucleosome, formed during RC nucleosomeassembly? Pull-down experiments have revealed that in mammaliancells, the Asf1-H3H4heterotrimer resides in a complexwith CAF-1 andHIRA [13]. We have shown that Asf1 is required for an efcientassociation of H3H4with the histone chaperones Rtt106 and CAF-1 inyeast [38]. Therefore, Asf1 may transfer histone H3H4 dimers to otherhistone chaperones for deposition onto chromatin [38,45]. Two possiblemodels help explain how newly synthesized (H3H4)2 tetramers areformed (Fig. 1B). First, two H3H4 dimers are deposited sequentially bya histone chaperone onto replicating DNA to form the (H3H4)2tetramer for nucleosome formation. Second, a histone chaperone maybind a single (H3H4)2 tetramer, which in turn, is deposited onto DNAto promote nucleosome formation. Several studies suggest that(H3H4)2 tetramers can be deposited by histone chaperones viaoligomerization of histone chaperone proteins. For instance, Xenopusp150, the large subunit of CAF-1, can formdimers in vitro, and the abilityof p150 to dimerize is important for nucleosome assembly [46]. Morerecently, two members of the NAP (nucleosome assembly protein)family, Nap1 and Vps75, bind histone H3H4 in a tetramer fashion [47];however, Nap1 in cells is a H2AH2B chaperone [48] and Vps75 is acomponent of Rtt109-Vps75 complex [49]. Therefore, the signicance ofNap1 and Vps75 binding to H3H4 remains to be further investigated.Recent studies from our lab indicate that Rtt106 dimerizes and binds a(H3H4)2 tetramer (Fazly et al., unpublished), further supporting thepossibility that (H3H4)2 tetramers are formedprior to thedeposition ofH3H4 onto DNA. Furthermore, Rtt106 directly interacts with CAF-1[37]. These studies highlight the complex interactions occurring amongthe histone chaperones for coordinated deposition of histones. Futurestudies are needed to address how eachhistoneH3H4 chaperone bindsdimeric or tetrameric forms of H3H4 and how the coordination amongthe histone chaperones and these differentH3H4 forms enables propernucleosome assembly (Fig. 1B).

2.2. Roles of modications on newly synthesized H3 and H4 in RCnucleosome assembly

Histone proteins contain a number of post-translational modica-tions. These modications aid in regulating a host of cellular processes[50]. It was observed long ago that newly synthesized histones wereacetylated and that these acetylationmarkswere removed shortly afterdeposition [51,52]. Three acetylation marks on newly-synthesizedhistones are relatively conserved among species: H4 lysine residues 5and 12 (H4K5,12Ac), H3 N-terminal tail acetylation and H3 lysine 56(H3K56Ac) [28,5355]. In addition to acetylation, monomethylation ofhistone H3 K9 (H3K9me) is present on H3.1 prior to deposition inmammalian cells [56]. These modications likely regulate RC nucleo-some assembly via one of the following mechanisms. First, histone

modications may regulate the interactions between histones and

histone chaperones. Second, histone modications on newly synthe-sized histones may be important for regulating histone import ashistones are shuttled fromthecytoplasm into thenucleus fordeposition.Finally, histone modications on newly synthesized histones mayfacilitate the inheritance ofmarksonhistones by aiding in the transfer ofinformation from parental histones to the newly synthesized histones(Fig. 2 and Table 1). Discussed below are the known and/or predictedfunctions of specicmodications foundonnewly synthesizedhistones.

The most highly conserved mark of newly synthesized histones isdiacetylationof histoneH4at lysine residues 5 and12 (H4K5,12Ac) [53].This mark is catalyzed by the HAT1 acetyltransferase complex [5759].While this modication has been known for a long time, only recentlyhas the function of this modication in RC nucleosome assembly cometo light. A detailed study of histone complexes formed from histonesynthesis to deposition indicate that H4K5, 12Ac occurs within thecytoplasm before histones associate with Asf1 and before newly-synthesized histones are imported into the nucleus [31]. Furthermore,an analysis of recombinant protein incorporation into the macropas-modia of Physarum revealed that the nonacetylable, H4K5,12R form ofH3H4 inhibits H3H4 nuclear import while the import of anacetylation mimic, H4K5,12Q, is improved over wild type H3H4 [60].In addition, H4K5,12Ac is present on H4 co-puried with histonechaperone CAF-1 [18,19]. These results suggest H4K5, 12Ac may havemultiple roles innucleosomeassembly. First, itmay facilitate thenuclearimport of H3H4. Second, it may regulate the function of CAF-1 innucleosome assembly. In addition, Hat1 also co-puries with H3.3,raising the possibility that this modication on H4 also impacts the

nucleosome assembly of H3.3 [13,61]. Future studies are needed todetermine towhat extent H4K5, 12Ac andHat1 regulate RC nucleosomeassembly via nuclear import of H3.1H4 and/or the regulation of theassociation between histone chaperones and histones.

In addition to acetylation of newH4, newly synthesizedhistoneH3 isalso acetylated. While acetylation of H3 lysine residues appears to beconserved among species, the acetylation patterns observed aredifferent [53]. In S. cerevisiae, the predominant acetylation on newlysynthesized histones is H3K56Ac, a residue located at the DNAentry/exit site of the nucleosome [28]. In the budding yeast, H3K56Acis catalyzed by the Rtt109-Vps75 complex that utilizes the Asf1H3H4complex as the substrate [2527,49,6264].WhileVps75 is not requiredfor H3K56Ac in vivo[49], Vps75 is required for the nuclear import ofRtt109 as well as the stability of Rtt109 [65,66]. Following deposition,H3K56Ac is deacetylated by Hst3 and Hst4, members of the NADdependent family of histone deacetylase (HDAC) enzymes, during late SorG2/Mphaseof the cell cycle [67,68]. Theprimary role ofH3K56Ac is toregulate RC nucleosome assembly. Supporting this idea, H3K56Ac peaksduring S phase [28]. Using chromatin-immunoprecipitation (ChIP)assay, we have shown that H3K56Ac is deposited only onto replicatingDNA, but not non-replicating DNA, during early S phase of the cell cycle[38]. This assay recapitulates early observations that nucleosomeassembly is coupled to DNA replication [4]. Therefore, this assay islikely to be useful in determining towhat extent a gene is involved in RCnucleosome assembly in budding yeast. Finally, H3K56Ac increases thebinding afnity of H3H4 with two histone chaperones, CAF-1 andRtt106, suggesting that H3K56Ac regulates RC nucleosome assembly by

leosA) Ad HK9m

241Q. Li et al. / Biochimica et Biophysica Acta 1819 (2012) 238246Fig. 2. Roles for post-translational modications on newly-synthesized histones in nucfunction associated with known modications observed on newly-synthesized H3H4. (domain) by Rtt109 regulates the interaction between histone chaperones (like CAF-1) anas a substrate for Suv39, and thereby contributing to the epigenetic inheritance of the H3

the cytoplasm into the nucleus. NPC: nuclear pore complex. Red circles are acetylation marome assembly. Described are the enzymes, post-translational modication (PTM) andcetylation of the H3 N-terminal tail by Gcn5 and Rtt109 and H3K56 (within the H3 core3H4. (B) H3K9me, catalyzed by SetDB1 (in complex with CAF-1 and HP1alpha), servese3 mark. (C) Hat1 catalyzes H4K5,12Ac, which may help regulate histone import from

ks, and yellow triangles are methylation marks.

242 Q. Li et al. / Biochimica et Biophysica Acta 1819 (2012) 238246increasing the binding afnity of H3H4 with specic histonechaperones [38].

Cells lacking Rtt109 or those expressing a non-acetylation stateH3K56Rmutant aremuchmore sensitive toward DNAdamaging agentsthan cells lacking both Rtt106 and CAF-1 [38]. This result suggests thatH3K56Ac may also promote nucleosome assembly mediated by otherhistone chaperones in addition to CAF-1 and Rtt106. Alternatively, itimplies that H3K56Ac has other functions in addition to its role in RCnucleosome assembly. Indeed, H3K56Ac is also important for nucleo-some assembly following DNA repair, which is important for therecovery from checkpoint activation [69]. Furthermore, H3K56Ac hasbeen shown to be involved in gene transcription [70,71] as well as theregulation of RI nucleosome assembly (see discussion below).

Inmammalian cells, H3K56Ac abundance is relatively low comparedto yeast cells [30,67,72], leading one to inquire to what extent thismodication plays a regulatory role in RC nucleosome assembly.Compared to H3K56Ac in budding yeast, the regulation of H3K56Ac inmammalian cells appears to be more complicated. For instance, whilethis modication is dependent on Asf1a and Asf1b in mammalian cells[29,30], there are at least two acetyltransferases, p300/CBP and Gcn5,reported to acetylate mammalian H3K56 [30,73], whereas Gcn5 inbudding yeast is not required for H3K56Ac [74]. In addition, severalH3K56Ac deacetylases including Sirt1, Sirt6, Hdac1 and Hdac2 havebeen reported [29,30,75,76]. Finally, there are conicting reports onwhether this modication is regulated during S phase of the cell cycleand how H3K56Ac levels change in response to DNA damage agents[29,30,73]. These conicting reports likely result from the followingpossibilities. First, since the levels of H3K56Ac are low in mammaliancells, much more specic antibodies are needed to detect thismodication by Western blot in mammalian cells. Therefore, theantibodies used by different groups from different sources maycontribute to such conicting results. Second, compared to yeast cellsthathave only one formofH3, humancells containH3.1 andH3.3,whichcannot be differentiated by Western blot. Therefore, different enzymescatalyzingH3K56Acmay targetH3.1 andH3.3differently. Future studiesare needed to resolve these issues.

In addition to H3K56Ac within the H3 core domain, acetylation oflysine residues at the N-terminus of H3 also regulates RC nucleosomeassembly [77]. Acetylation patterns on the N terminal tails of new H3vary among species. For instance, in yeast, H3K9 and K27 areacetylated, in Drosophila, H3K9 and K14, and in humans, H3K9 andH3K18 [53,55,72]. Both yeast Elp3 and Gcn5, two acetyltransferaseswith well-established roles in gene transcription, function withRtt109 to acetylate the H3 N-terminal tail of newly-synthesizedhistones and promote nucleosome assembly in a manner parallel toH3K56Ac [74,78]. Supporting this idea, Gcn5, although it does notdirectly regulate levels of H3K56Ac in yeast, is important for properdeposition of H3K56Ac at the replication fork. Furthermore, whileH3K56Ac is important for both CAF-1 and Rtt106 to bind H3H4, Gcn5and H3 N-terminal tail acetylation appear to only regulate theinteraction between CAF-1 and histones H3H4 [74].

In addition to a role in nucleosome assembly, Gcn5 and acetylationof the H3 N-terminus are likely to have a role in the initiation of DNAreplication [79,80]. The levels of acetylation at multiple lysineresidues on H3 and H4 in nucleosomes surrounding a replicationorigin increase during S phase. Interestingly, in the case of H3,multiple sites tend to be acetylated together. Mutational analysissuggests that acetylation at these sites functions redundantly in originring as the quantitative number of acetylation marks is moreimportant in regulating origin ring than acetylation at a particularsite [79]. Because nucleosome positioning at origins affects originring [81], it would be challenging to separate the roles for Gcn5 andacetylation of lysine residues at the H3 N-terminus in origin ringfrom their roles in nucleosome assembly.

Although histone lysine acetylation is predominantly a mark of

newly synthesized histones,mammalianH3.1 ismonomethylated at H3lysine 9 (H3K9me) by SETDB1 on more than one third of the H3.1 pool[56]. SETDB1 forms a complex with both CAF-1 and HP1 during S-phaseand preferentially methylates free histones over nucleosomal histones,suggesting it methylates histones prior to deposition. It is proposed thatH3K9me may serve as a precursor to set up H3K9me3, catalyzed bySuv39, at pericentric heterochromatin sites and thus play a role in thepropagation of this histonemark. Consistent with this idea, knockdownof SETDB1 results in lower levels of H3K9me3 [82]. Furthermore, kineticstudies on the levels of methylation of newly synthesized histonessuggest that monomethylation of many residues most likely occursconcurrent with or shortly after deposition to enable further higherorder methylation at particular sites [83]. A recent report suggests thatH3K9me1 is one of the rst modications observed on H3.1 followingsynthesis, and the levels ofH3K9me1are sharply reduced in conjunctionwith the appearance of H4K5,12Ac, suggestive of a role of thismodication in earlier histone processing [31]. Despite these advancesin understanding how this mark occurs on newly synthesized histones,the exact role of this abundant modication on newly-synthesized H3remains to be determined.

In summary, modications on newly synthesized H3 and H4 likelyplay multiple roles in the regulation of RC nucleosome assembly,whether it be through the regulation of histone nuclear import, histonechaperone and histone interactions, and/or other functions.

3. Replication independent (RI) nucleosome assembly

While canonical histone H3 is strictly incorporated into chromatinduring S phaseof the cell cycle, themajority of histoneH3variantH3.3 isassembled into nucleosomes in a replication independent (RI) manner[15,84]. This RI process likely involves the exchange of parental H3,including both canonical H3 and H3 variant H3.3, with new H3.3 tocompensate for nucleosome loss and/or replacement of damagedhistones in non-dividing cells such as neurons. In addition, RInucleosome assembly is also important during gene transcription andother cellular processes.

Most of the early studies on RI histone exchange focused on histoneH2AH2B exchange. H2AH2B exchange occurs namely at activelytranscribed regions and the 5 end of the genes, raising the possibilitythat histone exchange may be an important form of transcriptionalregulation [85,86].While the frequency ofH3H4 incorporation ismuchlower thanH2AH2B,H3H4exchangedoesoccur. The observation thatH3H4 incorporation independent of DNA replication occurs arose froman elegant study using a chromatin fractionation assay in immatureerythrocytes [87]. In addition, histone H3H4 exchange can be detectedin the presence of the transcription inhibitor actinomycin D [88],suggesting that mechanisms other than gene transcription are likelyinvolved in RI histone exchange. Supporting this idea, genome widemapping of H3.3 shows that H3.3 is enriched at both active and inactivegene promoters [89,90]. Increased histone exchange is also observed atboundary elements that block the spread of chromatin states in yeastand Drosophila[91,92]. Together, these studies suggest that histoneexchange may have an important regulatory role in chromatindynamics.

Similar to the problem arising during DNA replication, nucleosomesserve as barriers for gene transcription, and therefore, changes inchromatin structure are often a prerequisite for the initiation of genetranscription and transcriptional elongation. Genome wide nucleosomepositioningmappinghas revealed that twowell-positionednucleosomesank the transcription start site (TSS) with a nucleosome-depletedregion (NFR) in between (reviewed in [93]). Inmammalian cells, theNFRis likely to be marked by an unstable nucleosome containing H3.3 andH2AZ [90]. Thedynamics of nucleosomes at thepromoter likely provide amechanism for regulating gene expression.

During transcriptional elongation, nucleosomes are temporallydisassembled in front of the RNA Polymerase (Pol) II complex to

facilitate passage of the transcriptional machinery. Following gene

243Q. Li et al. / Biochimica et Biophysica Acta 1819 (2012) 238246transcription, nucleosomes must be reassembled by recycling theparental histone (H3H4)2 and/or incorporation of newly synthesizedhistones (replacement/exchange). Two different mechanisms areproposed to efciently restore the chromatin structure followingpassage of RNA Pol II [94]. First, partial loss or exchange of core histoneswas observed at highly transcribed gene regions [95100]. Second,H2AH2B exchange, but not H3H4 exchange, was detected atmoderately transcribed gene regions [101,102]. As mentioned above,genome wide mapping of H3.3 occupancy reveals that H3.3 is enrichedat gene bodies of both active and inactive genes in Drosophila andmammalian cells [84,90], suggesting that nucleosome assembly and theexchange of H3.3 occurs in both a transcription dependent andindependent manner. Consistent with this idea, despite having onlyone form of H3, histone H3H4 replacement/exchange also occurs atboth active and inactive gene promoter regions in budding yeast[103]. Collectively, these studies suggest that histone depositionand/replacement during gene transcription may regulate gene expres-sion and/or maintain gene expression state.

3.1. Histone chaperones involved in replication-independent nucleosomeassembly

Like RC nucleosome assembly, RI nucleosome assembly/exchange ismediated byhistone chaperones.Multiple histone chaperones includingAsf1, Hir1, Rtt106 and Spt6 have been shown to be involved in RInucleosomeassembly in budding yeast. TheHir1 complex (consisting ofHir1, Hir2, Hir3, and Hpc2 in budding yeast) is the major histonechaperone involved in RI nucleosome replacement [104,105]. In highereukaryotes, HIRA, the sequence homolog of Hir1, is well known topromote the assembly of histone H3.3 into chromatin independently ofDNA replication [13,106,107]. Similar to RC nucleosome assemblymediated by CAF-1, Asf1 is proposed to deliver H3H4 to HIRA forsubsequent nucleosome assembly.

In human cells, Asf1a and Asf1b appear to have distinct functions inregard to the regulation of RI nucleosome assembly. For instance, HIRApreferentially bindsAsf1aoverAsf1b [108].Moreover, depletionofAsf1b,butnotAsf1a, results in cell proliferationdefects [109]. Interestingly, bothAsf1a and HIRA promote the formation of senescence associatedheterochromatin foci [110]. These results raise the possibility thatAsf1a is theprimaryhistonechaperone that transfers histones toHIRA fornucleosome assembly.

While it is still not clear why cells need so many histone chaperonesfor RI nucleosome assembly, based on studies of HIRA and Daxx (Deathdomain containing protein) inmammalian cells, it is possible that thesehistone chaperones promote RI nucleosome assembly at distinctchromatin regions to regulate gene expression at different chromatindomains. Daxxhas been identied as anotherH3.3 histone chaperone inmammalian cells [61,89,111]. In contrast to HIRA which is required fordeposition of H3.3 at genic regions, Daxx is required for the localizationofH3.3 at telomeric repeats [89]. Interestingly, Daxx contains anRtt106-like domain at the C terminus, and this domain contributes to the H3.3binding afnity [61]. Therefore, it is possible that while Daxx does notshare signicant homology with Rtt106, Daxx may be the functionalhomolog of Rtt106 in human cells. In addition to Daxx and HIRA, otherH3.3 chaperoneswill likely be identied because it has been shown thatthe H3.3 occupancy at regulatory elements is independent of eitherDaxx or HIRA [89].

Unlike RC nucleosome assembly where the coordination betweenDNA replication and nucleosome assembly is carried out by therecruitment of histone chaperone complexes via direct interactionswith the replication machinery, it is still unclear as to how the histonechaperones involved in RI nucleosome assembly are recruited tochromatin. Accumulating evidence suggests that histone chaperoneslikely interact with transcriptional machinery and chromatin remod-eling complexes to promote RI nucleosome assembly and regulate gene

expression. For instance, Spt6 interacts with RNA Pol II physically andpromotes nucleosome disassembly in order to facilitate the passage ofRNA Pol II followed by nucleosome reassembly [112]. Similarly, thehistone chaperone complex FACT, which has multiple roles, was rstindentied through co-purication with RNA Pol II and is important forboth transcription initiation and elongation (Reviewed in [113]).Second, Drosophila Asf1 interacts with the Brahma chromatin remodel-ing complex [114]. Third, HIRA interactswith the chromatin remodelingcomplex Chd1 [115]. Lastly, Daxx interacts with the SWI/SNF typechromatin-remodeling factor ATRX (-thalassemia/mental retardationsyndrome protein), and the deposition of H3.3 onto telomere andpericentric DNA repeat regions depends on ATRX [61,89]. Supportingthis idea, it has been shown that ATRX is localized at repetitive DNAsequences [116]. It is worth noting that some histone chaperones, suchas DEK, can serve as transcription coactivators [117]. These studiessuggest that multiple factors may aid in recruiting histone chaperonecomplexes for RI nucleosome assembly, and such events may serve asanother form of regulation. Further studies should be carried out todetermine how these different modes of RI nucleosome assemblypathway impact gene transcription.

3.2. Role of histone modications in RI nucleosome assembly

Unlike modications on H3 and H4 that function in RC nucleosomeassembly, the roles ofmodications onnewH3andH4 inRI nucleosomeassembly has not been well explored. However, several studies suggestthat posttranslationalmodications onH3 andH4 also likely regulate RInucleosome assembly as well. For instance, genome wide measure-ments of H3K56Ac turnover in yeast cells reveal that H3K56Ac marksthe most dynamic nucleosomes [103]. This result suggests that inaddition to its role in RC nucleosome assembly, H3K56Ac likely alsoimpacts RI nucleosome assembly. Supporting this idea, Rtt109 and Asf1,two regulators of H3K56Ac, promote histone replacement/exchangeduring gene transcription. Interestingly, Vps75, a histone chaperonefacilitating the enzymatic activity of Rtt109, inhibits histone replace-ment, suggesting a possible feedback mechanism for nucleosomereplacement [103]. Inmammalian cells, quantitativemass spectrometryanalysis of histonemodications uncovered that histone turnover ratesdepend upon both site-specic histone modications and the aminoacid sequence of histone variants [118]. In general, histones containingactive marks have a faster turnover rate than histones containingrepressive marks, consistent with the idea that gene transcriptioninuenceshistone turnover and/or assembly. Interestingly, the turnoverrate of the H3K56Ac peptide is faster than many other histonemodications [118]. Despite these interesting observations, detailedstudies on the role of modications on H3 and H4 in RI nucleosomeassembly are needed to understand to what extentmodications on H3and H4 function in RI nucleosome assembly/exchange. Furthermore,since specic chromatin domains have distinct nucleosomal histonemodications [119,120], it is also possible that these histone modica-tions also regulate histone turnover rates and drive the histonedeposition pathways. Recently, our laboratory has found that phos-phorylation of histone H4 S47 (H4S47ph), catalyzed by the Pak2 kinase,promotesHIRAmediated nucleosome assembly of H3.3H4and inhibitsCAF-1 mediated nucleosome assembly of H3.1H4. This suggests thatmodications on histone H4 can help specify nucleosome assembly ofH3.1 and H3.3 [121].

4. Summary and future directions

Over the last several years, we have witnessed explosive interest inaddressing the regulation of nucleosome assembly pathways by histonechaperones andhistonemodications and the functional implicationsofRC and RI nucleosome assembly in the inheritance of chromatinstructure and regulation of gene expression. However, there is still a

plethora of unanswered questions waiting to be addressed.

244 Q. Li et al. / Biochimica et Biophysica Acta 1819 (2012) 238246Emerging evidence indicates that nucleosomes are much moredynamic thanpreviously recognized. In addition, deposition of newH3H4 and transfer of parental H3H4 during DNA replication and DNArepair proceed with rapid speed. Therefore, technical limitations havemade it extremely difcult to spatially and temporally monitor thedynamics of histone deposition in live cells. Hopefully, advances insingle cell imaging will enable us to design approaches to analyze howparental histones H3H4 are transferred onto replicated DNA during Sphase of the cell cycle and how multiple histone deposition pathwaysfunction coordinately to promote nucleosome assembly [122].

Second, in addition to regulating gene transcription, H3.3 likelyhas roles in heterochromatin formation [123]. Studies are needed toaddress how H3.3 performs these functions and to what extent thesedifferent functions are linked to histone chaperones that deposit H3.3to distinct chromatin domains.

Third, yeast models have aided immensely in understandingregulatory mechanisms for nucleosome assembly, such as H3K56Acand the regulation of histone chaperone interactions with H3H4. Itremains not well explored on how RC and RI nucleosome assemblypathways are regulated in higher eukaryotes. Given that nucleosomeassembly in mammalian cells is much more complex, it is likely thathistone modications play a signicant role in regulating RC and RInucleosome assembly in mammalian cells. Indeed, we have recentlydiscovered thatphosphorylationofhistoneH4promotesHIRAmediatednucleosome assembly and inhibits CAF-1 mediated nucleosomeassembly [121]. Moreover, covalent modications on histone chaper-ones are also detected and these modications likely impact thefunction of histone chaperones [124].

Lastly, several histone chaperones have been shown to interact withATP dependent nucleosome remodeling complexes for nucleosomeassembly and/or gene regulation. We suspect that future studies willreveal additional interactions amonghistone chaperones and chromatinremodeling complexes. The challengewill be to determine how histonechaperones coordinate with chromatin remodeling complexes duringdifferent cellular processes.

Acknowledgments

This work is supported by NIH grants to ZZ. ZZ is a scholar of theLeukemia and Lymphoma Society. RB is supported by an AmericanHeart Association Predoctoral Fellowship. We apologize to thosewhose publications have not been cited in this review.

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246 Q. Li et al. / Biochimica et Biophysica Acta 1819 (2012) 238246

All roads lead to chromatin: Multiple pathways for histone deposition1. Introduction2. Replication coupled (RC) nucleosome assembly2.1. Histone chaperones involved in de novo nucleosome assembly of H3H42.2. Roles of modifications on newly synthesized H3 and H4 in RC nucleosome assembly

3. Replication independent (RI) nucleosome assembly3.1. Histone chaperones involved in replication-independent nucleosome assembly3.2. Role of histone modifications in RI nucleosome assembly

4. Summary and future directionsAcknowledgmentsReferences