histone chaperone jun dimerization protein 2 (jdp2): role in … · 2016. 12. 12. · nonspecific...

The structure of chromatin, which influences numerousDNA-associated phenomena, such as transcription, re-plication, recombination and repair, is controlled by acomplex combination of histone modifications, ATP-dependent chromatin-remodeling enzymes, and nucle-osome-assembly factors. The modification of histonesoccurs at their N-terminal tails, which can be modifiedby acetylation, phosphorylation, methylation, ubiq-uitination, sumoylation, and ADP-ribosylation [1,2].

Kaohsiung J Med Sci October 2010 • Vol 26 • No 10 515© 2010 Elsevier. All rights reserved.

Received: Jun 2, 2010 Accepted: Jun 22, 2010Address correspondence and reprint requests to:Dr Kazunari Kzaushige Yokoyama, Center ofExcellence for Environmental Medicine, GraduateInstitute of Medicine, Kaohsiung MedicalUniversity, 100 Shih-Chuan 1st Road, San MingDistrict, 807 Kaohsiung, Taiwan.E-mail: [email protected]

HISTONE CHAPERONE JUN DIMERIZATION PROTEIN 2(JDP2): ROLE IN CELLULAR SENESCENCE AND AGING

Yu-Chang Huang,1 Shigeo Saito1,2 and Kazunari Kzaushige Yokoyama1,3,4

1Center of Excellence for Environmental Medicine, Graduate Institute of Medicine, Kaohsiung MedicalUniversity, Kaohsiung, Taiwan; 2Saito Laboratory of Cell Technology, Yaita, Tochigi, 3Department ofMolecular Preventive Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, and

4Gene Engineering Division, RIKEN BioResource Center, Koyadai, Tsukuba, Ibaraki, Japan.

Transcription factor Jun dimerization protein 2 (JDP2) binds directly to histones and DNA, andinhibits p300-mediated acetylation of core histones and reconstituted nucleosomes that containJDP2-recognition DNA sequences. The region of JDP2 that encompasses its histone-binding domainand DNA-binding region is essential to inhibit histone acetylation by histone acetyltransferases.Moreover, assays of nucleosome assembly in vitro demonstrate that JDP2 also has histone-chaperoneactivity. The mutation of the region responsible for inhibition of histone acetyltransferase activitywithin JDP2 eliminates repression of transcription from the c-jun promoter by JDP2, as well asJDP2-mediated inhibition of retinoic-acid-induced differentiation. Thus JDP2 plays a key role asa repressor of cell differentiation by regulating the expression of genes with an activator protein 1(AP-1) site via inhibition of histone acetylation and/or assembly and disassembly of nucleosomes.Senescent cells show a series of alterations, including flatten and enlarged morphology, increase innonspecific acidic β-galactosidase activity, chromatin condensation, and changes in gene expres-sion patterns. The onset and maintenance of senescence are regulated by two tumor suppressors,p53 and retinoblastoma proteins. The expression of p53 and retinoblastoma proteins is regulatedby two distinct proteins, p16Ink4a and Arf, respectively, which are encoded by cdkn2a. JDP2 inhibitsrecruitment of the polycomb repressive complexes 1 and 2 (PRC-1 and PRC-2) to the promoter of thegene that encodes p16Ink4a and inhibits the methylation of lysine 27 of histone H3 (H3K27). The PRCsassociate with the p16Ink4a/Arf locus in young proliferating cells and dissociate from it in senescentcells. Therefore, it seems that chromatin-remodeling factors that regulate association and dissoci-ation of PRCs, and are controlled by JDP2, might play an important role in the senescence program.The molecular mechanisms that underlie the action of JDP2 in cellular aging and replicativesenescence by mediating the dissociation of PRCs from the p16Ink4a/Arf locus are discussed.

Key Words: Arf, Jun dimerization protein 2, p16Ink4a, polycomb repressive complex,replicative senescence

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Among these modifications, the acetylation of histonesoccurs at highest frequency and has been studied mostextensively, with the resultant identification of a num-ber of histone acetyltransferases (HATs) and histonedeacetylases (HDACs), together with their target lysineresidues within specific histones in the chromatin. Theactivities of HATs are not only antagonized by HDACsbut are also regulated by cellular and viral regulatoryfactors that include histone chaperones and enzymesthat catalyze posttranslational modification [3,4].

Chromatin consists of structural units known asnucleosomes. Each nucleosome consists of two histoneH2A–H2B dimers, a histone (H3–H4)2 tetramer, andDNA that is wrapped around the resultant histoneoctamers. During chromatin assembly, a histone(H3–H4)2 tetramer is formed before the two hetero-dimers H2Aand H2B are incorporated to form a nucleo-some [5,6]. Regulation of transcription is associated withalterations in chromatin structure that include histonemodifications and changes in nucleosome structure[7–10]. Compaction of the chromatin and organizationof nucleosomes represent a barrier that has to be over-come prior to activation of transcription. The N-terminalhistone tails that protrude from the nucleosomes do notplay a significant role in nucleosome formation, but theyappear to act as docking sites for other proteins and pro-tein complexes to regulate chromatin compaction [9].

The structure of chromatin changes to allow greateraccessibility by transcription factors when a gene isto be activated [9]. It has been suggested that thechange to a more accessible state not only involves themodification of histones and alterations in nucleoso-mal arrays, but also results from changes in nucleo-some integrity that are due to displacement of histones[11]. Furthermore, it has been demonstrated that his-tone chaperones play a critical role in these processes[12–15]. Thus it is tempting to speculate that histonechaperones might be important for the compaction of chromatin, and it is now important to determinewhether certain co-repressors of transcription mightinfluence the deposition and assembly of nucleosomesvia regulation of histone-chaperone activity.

Primary cultures of untransformed cells stopgrowing after several weeks and undergo senescence,a phenomenon that is related to so-called cellularaging. Senescence protects normal cells from abnor-mal growth signals and oncogenic transformation, andimpairs their reprogramming to pluripotent stem cells[16] by interrupting the cell cycle. Senescence is induced

not only by cellular aging, but also by the forced activation of the mitogen-activated protein (MAP)kinase pathway and by genotoxic stressors, such ashydrogen peroxide and certain DNA-damaging com-pounds. Senescence increases the activities of two well-known tumor suppressors, retinoblastoma protein (Rb)and p53 protein. The expression of Rb and p53 is regu-lated by two distinct proteins, p16Ink4a and Arf, respec-tively, which are encoded by the cdkn2a locus [17].The expression of p16Ink4a increases dramatically withincreasing numbers of cell divisions in primary fibro-blasts in culture, and in rodent and human models in vivo. In this review, we summarize the roles ofp16Ink4a and Arf in the cell cycle, and describe a novelmechanism for the regulation of their expression.

P16INK4A AND THE RB PATHWAY

Genes that are essential for cell-cycle progression aretranscribed at the beginning of G1 phase by the E2Ffamily of transcription factors. E2F is controlled by theRb family of proteins, pRb, p107, and p130 [18,19].Early in G1, unphosphorylated Rb proteins bind tothe E2F family of proteins and inactivate their func-tion [20,21]. During G1, the Rb proteins are inacti-vated by phosphorylation by cyclin dependent kinase4/6 (Cdk4/6)–cyclin D complexes, thereby allowingthe transcription of E2F-dependent genes, includingcyclin E. Upregulated cyclin E forms a complex withcdk2 that mediates the hyperphosphorylation of theRb proteins, which is an essential requirement forG1/S transition. p16Ink4a is an allosteric inhibitor ofcdk4/6. Binding to p16Ink4a changes the conformationsof cdk4/6, which prevents its interaction with cyclin D[22,23]. Therefore, p16Ink4a acts as an inhibitor of the cellcycle at G1 by modulating the Rb pathway. p16Ink4a isoften lost in a variety of human malignancies, includ-ing glioblastoma, melanoma, and pancreatic adenocar-cinoma [24]. In contrast, the upregulation of p16Ink4a

induces cell-cycle arrest and senescence [23,24].

ARF AND THE P53 PATHWAY

p53 is known to mediate cell-cycle arrest in G1 and G2,and apoptosis. A number of downstream targets of p53are involved in these processes, including p21Cip/waf1

in G1 arrest [25], 14-3-3 sigma and growth arrest and

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DNA damage inducer gene 45 (GADD45) in G2 arrest[26,27], and p21, Bax, PI-3 up-regulation modulatorof apoptosis (PUMA), Fas/Apo1, and Killer/DR5 inapoptosis [28–32]. p53 is regulated at the levels of pro-tein stability and activity, and to some extent at tran-scription and translation [33,34]. In unstressed cells,p53 protein levels are very low because its degrada-tion is mediated by the E3 ubiquitin ligase activity ofmurine double minute 2 (MDM2), which targets p53for ubiquitin-dependent proteolysis [35]. MDM2 is atranscriptional target of p53, therefore, p53 directlyactivates expression of its own negative regulator,which produces a potent negative feedback regulatoryloop [36]. There are several stress-responsive kinases,which, by phosphorylating p53, inhibit p53 degrada-tion by MDM2 and increase its transcriptional activ-ity [37–39]. DNA damage rapidly activates the ataxiatelangiectasia mutated and ataxia telangiectasia relatedproteins, which phosphorylate the checkpoint kinases1 and 2 (Chk1 and Chk2), which in turn propagate thesignal to downstream effectors such as p53 [40,41].Chk1 and Chk2 phosphorylate p53 at Ser 20, whichprevents the efficient recruitment of MDM2. Thusp53 is stabilized and its expression level is increasedin response to stress signaling. Arf is predominantlylocalized in the nucleoli and is stabilized by bindingto nucleophosmin. In response to stress signaling, Arfis released from nucleophosmin and translocates tothe nucleoplasm, where it interacts with MDM2,inhibits its E3 ubiquitin ligase activity, and blocks thenucleocytoplasmic shuttling of the MDM2–p53 com-plex. Therefore, the consequences of the activation ofArf are stabilization and activation of p53 [42,43].

TRANSCRIPTIONAL REGULATION OF P16INK4A

The transcriptional regulation of the p16Ink4a gene is animportant event in cellular senescence. Expression ofp16Ink4a is increased during replicative senescence andin the premature senescence induced by oncogenicactivation. Its expression is regulated by transcriptionalactivators such as Ets1/2 and the basic helix-loop-helix(b-HLH) protein E47, as well as by transcriptionalinhibitors including the Id-1 HLH protein [44–47]. Thep16Ink4a locus is also epigenetically repressed by thepolycomb repressive complexes 1 and 2 (PCR-1 andPCR-2, respectively), which methylate lysine 27 of his-tone H3 (H3K27) [48]. In transformed cells, in which

the cell cycle is not arrested by a senescence program,the CpG islands in the p16Ink4a promoter and exon 1 aremethylated and the p16Ink4a gene is silenced [49,50].

Understanding the role of all the factors that regu-late the expression of p16Ink4a is important in clarifyingthe molecular mechanism of cellular aging. We describehere some of these factors, including transcriptionalactivators and inhibitors, and epigenetic regulators.

Transcriptional activatorsBoth Ets1 and Ets2 activate p16Ink4a expression throughactivation of the Ras-MEK-MAP kinase pathway bydirectly binding to the ETS consensus sites in its pro-moter [47]. In human fibroblasts, the hyperactivationof Ets2 by overexpression of the Ras oncogene inducesG1 arrest, premature senescence, and increased expres-sion of p16Ink4a. Ets2 seems to be the main regulatorof p16Ink4a expression during oncogenic prematuresenescence, whereas Ets1 plays a role in replicativesenescence [51,52]. The basic helix-loop-helix (b-HLH)protein E47 binds to DNA and proteins through itsbasic and HLH domains, respectively. The E47 homod-imer binds specifically to the E box (CANNTG) in thep16Ink4a promoter [46]. Overexpression of E47 inhibitsthe proliferation of some tumor cell lines by inducingexpression of p16Ink4a. Inhibition of E47 by RNA inter-ference significantly reduces expression of p16Ink4a

and delays the onset of senescence [44]. Similarly, theheterodimerization of E47 with ectopically expressedTal1 inhibits expression of p16Ink4a [53].

Transcriptional inhibitorsThe expression of inhibition of differentiation protein-1(Id-1) correlates negatively with p16Ink4a expressionduring the process of senescence [44,47]. The expres-sion of p16Ink4a in mouse embryonic fibroblasts (MEFs)is higher in those from Id-1-deficient mice than inwild-type MEFs [38]. Overexpression of Id-1 delaysreplicative senescence by inhibiting p16Ink4a in humankeratinocytes and endothelial cells [54,55]. Id-1 doesnot have a basic DNA-binding domain, unlike the b-HLH protein E47. Instead, Id-1 inhibits transcrip-tion of p16Ink4a by heterodimerization with Ets2 [47].Id-1 also heterodimerizes with E47, and inhibits itstranscriptional activity [56].

Epigenetic regulatorsThe p16Ink4a locus is also regulated epigenetically. Thelocus is transcriptionally silenced by the trimethylation

Control of senescence by JDP2

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of lysine 27 of histone H3 (H3K27) in young proli-ferating primary cells. In contrast, the expression ofp16Ink4a increases in aged and senescent cells with theloss of H3K27 trimethylation [57]. Methylation ofH3K27 and silencing of the p16Ink4a locus are medi-ated by PRC1 and PRC2. PRC1 contains a number of subunits, including polycomb (CBX2, 4, 6, 7 or 8 inhumans), polyhomeotic (PH1 or PH2 in humans), B lymphocyte molony murine leukemia virus insertionregion 1 homology (Bmi1), Ring1B, and other sub-units [58], whereas PRC2 is composed of enhancer ofZeste 2 (Ezh2), suppressor of Zeste 12 protein homolog(Suz12), and embryonic ectoderm development (Eed)subunits [48,59]. In PRC2, Ezh2 is the catalytic subunitthat methylates H3K27 [48], whereas the other com-ponents are indispensible for the function of the com-plex. Suz12 is essential for complex formation and di- and trimethylation of H3K27 in vivo [60,61]. In con-trast, Eed is required for global H3K27 methylation,including its monomethylation [62]. PRC1, the CBXsubunit recognizes and binds to trimethylated H3K27[63,64]. Ring1B has E3 ligase activity for the ubiquity-lation of histone H2A, whereas Bmi1 acts as a cofactor[65,66]. Ubiquitylation of H2A by PRC1 preventstranscript elongation by RNA polymerase II [67].

PRC1 has been shown by electron microscopy tocontribute to chromatin compaction [68]. Therefore, a possible molecular mechanism of PRC-mediatedgene silencing might involve trimethylation of histoneH3K27 by Ezh2 and the other subunits of PRC2. Then,this acts as a binding site for PRC1, which ubiquitylatesH2A and compacts the chromatin, and leads to inhi-bition of transcript elongation by RNA polymerase II.

PRCs might also inhibit earlier steps in transcrip-tion, because PRC1 interacts with components of thebasal transcription machinery, the TATA-box-bindingprotein-associated factors [69,70]. This inhibition doesnot appear to involve blocking the access of RNA poly-merase to the promoter, because the PRCs and tran-scription factors bind to the target genes at the sametime [68,70–73]. In summary, p16Ink4a transcription isinhibited by PRC2, which methylates histone H3K27,which in turn recruits PRC1. PRC1 represses p16Ink4a

expression in young proliferating cells. In aged andstressed cells, H3K27 trimethylation markers of PRC1are lost and PRC1 dissociates from the p16Ink4a locus,which results in transcriptional activation of p16Ink4a

by Ets1 and/or Ets2, and the entry of the cells intosenescence.

An important aim is to understand how the H3K27trimethylation signal is lost in senescent cells. The pos-sible explanations are as follows: (1) the action of as-yet-unidentified histone demethylases and/or inhibitorsof the methyl transferases involved in H3K27 trimethy-lation; or (2) histone chaperones recruit histone vari-ants to the chromatin to alter gene expression. A recentstudy has shown that the H3K27-specific demethy-lase, Junonji domain-containing protein 3 (JMJD3) isinduced by Ras–Raf signaling, as well as by environ-mental stresses. JMJD3 is recruited to the p16Ink4a locusand contributes to the transcriptional activation ofp16Ink4a [74,75]. We propose that senescence is regu-lated by Jun dimerization protein 2 (JDP2) of p16Ink4a,which has histone-binding and chaperone activities[74,75]. JDP2 is a member of the activator protein 1(AP1) family of transcription factors, and activatesthe transcription of p16Ink4a, as described below [76].

REGULATION OF ARF

The contribution of Arf to senescence is still controver-sial. In general, it seems that p16Ink4a plays a centralrole in senescence and tumor suppression in humancells, whereas Arf has a relatively more prominent rolein mouse cells. In human cells, mutations are specifi-cally found in p16Ink4a, rather than in Arf. Mutations ofp16Ink4a are frequently observed in primary cancers,and occur during the establishment of immortal celllines [24,77]. Signaling by the Ras oncogene and telom-ere shortening also induce p53- and Arf-independentgrowth arrest [78,79]. In contrast, in MEFs, expressionof Arf correlates with the onset of senescence, and cellsthat lack Arf do not become senescent in culture [80,81].Mouse strains with targeted deletions in p16Ink4a orArf are tumor prone, whereas animals that lack p16Ink4a

and Arf have a more severe phenotype [53,81–84].Signaling from oncogenic Ras activates transcription ofthe cyclin-D-binding Myb-like protein 1 gene (Dmp1)via the MAP kinase pathway and AP1 transcriptionfactors, such as c-Jun and Jun-B. DMP1 binds to andactivates transcription of the Arf promoter [85]. Thispathway is important, because oncogenic Ras fails toactivate Arf in MEFs from Dmp1-null mice [86].

Curiously, factors that activate Arf expression canhave different phenotypic effects: Ras induces sen-escence, whereas Myc induces apoptosis. The over-expression of Myc in B lymphocytes augments cell



proliferation, which is counteracted by the Arf–p53–MDM2 pathway. Suppression of this pathway inhibitsMyc-induced apoptosis and facilitates formation ofB-cell lymphoma [87]. However, another study hasshown that induction of Arf requires high and con-tinuous Myc activity, and that physiological levels ofMyc are insufficient to stimulate the Arf promoter [88].

E2F TRANSCRIPTION FACTORS ACTIVATETHE ARF PROMOTER

E2F1 stimulates expression of Arf and activates theArf–p53–p21WAF1 axis, which blocks cell proliferation.This block is removed by the loss of function of theArf–MDM2–p53 pathway, which results in E2F1-induced S-phase entry [89].

E2F family members bind directly to the Arf pro-moter, as shown by chromatin immunoprecipitationassay [90–92]. Ectopic expression of E2F1, E2F2 andE2F3 activates the Arf promoter in human cells [90)]In contrast, an isoform of E2F3, E2F3b, represses theArf promoter in MEFs [91]. Among the AP1 family of transcription factors, the c-Jun- and Fos-relatedantigen-1 (Fra1) heterodimer is an activator of Arftranscription in human and mouse cells. The knock-down of Fos-related antigen-1 in human cells or adeficiency of c-Jun in MEFs results in reduced expres-sion of Arf [93]. In contrast, JunD seems to be a repres-sor of Arf, because MEFs that lack JunD expresselevated levels of Arf, and display p53-dependentgrowth arrest and premature senescence [94]. Arf isalso repressed transcriptionally by early growth re-sponse protein 1 (Egr1) and Zbtb7a (pokemon). Egr1-null MEFs express increased levels of Arf, but escapereplicative senescence with reduced expression of p53,p21Cip1/Waf1 and other p53 downstream proteins [95].Zbtb7a-null MEFs become senescent prematurelybecause of the upregulation of Arf expression, becausesenescence can be overcome by mutation of Arf [96].

SENESCENCE AND AGING IN HUMANSAND MICE

Cellular senescence appears to be related to organismaging. Cellular senescence involves processes thatinclude telomere shortening, accumulation of DNAdamage, and activation of the p16Ink4a/Arf locus. The

contributions of these factors to senescence seem todiffer in humans and mice. Cultured mouse fibrob-lasts undergo senescence even when they have longtelomeres and high telomerase activity. Senescence isabrogated by the loss of the p16Ink4a/Arf locus [82]. Inhuman cell cultures, the ectopic expression of telom-erase is sufficient to overcome senescence by main-taining the length of the telomeres [97]. In mice, themaintenance of telomere length is important becausetelomerase deficiency shortens their lifespan andleads to premature aging [98–100]. The age-dependentaccumulation of INK4A has been observed in humankidneys and skin [101,102], as well as in the majorityof mouse tissues [100,103]. In oncogene-induced senes-cence, there is in vivo evidence that Arf is the impor-tant factor in the activation of p53 tumor suppression[103,104]. However, another study has shown thatcomponents of the DNA-damage signaling cascade,including ataxia telangiectasia mutated (Atm) and Chk2proteins, are crucial for activation of p53 in responseto oncogenic signals [105–107]. These differencesbetween humans and mice could be attributable tospecies specificity and/or experimental conditions.Cellular senescence appears to be related to organismaging because the same processes appear to be in-volved. Genetic variants of the p16Ink4a/Arf locus arelinked to age-associated disorders, such as generalfrailty, heart failure, and type 2 diabetes [108–113].Mutations in telomerase or proteins that affect telom-erase activity are linked to premature human aging syn-dromes, including congenital dyskeratosis and aplasticanemia [114]. There are increases in DNA mutations,DNAoxidation, and chromosome loss during organismaging. It seems reasonable to assume that all threefactors, activation of the p16Ink4a/Arf locus, telomereshortening, and accumulation of DNA damage, havecooperative effects on aging in physiological situations.Understanding the mechanisms of cellular senescenceis currently of wide interest and it is important that weidentify new components of this process, such as JDP2.

HISTONE CHAPERONES

Activation and repression of gene expression at thechromatin level are subject to regulation by enzymesthat reorganize and rearrange the chromatin template[10]. Both ATP-dependent chromatin-remodeling com-plexes and histone chaperones mediate an increase in



the accessibility of promoters to transcription factors,and facilitate histone-exchange reactions within thenucleosomes [115,116]. Histone-exchange reactions areimportant because they allow histone variants to beincorporated into nucleosomal arrays independentlyof the synthesis of DNA. The deposition of histonevariants, such as H2A.Z, has a major impact on geneexpression because these variants are known to belocalized within specific transcriptional domains andwithin the heterochromatin [117,118].

In yeast cells, transcription is accompanied by thedisruption of nucleosomal structures, which suggestsan inverse correlation between gene activation andnucleosome assembly [119]. Although sliding mecha-nisms have been proposed to explain this phenome-non, experimental evidence suggests that nucleosomedisplacement can occur via disassembly of the nucle-osome that involves the removal of histones [7–10].Analysis of the expression of an inducible gene forinterleukin-2 also suggests the removal of histones fromthe promoter of this mammalian gene upon activationof transcription [120]. Histone chaperones are key play-ers in the removal of histones in this context [121].For example, nucleosome disassembly is mediatedby anti-silencing function 1 (ASF1)/CCG-1-interactingfactor (CIA), which is a histone chaperone and is essen-tial for activation of gene transcription [121]. Duringgene repression, however, ASF1/CIA-independentreassembly of nucleosomes is observed in the samesystem [121]. Thus regulation of transcription includesthe disassembly of nucleosomes for gene activation,and deposition of histone nucleosomes and assemblyof nucleosomes for establishment of the basal or re-pressed state. Some histone chaperones function withcomponents of several chromatin-related complexes,which include HATs [122], HDACs [123], and theATP-dependent chromatin-remodeling complex [124].Thus, acting in concert with other chromatin-relatedfactors, histone chaperones play important roles innuclear events that involve chromatin templates.

The histone chaperone known as nucleosome-assembly protein-1 (NAP-1) acts autonomously toexchange H2A–H2B dimers and to incorporate his-tone variants into intact nucleosomes [118,125]. In vitro,NAP-1 has been shown to facilitate the binding oftranscription factors to sites of nucleosomes, and thisprocess requires the disassembly of nucleosomes [126].NAP-1 also associates with the co-activator p300/CBP,with the resultant enhancement of p300-mediated

activation of transcription [127]. NAP-1 is, to date,the best characterized histone-chaperone protein. Thecrystal structure of NAP-1 reveals that it includes along α helix, which is responsible for homodimeriza-tion, and a four-stranded antiparallel β sheet, as doother histone-chaperone proteins [128].

A protein that is involved in the sorting of proteinsto vacuoles, designated Vps75, has been identified asa member of the NAP-1 family of histone-chaperoneproteins in yeast [129]. It has a preference for (H3–H4)2

tetramers and forms a complex with Rtt109 (a proteinthat participates in the regulation of Ty1 transposition)[130]. The complex is composed of acetylated-onlyfree histone H3 in vitro, with no detectable activitytowards nucleosomal H3 [131]. Thus we speculatethat acetylation of lysine 56 of H3 is, at least in part,regulated by the inability of Rtt109–Vsp75 HAT com-plexes to acetylate nucleosomal H3 during the G2/Mphase of the cell cycle [131]. During DNA replication,Rtt109 acetylates lysine 56 of histone H3 to promotegenome stability [132,133] in the premeiotic and mei-otic S phases, and its activity is detectable when DNAis damaged [134,135]. Efficient acetylation by Rtt109requires a histone-chaperone protein, either ASF1 orVsp75, but not chromatin-assembly factor-1 (CAF-1)[130]. By contrast, chromatin-assembly factor-1 andRtt106p mediate the formation of heterochromatin bycontributing to the spreading of Sir protein during theearly stages of heterochromatin formation [136].

English et al [137] have determined the crystal struc-ture of a histone chaperone (ASF1/CIA) that binds toa histone H3–H4 heterodimer. ASF1/CIA physicallyblocks formation of (H3–H4)2 tetramers and the Cterminus of H4 undergoes a dramatic conformationalchange upon binding to ASF1. Within nucleosomes, theC terminus of H4 (residues 93–102) makes contactwith H2A and is bound within the nucleosome [5].During nucleosome disassembly, H2A–H2B dimersare removed by other histone chaperones and ATP-dependent remodeling complexes, which exposes theC terminus of H4 in the tetrasome complex [5,137,138].

ASF1 recognizes and captures the C terminus ofH4 in the tetrasome, which is the site of complexchanges in the conformation of the H4 C terminus inthe ASF–(H3–H4)2 complex. ASF1 then dissociates theH3–H4 dimer that is bound to ASF1. Other histonechaperones and chromatin-remodeling complexesmight also be involved in the dissociation of the(H3–H4)2 tetramer. The reverse relationship might be a



feature of nucleosome assembly. The histone (H3–H4)2

tetramer-disrupting activity of ASF1/CIA and the cry-stal structure of the ASF1/CIA–histone H3–H4 dimercomplex provide some insight into the mechanismsof both the assembly and disassembly of nucleosomes.

Recently Muto et al [139] have reported a compar-ison of the crystal structures of template-activatingfactor (TAF)-1β and NAP-1. They have found that thetwo proteins are folded similarly, with the exception ofthe inserted helix. These backbone helices are shapeddifferently, and the relative disposition of each back-bone helix and the “earmuff” domains between thetwo proteins differ significantly. The histone-chaperoneactivity is associated with the lower surface of theearmuff domains [139]. Akai et al have reported thatthe molecular complex between CIA/ASF-1 and thedouble bromodomain complex plays a key role in site-specific histone eviction at the active promoter [140].

DOMAINS THAT INTERACT WITHCHROMATIN

We have defined five groups of nucleosome-bindingdomains: bromodomains, chromodomains, WD40repeats, tudor domains, and PHD fingers. Bromo-domains recognize and bind acetylated lysine residues[141,142]. For example, general control nonrepressive5 (Gcn5) contains a bromodomain that is importantfor the binding, recognition and retention of SAGAon acetylated promoter nucleosomes [143]. Chromod-omains bind methylated lysine tails specifically. Thechromodomain of heterochromatin protein 1 bindsmethylated lysine 9 of H3, whereas the chromod-omain of Polycomb binds methylated lysine 27 of H3[144–148]. The chromodomain of Eaf3, a protein sub-unit shared by the HAT complex NuA4 and the HDACcomplex Rpd3S, is important for the recognition of andbinding to methylated lysine 36 of H3 [149–151]. Lossof this binding leads to increased acetylation in tran-scribed regions and the formation of spurious tran-scripts that are initiated within open reading frames[151]. Unlike acetylation, methylation can occur once,twice or three times, generating mono-, di- andtrimethylated histones, and the protein domains thatbind to methylated histones associate differentiallywith histones with varying levels of methylation. TheWD-domain-containing protein WDR5 binds dimethy-lated lysine 4 of H3 preferentially in vitro, in addition to

associating with a complex that also has HAT activity[152]. The crystal structure that demonstrates recogni-tion by WDR5 of lysine 4 dimethylated H3 shows howthe WDR repeats distinguish between the di- andtrimethylated lysine 4 residues of H3 [153,154]. Bothso-called tudor domains and WD-domain-containingproteins are present in various HAT complexes, includ-ing SAGA. Recently, a plant homeodomain, the PHDfinger, which has been found in a number of proteinsin various HAT complexes, has been shown to bindto trimethylated lysine 4 of H3 [155–157]. An emergingtheme for these various chromatin-binding domainsis that they are often associated with enzymes thatmodify or alter chromatin, such as Eaf3 and WDR5[157,158], in the case of Gcn5 HAT [149]. To under-stand fully the functions of these domains, we needto study their locations within relevant protein complexes.

INTERACTION OF TRANSCRIPTION FACTORSWITH HISTONES AND NUCLEOSOMES

Transcription factor IIIA (TFIIIA) is a 40-kDa protein,with nine zinc finger domains, that binds specificallyto the internal promoter of the gene for 5S RNA and tothe N-terminal tail domains of histones H3 and H4,but not those of H2A and/or H2B, and directly mod-ulates its ability to bind nucleosomal DNA [159,160].

A new class of HAT-regulatory proteins has beenidentified. These proteins block HAT activity via bind-ing to and masking of the histone themselves. Thisclass includes the subunits of the inhibitor of histoneacetyltransferase (INHAT) complex; TAF-1α, TAF-1β,and pp32, as well as ataxin 3; silencing mediator orretinoid receptor corepressor/nuclear hormone recep-tor corepressor; and proline-, glutamate- and leucine-rich protein 1 [161–167]. Thanatos-associated protein 7is known to associate with TAF-1β and to represstranscription by inhibition of histone acetylation[168,169]. This is a novel co-repressor that activatesINHAT binds directly to nucleosomes and core his-tones, and prevents acetylation by HATs, thus actingas a bona fide INHAT [170]. PU.1, a member of the Etsfamily of oncoproteins, inhibits CBP-mediated acety-lation of GATA-binding protein-1 (GATA-1) and ery-throid Krüppel-like factor 2 (EKLF2), as well as ofhistones, and disrupts acetylation-dependent tran-scriptional events [171]. Moreover, PU.1 recruits the



retinoblastoma tumor-suppressor protein, a histonemethyltransferase Suv39H, and heterochromatin pro-tein 1 [172]. Similarly, proliferating cell nuclear anti-gen binds to p300 to inhibit its HAT activity in vitroand to block HAT-dependent transcription in vivo[173]. TAF-1β and pp32 have also been shown tointeract with estrogen receptor-α (ER-α), to inhibit theERα-mediated activation of transcription, and activa-tion of ER-α acetylation by p300 [166,167]. Moreover,TAF-1β also interacts with the Sp-1 transcription fac-tor and with KLF5, negatively regulating binding toDNA and activation of transcription by these factors[174,175]. Transcription factor nuclear factor-κB p50can accommodate distorted, bent DNA within thenucleosome [176], whereas the DNA-binding domainof c-Myb binds to the N-terminal tails of H3 and H3.3,and binding of c-Myb facilitates acetylation of histonetails [177]. Moreover, kinetochore-null protein 2, whichhas a c-Myb-like DNA-binding domain is specificallyrequired for loading of centromere-specific variantsof histone H3 (centromere protein A) in nematodesand mammalian cells [178]. The recruitment of re-pressive macroH2A nucleosomes requires directinteractions between ATF-2 bound to the nearby AP-1 site and macroH2A and it is regulated by DNA-induced protein allostery [179]. Thus the above-mentioned sequence-specific DNA-binding factorsmight regulate transcription either via histone coresor tails, as well as via the structure of nucleosomes, in conjunction with other proteins that bind to the chromatin.

JDP2 REGULATES AP-1-MEDIATEDACTIVATION OF TRANSCRIPTION

JDP2 has been identified as a binding partner of c-Junin yeast two-hybrid screening experiments, based onthe recruitment of the SOS system [180]. JDP2 formsheterodimers with c-Jun and represses AP-1-mediatedactivation of transcription [180]. Similarly, JDP2 wasisolated by yeast two-hybrid screening with activa-tion transcription factor-2 (ATF-2) as the “bait” [181].JDP2 has also been shown to associate with theCAAT/enhancer-binding protein-γ [182] and theprogesterone receptor [183]. JDP2 is constitutivelyexpressed in many cell lines and represses the tran-scriptional activity of AP-1 [184]. Moreover, JDP2 israpidly phosphorylated at threonine residue 148 when

cells are exposed to UV irradiation, oxidative stress,or inhibitor-induced depressed levels of translationby JNK [185]. Although a novel JNK-docking domainis necessary for the p38-mediated phosphorylation ofJDP2 at threonine residue 148, this domain is not suf-ficient for this process [186]. JDP2 binds to both cAMP-and TRE-response elements on DNA as a homodimer,and as a heterodimer with ATF-2 and members of theJun family, respectively [180,181]. JDP2 inhibits UV-induced apoptosis by suppressing the transcription ofthe p53 gene [187]. Given the roles of AP-1 in cellulartransformation and the reported repression of Jun-and ATF-2-mediated transcription by JDP2, we havedemonstrated that JDP2 inhibits the oncogenic trans-formation of chicken embryonic fibroblasts [188]. JDP2also modulates the expression of cyclin D1 and p21,which have opposing effects on cell-cycle progression.JDP2 interferes with the progression of the cell cycleby reducing the levels of cyclin D1 and at the sametime, increases the expression of p21 [189,190]. Theforced expression of JDP2 promotes the myogenicdifferentiation of C2C12 cells, which is accompaniedby the formation of C2 myotubes and the strong expres-sion of major myogenic markers. Moreover, the ectopicexpression of JDP2 in rhabdomyosarcoma cells inducesincomplete myogenesis and the incomplete forma-tion of myotubes. These cells become committed todifferentiation via the p38-MAPK pathway [190]. Asimilar enhancement of cell differentiation has beenreported during induction of osteoclast formation bythe receptor activator of the nuclear factor κB ligand(RANKL) [191]. Unlike other members of the AP-1family, the levels of JDP2 remain constant in responseto a large variety of stimuli, such as UV, irradiation,and retinoic acid (RA), which affect the levels of otherfactors involved in cell-cycle control. The inductionof JDP2 expression has only been observed duringthe differentiation of F9 cells to muscle cells andosteoclasts. Therefore, JDP2 might provide a thresh-old for exit from the cell cycle and a commitment todifferentiation. Further studies on regulation of thecell cycle and differentiation of cells induced by JDP2should be instructive. It is also interesting that JDP2is one of the candidate oncoproteins that collaboratein the oncogenesis associated with the loss of p27, as aresult of insertional mutations [192]. Recent studies oftumor cells have demonstrated that JDP2 is a tumorsuppressor [193]. We have also found that JDP2 is arepressor of activation of transcription via AP-1, and



a negative regulator of RA-induced differentiation ofmouse embryonic F9 cells [194,195].

JDP2 INHIBITS HAT ACTIVITY

We have reported previously that JDP2 represses thetransactivation mediated by p300 [195]. Both p300and ATF-2 have HAT activity [196,197]. It was recentlyshown that p300 acetylates ATF-2 protein in vitro atlysine residues 374 and 357 and that ATF-2 is essen-tial for the acetylation of histones H4 and H2B in vivo[198,199]. We found that acetylation by p300 is inhibitedin a dose-dependent manner by JDP2, when addedexogenously. We also found that JDP2 was not acety-lated by p300 under our experimental conditions. Theinhibitory effect of JDP2 on histone acetylation is in-duced by p300, CREB binding protein (CBP), p300/CBPassociated factor (PCAF), and Gcn5. Overexpressionof JDP2 apparently represses RA-induced acetylationof lysine residues 8 and 16 of histones H4 and H3.

JDP2 HAS INTRINSIC NUCLEOSOME-ASSEMBLY ACTIVITY IN VITRO

The TAF-Iβ protein, which is a component of theINHAT complex identified by Seo et al [162,163], is ahistone chaperone that binds directly to core histonesand facilitates the assembly of nucleosomes in vitro.JDP2 interacts directly with all the core histones testedand inhibits the p300-mediated acetylation of those his-tones. To our surprise, JDP2 also introduces super-coils into circular DNA in the presence of core histones,to levels similar to those observed for yeast CCG1-interacting factor 1 protein (yCia1p) and CIA1. There-fore, JDP2 appears to have significant histone chaperoneactivity in vitro [195]. We have also shown that theHAT-inhibitory activity of JDP2 is involved, to someextent, in the repression of transcription by JDP2,whereas the maximal capacity of JDP2 to suppressthe RA-mediated activation of the c-Jun promoter(Figure 1) [195; Figure 1] and to suppress adipocytedifferentiation [200] requires recruitment of HDACs.



p300, pCAF, X2

HDAC3

NCoR/SMART

X1 = ATF-2, ATF-7, JunD, JunB, JDP2 etc.

+ RA

p38 a/b

PCAFIni1/SNF5

BRG1-basedSWI/SNFATPase

P P

P

PSWI/SNF

BAF60

RARE DRE

X1 = ATF-2, ATF-7, JunD, JunB etc.X2 = c-jun etc.

TATA

TAFs

IID/Pol II

TBPp300p160

X2 X1RXRRAR

P

JDP2, NCoR/SMART, HDAC3

DRE

K

Ac Ac Ac AcAc

Ac Ac

AcAcTAF12(TAF4)

AcAcAcAc

K KKK KK K

K K K K K K K K

K K K KK K

X1JDP2H3KH3KH4K16H4K8

c-Jun

Figure 1. Schematic representation of the signal pathways of retinoic acid-induced differentiation of mouse embryonic carcinoma F9cells. At the undifferentiated stage of F9 cells, histone deacetylase 3, nuclear hormone receptor corepressor, silencing mediator orretinoid receptor corepressor and Jun dimerization protein 2 are recruited to the differentiation response element in the promoter regionof the c-jun gene to induce heterochromatin. In response to retinoic acid, the signals of p38, BRG1-based SWI/SNF complex, Ini1/Snf5complex and p300/PCAF complex are recruited to the differentiation response element of the c-jun promoter, and the c-jun gene isfinally activated.

JDP2 REGULATES REPLICATIVE SENESCENCE

We have analyzed the aging-dependent proliferationof MEFs from Jdp2–/– transgenic mice in the pres-ence of environmental (20%) or low (3%) oxygen. TheJdp2–/– MEFs continued to divide, even after 6 weeks,whereas the wild-type MEFs almost stopped prolifer-ating and entered senescence under environmentaloxygen. Conversely, neither wild-type nor Jdp2–/–

MEFs succumbed to replicative senescence at loweroxidative stress. These results demonstrate that MEFsthat lack Jdp2 can escape from the irreversible growtharrest caused by environmental oxygen. The expres-sion of p16Ink4a and Arf were repressed in aged Jdp2–/–

MEFs (40 days) compared with their levels in wild-type MEFs. In 3% oxygen, at the equivalent time (40days), wild-type MEFs expressed lower levels ofp16Ink4a and Arf compared with those in 20% oxygen,whereas Jdp2–/– MEFs maintained low-level expres-sion of p16Ink4a and Arf. These observations indicatethat the aging-associated expression of p16Ink4a andArf is dependent on oxygen stress and that JDP2 con-trols the expression of both p16Ink4a and Arf. We foundno dramatic downregulation of the upstream repres-sors of p16Ink4a/Arf, Bmi1 and Ezh2, in the absence ofJDP2, which suggests that JDP2 does not regulatetheir expression. JDP2 expression in wild-type MEFsincreases in the presence of 20% oxygen, but not in

the presence of 3% oxygen, which suggests that itsexpression depends on oxygenic stress, and that accu-mulated JDP2 plays a role in the transcriptional acti-vation of p16Ink4a/Arf. Studies based on chromatinimmunoprecipitation have demonstrated that methy-lation of H3K27 at the p16Ink4a/Arf locus is higher inJdp2–/– than wild-type MEFs, and that the binding ofPRC1 and PRC2 to the p16Ink4a and Arf promoters ismore efficient in Jdp2–/– MEFs. These observationssuggest that, in the absence of JDP2, H3K27 is methy-lated by PRC2 and the p16Ink4a/Arf locus is silenced byPRC1, whereas the increased expression of JDP2 helpsto release PRC1 and PRC2 from the p16Ink4a/Arf locus,thereby reducing H3K27 methylation. Our data dem-onstrate that JDP2 is an important factor for regula-tion of cellular senescence. The loss of JDP2 allowsMEFs to escape senescence and conversely, overex-pression of JDP2 induces cell-cycle arrest. The absenceof JDP2 reduces the expression of both p16Ink4a andArf, which inhibit cell-cycle progression. We proposea model that takes into account these results. Theaccumulation of oxidative stress and/or other envi-ronmental stimuli during aging upregulate JDP2expression in primary untransformed cells. IncreasedJDP2 helps to remove PRC1 and PRC2, which areresponsible for the methylation of histone H3, fromthe p16Ink4a/Arf locus, which leads to increasedp16Ink4a and Arf expression and entry into senescence



Jdp2−/−

Jdp2−/−

WT

WT

Senescenced

Jdp2−/−

Proliferative

Proliferative

Proliferative

Ink4a

Arf

Up-regulation

Low oxygen (3%)

High oxygen (20%)

WTYoungMEF

Figure 2. Model for the epigenetic regulation of the p16Ink4a/Arf locus by Jun dimerization protein 2 (JDP2). Young primary cellsexposed to oxidative stress accumulate JDP2. In the presence of JDP2, polycomb repressive complexes 1 and 2 dissociate from thep16Ink4a/Arf locus, and histone H3 on the promoter is demethylated. Finally, p16Ink4a and Arf are expressed and the aged cells becomesenescent.

[76; Figure 2]. There is some evidence that Jdp2 acts as a tumor suppressor: Jdp2 inhibits the Ras-dependent transformation of NIH3T3 cells [193] andJdp2 gene disruptions are often found in lymphomasinduced by insertional mutagenesis caused by theMoloney murine leukemia virus in MYC/Runx2transgenic mice [201]. Here, we suggest that Jdp2 notonly inhibits the transformation of cells, but alsoplays a role in the induction of cell senescence as wellas cell cycle arrest [202]. These functions of JDP2might be important for its role in inhibiting tumorformation. Our findings also provide new insightsinto the molecular mechanisms by which senescenceis induced in the context of the epigenetic regulationof the p16Ink4a/Arf locus.

CONCLUDING REMARKS

Like differentiation and tumorigenesis, senescence isassociated with dynamic changes in gene expression,which are regulated by chromatin remodeling. Here,we have shown that the expression of p16Ink4a andArf is upregulated in response to accumulating envi-ronmental stresses, oncogenic signaling, and DNA-damaging signals, and that they in turn induce

irreversible cell-cycle arrest by activating the Rb andp53 pathways, respectively. The expression of p16Ink4a

and Arf is epigenetically regulated by PRC1 and PRC2,which associate with these loci, methylate histone H3in young cells, and dissociate in aged and senescentcells. Several factors that upregulate or downregu-late the expression of the p16Ink4a/Arf locus have beenreported. It is now important to establish how thesedifferent factors regulate senescence. Do they affectonly euchromatin or do they also regulate hetero-chromatin? Do they modify the chromatin structureby recruiting HDACs, HATs, histone methyltrans-ferases, histone chaperones, and/or other molecules?Addressing these precise functions in the context ofepigenesis should help us to understand how senes-cence and, in a broader context, aging, are regulated.

ACKNOWLEDGMENTS

The authors thank Drs K. Itakura, G. Gachelin, R. Chiu, R. Eckner, P.-T. Yao, T. Kouzarides, C. D. Allis,D.M. Livingston and M. Horikoshi for their helpfuldiscussions and reagents. This work was supportedby the BioResource Center of RIKEN (to K.K.Y) and a grant from Taiwan KMU (KMU-EM-99-3 to K.K.Y.).



Stress

JDP2up-regulation

PRC1

Bmi

PRC2

Ezh

Histone Histone

Histone

Ink4a/Arf locus of aged cells

p16Ink4a expressionp19Arf expressionGrowth arrestSenescence

On

PRC1

Bmi

PRC2

Ezh

Histone

Me

Histone

Me

Histone

Me

Ink4a/Arf locus of young cells

Cell growth

Off

Figure 3. Proposed model of the epigenetic regulation of the expression of the genes for p16Ink4a and Arf by Jun dimerization protein 2(JDP2). Exposure of young primary cell cultures to aging stress leads to the accumulation of JDP2. JDP2 binds to histones and inhibitsthe methylation of H3K27 at the p16Ink4a/Arf locus. As a result, polycomb repressive complexes 1 and 2 fail to form stable repressivecomplexes and are released from the locus. The consequent expression of p16Ink4a and Arf in the aged cells leads to senescence.

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