Molecular Dissection of Formation of Senescence-Associated Heterochromatin Foci

Senescence is characterized by an irreversible cell proliferationarrest. Specialized domains of facultative heterochromatin,called senescence-associated heterochromatin foci (SAHF), arethought to contribute to the irreversible cell cycle exit inmany senescent cells by repressing the expression of proliferation-promotinggenes such as cyclin A. SAHF contain known heterochromatin-formingproteins, such as heterochromatin protein 1 (HP1) and the histoneH2A variant macroH2A, and other specialized chromatin proteins,such as HMGA proteins. Previously, we showed that a complexof histone chaperones, histone repressor A (HIRA) and antisilencingfunction 1a (ASF1a), plays a key role in the formation of SAHF.Here we have further dissected the series of events that contributeto SAHF formation. We show that each chromosome condenses intoa single SAHF focus. Chromosome condensation depends on theability of ASF1a to physically interact with its depositionsubstrate, histone H3, in addition to its cochaperone, HIRA.In cells entering senescence, HP1 ${gamma}$ , but not the related proteinsHP1 ${alpha}$ and HP1ß, becomes phosphorylated on serine 93.This phosphorylation is required for efficient incorporationof HP1 ${gamma}$ into SAHF. Remarkably, however, a dramatic reductionin the amount of chromatin-bound HP1 proteins does not detectablyaffect chromosome condensation into SAHF. Moreover, abundantHP1 proteins are not required for the accumulation in SAHF ofhistone H3 methylated on lysine 9, the recruitment of macroH2Aproteins, nor other hallmarks of senescence, such as the expressionof senescence-associated ß-galactosidase activityand senescence-associated cell cycle exit. Based on our results,we propose a stepwise model for the formation of SAHF.

INTRODUCTION

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Introduction

Senescence was initially described as a stable cell proliferationarrest resulting from the progression of primary human fibroblaststhrough a finite number of population doublings in vitro (35).However, activated oncogenes, oxidative stress, DNA damage,and drug-like inhibitors of specific enzymatic activities alsoinduce senescence (14, 37, 82). In addition, senescence occursin other cell types, such as primary human epithelial cells.In vivo, senescence is an important tumor suppression mechanismthat restrains the proliferation of cells that harbor activatedoncogenes (12, 16, 17, 51). Also, by limiting the self-renewalcapacity of adult tissue stem cells, senescence is thought tocontribute to tissue aging of many multicellular adult animals(38, 42, 53).

Senescent cells are typically characterized by a large flatmorphology and the expression of a senescence-associated ß-galactosidase(SA ß-gal) activity of unknown function (16, 21).In the nucleus of senescent cells, the chromatin undergoes dramaticremodeling through the formation of domains of facultative heterochromatincalled senescence-associated heterochromatin foci (SAHF) (56,57, 86). Cytologically, SAHF appear as compacted punctate DAPI(4,6-diamidino-2-phenylindole)-stained foci of DNA in senescentcell nuclei. The formation of SAHF is also reflected in a generalincrease in the resistance of nuclear chromatin to digestionby nucleases (57). SAHF contain modifications and associatedproteins characteristic of transcriptionally silent heterochromatin,such as methylated lysine 9 of histone H3 (H3K9Me), heterochromatinprotein 1 (HP1), and the histone H2A variant macroH2A. In addition,Narita et al. recently showed that high-mobility group A (HMGA)proteins, a family of abundant non-histone chromatin proteins,are essential structural components of SAHF (56). Proliferation-promotinggenes, such as E2F target genes (e.g., cyclin A), are recruitedinto SAHF, dependent on the pRB tumor suppressor protein, therebyirreversibly silencing expression of those genes.

Recently, we showed that two chromatin regulators, histone repressorA (HIRA) and antisilencing function 1a (ASF1a), drive the formationof SAHF in human cells (86). HIRA and ASF1a are the human orthologsof proteins known to create transcriptionally silent heterochromatinin yeasts, flies, and plants (9, 29, 39, 54, 63, 70-73, 78).In Saccharomyces cerevisiae, Hir1 and Hir2 are required forheterochromatin-mediated silencing of histone genes, telomeres,and mating loci, and the formation of pericentromeric chromatinstructure (39, 70-73). Likewise, yeast Asf1p is required forheterochromatin-mediated silencing of telomeres, mating loci,and histone genes (40, 50, 70, 73, 75, 78) but also mediatesnucleosome disassembly (2, 3, 68). Both HIRA and ASF1a bindto histones and exhibit histone chaperone activity in vitro(28, 64, 70, 78, 79). The HIRA/ASF1a-containing complex preferentiallydeposits the histone variant histone H3.3 into nucleosomes (46,65, 76). Canonical human histone H3.1 and histone H3.3 differin their primary amino acid sequences by only five amino acids.However, histone H3.1 is expressed periodically in the S phaseof the cell cycle and is incorporated into chromatin duringreplication-coupled chromatin assembly (5, 36, 76). In contrast,histone H3.3 is expressed throughout the cell cycle and is incorporatedinto chromatin by the HIRA/ASF1a complex in a DNA replication-and repair-independent manner (5, 36, 76). Consistent with theirpartially overlapping biological and biochemical properties,yeast Asf1p and Hir proteins physically interact, and this interactionis necessary for telomeric silencing (19, 70). Likewise, theformation of SAHF in human cells by HIRA and ASF1a depends upona physical interaction between these two proteins (76, 77, 86).

A previous careful kinetic analysis of SAHF formation from ourlaboratory indicated that formation of SAHF is likely a multistepprocess (87). In the earliest defined step, the histone chaperoneproteins HIRA and HP1 are both recruited to a specific subnuclearorganelle, the acute promyelocytic leukemia (PML) nuclear body(10, 67). Most human cells contain 20 to 30 PML nuclear bodies,which are typically 0.1 to 1 µm in diameter and are enrichedin the protein PML, as well as many other nuclear regulatoryproteins (10, 67). PML bodies have been previously implicatedin various cellular processes, including tumor suppression andcellular senescence (20, 23, 61). At a molecular level, theyhave been proposed as sites of assembly of macromolecular regulatorycomplexes and protein modification (24, 31, 61). After HIRA'stranslocation into PML bodies, chromatin condensation occurs,as defined by the appearance of DAPI-stained foci. Finally,H3K9Me accumulates, and HP1 and macroH2A proteins are recruitedto SAHF.

In this study, we set out to understand the series of eventsthat contribute to the formation of SAHF in more detail and,in particular, to identify the molecular requirements for thedifferent steps that were previously temporally defined. Herewe report that during SAHF formation, each chromosome condensesinto a single DAPI focus. Chromosome condensation mediated bythe histone chaperone ASF1a depends on its binding to histoneH3, as well as HIRA. Interestingly, HP1 ${gamma}$ , but not HP1 ${alpha}$ and HP1ß,is phosphorylated on serine 93 in senescent cells. This phosphorylationis not required for the protein's localization to PML bodies,but is required for its binding to SAHF. Remarkably, a largereduction in the amount of chromatin-bound HP1 proteins doesnot affect chromosome condensation, recruitment of histone variantmacroH2A to SAHF, expression of SA ß-gal, or senescence-associatedcell cycle exit. Based on these data, we propose a multistepmodel of dependent and independent steps that culminate in theformation of mature SAHF.

MATERIALS AND METHODS

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Materials and Methods

Cell culture and retroviral infection. Cells were cultured according to ATCC. Retrovirus infectionof WI38 cells was performed as described previously using phoenixpackaging cells to make infectious virus (86). The followingplasmids were used to produce viruses: pBabe-neo-H-Ras^V12 (agift of Bill Hahn and Bob Weinberg), pQCXIP-HA-HP1 ${gamma}$ and its mutants,pQCXIP-HA-ASF1a and its mutants, pQCXIN-myc-ASF1a and its mutants,pQCXIP-myc-HP1ß (103-185), and pQCXIP-HA-HP1ß(103-185).

Immunofluorescence, antibodies, SAHF, and SA ß-gal staining. Two color indirect immunofluorescence assays were performedas described previously (86). Antihemagglutinin (anti-HA) (Y11)(Santa Cruz), anti-myc (9E10) (Santa Cruz), anti-HP1 ${gamma}$ (Chemicon),anti-histone H3 (Abcam), anti-glutathione S-transferase (anti-GST)(Santa Cruz), and anti-PML (AB1370) (Chemicon) were from theindicated suppliers. Anti-macroH2A and anti-HIRA antibodieswere described previously (18, 33). Additional antibodies wereraised to the macrodomain of macroH2A1.2 fused to GST, followinga protocol described previously (34). Anticentromere antibody(ACA) was a gift from J. B. Rattner, University of Calgary.DAPI staining for SAHF and SA ß-gal staining in senescentcells were performed essentially as described previously (86).

Chromosome painting and fluorescence in situ hybridization. The protocol was adapted from that of Mahy and coworkers (48).Growing or senescent WI38 cells were cultured on coverslips,washed twice with phosphate-buffered saline (PBS), and incubatedin 0.075 M KCl at room temperature for 20 min. The slides werefirst fixed in a 3:1 solution of methanol:acetic acid for 10min at room temperature, followed by overnight fixation in 3:1methanol:acetic acid at –20°C. The slides can be keptat –20°C for up to 1 week. After overnight fixation,the slides were washed three times in fresh 3:1 methanol:aceticacid and dried by steaming. Steaming was immediately stoppedonce the slides had dried. The slides were then treated withRNase (100 µg/ml in 2x SSC [1x SSC is 0.15 M NaCl plus0.015 M sodium citrate]) at 37°C for 1 h, followed by pepsintreatment (0.1 mg/ml in 0.01 M HCl) for 3 min at 37°C. Afterpepsin treatment, the slides were washed with PBS and postfixedin 1% paraformaldehyde in PBS with 50 mM MgCl₂ for 10 min atroom temperature. After washing in PBS, the slides were dehydratedfor 2 min each in 70%, 80%, and 100% ethanol at room temperature.The slides were dried completely at 37°C and then denaturedin 70% formamide in 2x SSC in 73°C for 3 min. Immediatelyafter denaturation, the slides were dehydrated with 70%, 80%,and 100% ethanol at –20°C for 2 min each. The slideswere dried completely again and hybridized overnight in chromosomepaint hybridization buffer with chromosome paint probes directlylabeled with fluorescein isothiocyanate (whole chromosome paintprobes were from Vysis) or together with a biotin-labeled probefor the cyclin A gene at 37°C. Biotin labeling of the cyclinA bacterial artificial chromosome probe CTD-2217D23 (Invitrogen)was performed using a Bioprimer DNA-labeling kit from Invitrogen.Hybridized biotin-labeled cyclin A probe was detected by thebinding of Texas Red-avidin DCS (Vector Laboratories) and amplifiedby the binding of biotinylated anti-avidin D9 (Vector Laboratories),followed by another layer of binding of Texas Red-avidin DCS.The slides were counterstained for SAHF using 0.125 µg/mlDAPI for 5 min at room temperature before being visualized byepifluorescence.

GST pulldown and coimmunoprecipitation assays. GST or GST-tagged wild-type ASF1a or its mutants were preboundto glutathione-Sepharose resin (Amersham Biosciences) and incubatedwith 1 µg histone H3 (a gift of Takashi Sekiya and KennethZaret) in binding buffer (25 mM HEPES-NaOH [pH 7.5], 200 mMKCl, 13 mM MgCl₂, 10% glycerol, 0.1% NP-40, and 0.3% ß-mercaptoethanol)at 4°C for 2 h. After incubation, the resin was washed fivetimes with binding buffer, and bound proteins were separatedby sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). Coimmunoprecipitation was performed as describedpreviously (1, 33).

RESULTS

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Results

Single chromosomes condense into a single SAHF. We reasoned that an individual chromosome might condense toform a single SAHF focus. Alternatively, a single chromosomemight contribute to more than one SAHF focus. To distinguishbetween these two possibilities, we performed chromosome paintingof specific chromosomes in WI38 primary human fibroblasts inducedto undergo senescence by expression of an activated Ras oncogene.In growing cells, each copy of chromosome 4 occupied a dispersednuclear territory, as described previously (27, 60) (Fig. 1A).In striking contrast, in senescent WI38 cells, each copy ofthese chromosomes was condensed into a single SAHF focus. Chromosomepainting showed that the interior of the SAHF focus did nothybridize efficiently to the probe, presumably because the denseheterochromatin is inaccessible to the probe. When chromosome4 painting was combined with cyclin A2 gene fluorescence insitu hybridization, this gene occupied the diffuse territoryof chromosome 4 in growing cells. However, in senescent WI38cells, the cyclin A2 gene either was localized at the peripheryof the dense heterochromatin domain or was undetectable, presumablybecause it was localized to the nonhybridizing interior of thefocus (Fig. 1A and data not shown). Similarly, in senescentcells, both copies of chromosome X and chromosome 6 were eachcondensed into a single SAHF focus (Fig. 1B). Recently, Funayamaand coworkers also reported that SAHF result from the condensationof single chromosomes, based in part on chromosome paintingwith a chromosome 12-specific probe (25). Interestingly, asoriginally reported by Narita and coworkers (57), we found thattelomeric and centromeric chromatin is located predominantlyat the periphery of SAHF. In our experiments, telomeric DNAwas detected with a telomeric DNA probe (84), and centromericDNA localization was found by staining with ACA which recognizecentromere-bound kinetochore proteins (Fig. 1C) (22). Funayamaand coworkers also found centromeric chromatin to be peripheralto SAHF (25). Together, the data indicate that during SAHF formation,individual chromosomes condense to form a single SAHF focus.Domains of constitutive heterochromatin, such as pericentromericand telomeric heterochromatin, do not appear to be integralto SAHF.

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FIG. 1. Individual chromosomes condense into a single SAHF focus. (A) Chromosome (Chr.) 4 painting combined with cyclin A2 gene fluorescence in situ hybridization (FISH) in growing and senescent WI38 cells. DNA was stained using DAPI. (B) Chromosome 6 and X chromosome painting and DAPI staining of senescent WI38 cells. Images were collected by confocal microscopy. (C) Senescent WI38 cells were stained with DAPI to visualize DNA (blue) and with ACA to visualize kinetochore proteins bound to centromeric DNA (red).

Formation of SAHF requires ASF1a histone H3 binding activity. ASF1a binds to the histone chaperone HIRA and also to histoneH3 (55, 70, 76, 77, 86). We previously showed that the formationof SAHF by ASF1a depends on its ability to bind to HIRA (86).To assess whether the histone H3 binding activity of ASF1a isalso required for formation of SAHF, we introduced point mutationsonto the surface of ASF1a used for histone H3 binding, basedon the published nuclear magnetic resonance structure of humanASF1a bound to a histone H3-derived peptide (55) (Fig. 2A).We also made use of our recently solved X-ray crystal structureof ASF1a bound to a fragment of HIRA, to ensure that these mutantsleave the HIRA interaction surface intact (77). Specifically,we generated ASF1a(D54R) and ASF1a(V94D). We tested the bindingof these ASF1a mutants to histone H3.1. Although the HIRA/ASF1acomplex preferentially uses histone H3.3 as a substrate (46,76), ASF1a binds to both isoforms and makes key contacts witha peptide that is 100% conserved between histone H3.1 and H3.3(55, 76). First, we tested the binding of the mutant proteinsto histone H3.1 by GST pulldown assay, using purified recombinanthistone H3.1 and GST-tagged wild-type ASF1a or its mutants.As predicted, both of the ASF1a mutants failed to bind to histoneH3.1 in vitro, whereas wild-type ASF1a efficiently bound tohistone H3.1 under identical conditions (Fig. 2B). To confirmthese results, we tested the binding of wild-type ASF1a andits mutants to histone H3.1 in vivo by coexpressing myc epitope-taggedwild-type ASF1a or its mutants together with HA-tagged histoneH3.1 in WI38 primary human fibroblasts and then testing fora physical interaction between the epitope-tagged proteins byimmunoprecipitation and Western blotting analysis (Fig. 2C).Consistent with the in vitro binding results, both of the ASF1amutants failed to bind to HA-histone H3.1, while wild-type ASF1aefficiently bound HA-histone H3.1. To eliminate the possibilitythat the ASF1a mutants failed to bind to histone H3.1 due toa gross disruption of protein folding, we tested the bindingof wild-type ASF1a and the mutants to endogenous HIRA in WI38cells, also by immunoprecipitation analysis (Fig. 2D). We foundthat, as intended, both of the ASF1a mutants still bound efficientlyto HIRA, showing that the targeted mutations primarily disruptthe histone H3-binding surface and not the HIRA-binding surface.

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FIG. 2. Design of ASF1a mutants deficient in histone H3 binding. (A) Mutated residues (space fill, CPK coloring) on a modeled human ASF1a/H3 structure. Residues 1 to 156 of ASF1a are shown as the green ribbon. An ASF1a-binding H3 peptide (residues 122 to 135) is shown as the blue ribbon (data are taken from reference 55; see also reference 77). (B) GST pulldown assay using GST alone or GST-tagged wild-type (WT) ASF1a and its mutants and purified recombinant histone H3. After pulldown, the washed proteins bound to resin were separated on SDS-PAGE gels and visualized either by Coomassie blue staining or by Western blotting using the indicated antibodies. (C) Myc-tagged wild-type ASF1a or its mutants were coexpressed with HA-tagged histone H3 in WI38 cells, immunoprecipitated with anti-myc antibody (9E10), and Western blotted with the indicated antibodies. Note that Myc-ASF1aV94D comigrates with the light chain of the antibody used for immunoprecipitation (IP; marked with *) and that there is a background band in the control lane of the anti-Myc input. (D) Myc-tagged wild-type ASF1a or its mutants were expressed in WI38 cells, immunoprecipitated with the anti-myc antibody (9E10), and Western blotted with the indicated antibodies.

Using these ASF1a mutants, we tested whether the formation ofSAHF by ASF1a requires its histone H3-binding activity, by ectopicallyexpressing wild-type ASF1a or the mutants deficient in histoneH3 binding in WI38 cells, using retroviruses. Compared to wild-typeASF1a, both ASF1a mutants deficient in histone H3 binding weregrossly impaired in formation of SAHF, as judged by chromatincondensation using DAPI staining and the deposition of macroH2Aproteins (Fig. 3A). The expression level of each of the HA-taggedASF1a mutants was comparable to that of wild-type ASF1a, indicatingthat impaired SAHF formation by the mutants was not due to theirunderexpression (Fig. 3B). We conclude that formation of SAHFdriven by ASF1a depends on its histone H3 binding activity.

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FIG. 3. Formation of SAHF by ASF1a depends on histone H3-binding activity. (A) HA-tagged wild-type (WT) ASF1a or its mutants deficient in histone H3 binding were expressed in WI38 cells by retrovirus infection and drug selected with 3 µg/ml puromycin. Ten days postinfection, drug-selected cells were stained with DAPI and antibodies to macroH2A1.2. The white arrow marks the inactive X chromosome. (B) One hundred cells shown in panel A were scored for the indicated nuclear foci. The inactive X chromosome was excluded when scoring cells with macroH2A foci. The bottom panel shows whole-cell lysates Western blotted with anti-HA and antiactin antibodies.

Phosphorylation of HP1 ${gamma}$ is required for its deposition in SAHF but not its localization to PML bodies. Next, in an attempt to understand how HP1 proteins are targetedto SAHF, we set out to identify posttranslational modificationsof HP1 proteins that are regulated between young and senescentWI38 cells. Indicative of such a modification, we found by SDS-PAGEthat HP1 ${gamma}$ exhibited reduced mobility in senescent cells comparedto that in growing cells (Fig. 4A). This apparent posttranslationalmodification was not observed for HP1 ${alpha}$ and HP1ß (datanot shown). To test whether this modification of HP1 ${gamma}$ is dueto phosphorylation, HP1 ${gamma}$ was immunoprecipitated from growingor senescent WI38 cells and treated with or without ${lambda}$ -phosphatase.We found that phosphatase treatment completely abolished themodified form of the protein, confirming that HP1 ${gamma}$ is phosphorylatedin senescent cells (Fig. 4A).

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FIG. 4. HP1 ${gamma}$ is phosphorylated on serine 93 in cells approaching senescence. (A) Control or HP1 ${gamma}$ immunoprecipitates (IP) from growing (control-infected) or senescent (activated Ras-infected) WI38 cells were treated with or without ${lambda}$ -phosphatase, separated by SDS-PAGE, and Western blotted. (B) Alignment of regions of HP1 ${alpha}$ , -ß, and - ${gamma}$ . Residues shaded gray are nonidentical but conserved; residues shaded black are identical. The numbers across the top indicate the residue numbers of candidate phosphoacceptor sites in HP1 ${gamma}$ which are not conserved in both HP1 ${alpha}$ and -ß. Residue numbers are taken from sequences in the Swiss-Prot database. (C) HA-tagged wild-type HP1 ${gamma}$ and its mutants were ectopically expressed in WI38 cells, together with an activated Ras oncogene, and then Western blotted with anti-HA antibody.

Next, we wanted to identify the residue of HP1 ${gamma}$ that is phosphorylated.Based on the observation that only HP1 ${gamma}$ , and not HP1 ${alpha}$ or -ß,becomes phosphorylated in senescent cells, each of the nonconservedserine or threonine residues in HP1 ${gamma}$ was mutated to alanine togenerate HP1 ${gamma}$ (T89A), HP1 ${gamma}$ (S93A), HP1 ${gamma}$ (S99A), HP1 ${gamma}$ (S102A), and HP1 ${gamma}$ (S104A)(Fig. 4B and C). HA-tagged wild-type HP1 ${gamma}$ or the mutants wereexpressed in WI38 cells, together with activated Ras to inducesenescence. Western blotting analysis of the ectopically expressedHP1 ${gamma}$ mutants showed that phosphorylation of HP1 ${gamma}$ was completelyabolished by the HP1 ${gamma}$ (S93A) mutant (Fig. 4C). In contrast, therewas no effect for any of the other mutations. We conclude thatHP1 ${gamma}$ is phosphorylated on serine 93 in senescent cells.

To test whether HP1 ${gamma}$ phosphorylation is required for localizationof HP1 ${gamma}$ into PML bodies and/or SAHF, HA-tagged wild-type HP1 ${gamma}$ and HP1 ${gamma}$ (S93A) were coexpressed with activated Ras in WI38 cells.We found that both wild-type HP1 ${gamma}$ and HP1 ${gamma}$ (S93A) localized equivalentlyto PML bodies (Fig. 5A and B, yellow arrows). However, comparedto wild-type HP1 ${gamma}$ , the HP1 ${gamma}$ (S93A) mutant was impaired in its depositionin SAHF (Fig. 5A, yellow arrowheads). We conclude that HP1 ${gamma}$ isphosphorylated on serine 93 in senescent cells and that thisphosphorylation is required for its efficient deposition inSAHF but not for its localization to PML bodies.

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FIG. 5. Phosphorylation of HP1 ${gamma}$ is required for recruitment of HP1 ${gamma}$ to SAHF but not its localization to PML bodies. (A) HA-tagged wild-type (WT) HP1 ${gamma}$ or HP1 ${gamma}$ (S93A) was coexpressed with an activated oncogene Ras (H-RasV12) in WI38 cells by retrovirus infection and drug-selected with 3 µg/ml puromycin and 500 µg/ml G418. Eights days postinfection, drug-selected cells were stained with the indicated antibodies and DAPI. Yellow arrows mark HP1 ${gamma}$ wild type and HA-HP1 ${gamma}$ (S93A) in a PML body, and yellow arrowheads mark HP1 ${gamma}$ wild type in SAHF. The latter is largely absent from SAHF. Images were collected by confocal microscopy. (B) One hundred cells shown in panel A were scored for the colocalization of PML and HP1 ${gamma}$ wild type or HP1 ${gamma}$ (S93A).

Abundant HP1 proteins are not required for the formation of SAHF or senescence-associated cell cycle exit. Given that all three HP1 family members are localized to SAHFin senescent cells (57, 86), we next asked whether these proteinsare required for formation of SAHF and other features of thecellular senescence program. To do so, we took advantage ofa dominant-negative HP1ß mutant that we had developedpreviously (85). This dominant-negative mutant was created basedon the prediction made by Lechner and coworkers that the deletionof the N-terminal chromodomain of HP1 proteins should preventtheir binding to chromatin but not affect their ability to homo-or heterodimerize, thereby sequestering endogenous HP1 proteinsfrom chromatin (43). Previously we showed that the ectopic expressionof this mutant HP1ß(103-185) (designated HP1ß ${Delta}$ N)in WI38 cells does indeed deplete all three endogenous chromatin-boundHP1 subtypes by 70 to 80% (85) (see Fig. S1 in the supplementalmaterial). Remarkably, ectopic expression of HP1ß ${Delta}$ Nin WI38 cells had no discernible effect upon cell viabilityor proliferation (85), so we were able to generate a polyclonalpopulation of WI38 cells stably expressing HP1ß ${Delta}$ N andmarkedly deficient in chromatin-bound HP1 proteins, or the appropriateempty vector-infected and drug-selected cells as a control.

To test the requirement for chromatin-bound HP1 proteins forformation of SAHF and other aspects of the senescence program,we infected both control and HP1ß ${Delta}$ N-expressing cellswith a retrovirus encoding activated Ras and scored the effecton SAHF and other features of senescence. As expected, HP1ß ${Delta}$ Nefficiently removed the bulk of all three endogenous HP1 proteinsfrom chromatin in WI38 cells coexpressing activated Ras. Thisis apparent from the almost complete absence of HP1 proteinsfrom SAHF (Fig. 6A; also see Fig. S2 in the supplemental material).Interestingly, HP1ß ${Delta}$ N also blocked the recruitmentof endogenous HP1 proteins to PML bodies, a further indicationof the ability of this mutant to disrupt the function of endogenousHP1 proteins (Fig. 6A and B, yellow arrowheads). However, HP1ß ${Delta}$ Ndid not affect Ras-induced relocalization of HIRA to PML bodies(Fig. 6C and D). We previously showed that localization of HIRAto PML bodies is an early step in the formation of SAHF (86).Therefore, HP1ß ${Delta}$ N did not impair early signaling eventsactivated by oncogenic Ras. Remarkably, however, depletion ofchromatin-bound HP1 proteins had no effect on chromosome condensation,as visualized by DAPI staining and the accumulation of H3K9Me2and the macroH2A histone variant in SAHF (Fig. 6A, yellow arrows,and Fig. 7A to D; also see Fig. S2 in the supplemental material).No effect of HP1ß ${Delta}$ N was observed on the rate of formationof SAHF (data not shown). We conclude that visible enrichmentof HP1 proteins is not required for chromosome condensationand formation of SAHF.

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FIG. 6. HP1ß ${Delta}$ N blocks the relocalization of HP1 proteins into PML bodies but has no effect on recruitment of HIRA into PML bodies. (A) WI38 cells were infected with vector control (Cont or Con) or HP1ß ${Delta}$ N-expressing virus, together with a virus containing an activated Ras oncogene. The infected cells were selected with 3 µg/ml puromycin and 500 µg/ml G418 cells. Eight days postinfection, the cells were stained with antibodies to HP1ß and PML. Yellow arrowheads indicate colocalizing HP1ß and PML. Yellow arrows indicate HP1ß in SAHF. (B) One hundred cells shown in panel A were scored for colocalizing HP1ß and PML. (C) As in panel A but stained with antibodies to HIRA and PML. (D) One hundred cells shown in panel C were scored for colocalizing HIRA and PML.

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FIG. 7. HP1ß ${Delta}$ N blocks deposition of HP1 proteins in SAHF, but has no effect on chromosome condensation, deposition of macroH2A protein, or accumulation of H3K9Me in SAHF. (A) WI38 cells were infected with vector control (Cont or Con) or HP1ß ${Delta}$ N-expressing virus, together with a virus containing an activated Ras oncogene, as indicated. The infected cells were selected with 3 µg/ml puromycin and 500 µg/ml G418. Eight days postinfection, the cells were stained with antibodies to HP1ß and macroH2A1.2. (B) One hundred cells shown in panel A were scored for chromosome condensation (DAPI foci) and deposition of macroH2A1.2 and HP1ß in SAHF (colocalization of macroH2A1.2 and HP1ß, respectively, with DAPI). Cells with macroH2A foci do not include cells with a visible inactive X chromosome only. (C) As in panel A but stained with antibodies to H3K9Me2 and HP1ß. (D) One hundred cells shown in panel C were scored for incorporation of H3K9Me2 and HP1ß in SAHF (colocalization of H3K9Me2 and HP1ß, respectively, with DAPI). coloc., colocalization.

We previously showed that the histone chaperone ASF1a is aneffector of Ras-mediated SAHF formation (86). We likewise foundthat the removal of chromatin-bound HP1 proteins by HP1ß ${Delta}$ Ndid not affect chromosome condensation induced by ASF1a (Fig.8A and B). Although we cannot exclude the possibility that therelatively small amount of remaining chromatin-bound HP1 proteinsis sufficient for the observed changes in chromatin structure,we can conclude that a large depletion of chromatin-bound HP1proteins does not obviously affect chromatin remodeling in senescentcells. Most notably, there is no effect on chromosome condensationto form morphological SAHF.

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FIG. 8. HP1ß ${Delta}$ N has no effect on ASF1a-induced chromosome condensation. (A) WI38 cells were infected with vector control (con) or HP1ß ${Delta}$ N-expressing virus, together with a virus encoding ASF1a, as indicated. The infected cells were selected with 3 µg/ml puromycin and 500 µg/ml G418. Ten days postinfection, the cells were stained with antibodies to HP1 ${gamma}$ . The circles and arrow indicate HP1 ${gamma}$ in SAHF and PML bodies, respectively. (B) One hundred cells shown in panel A were scored for chromosome condensation (DAPI foci) and HP1 ${gamma}$ colocalized with SAHF (HP1 ${gamma}$ coloc. with DAPI).

In light of this conclusion, we asked whether depletion of HP1proteins affects other senescence phenotypes, specifically senescence-associatedcell cycle exit and expression of SA ß-gal activity.Remarkably, depletion of chromatin-bound HP1 proteins by HP1ß ${Delta}$ Nhad no effect on either phenotype (Fig. 9A to C). We concludethat a large decrease in the binding of these proteins to chromatinand the resultant complete failure of these proteins to enrichin SAHF have no effect on any of the tested hallmarks of thesenescence phenotype.

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FIG. 9. HP1ß ${Delta}$ N does not affect senescence-associated cell cycle arrest and expression of SA ß-gal. (A) WI38 cells stably infected with an HP1ß ${Delta}$ N-expressing retrovirus or a control (Cont or Con) virus were infected with viruses encoding activated Ras or a control virus. The infected cells were selected with 3 µg/ml puromycin and 500 µg/ml G418. Eight days later, the cells were pulse-labeled with 5'-bromodeoxyuridine (BrdU) for 1 h and stained with antibodies to 5'-BrdU. (B) One hundred cells shown in panel A were scored as 5'-BrdU positive or negative. (C) Cells shown in panel A were stained to detect expression of SA ß-gal (blue).

DISCUSSION

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Discussion

In this article, we report the following important findingsregarding the mechanism of assembly of SAHF. First, SAHF resultsfrom condensation of individual chromosomes, each chromosomecondensing into a single SAHF focus. Second, formation of SAHFby the histone chaperone ASF1a depends on its ability to bindto histone H3, in addition to the requirement for HIRA bindingthat we showed previously (86). Third, HP1 ${gamma}$ is phosphorylatedon serine 93 in senescent cells, and this modification is requiredfor its deposition in SAHF but not for its localization to PMLbodies. Fourth, high levels of chromatin-bound HP1 proteinsare not required for chromosome condensation, deposition ofmacroH2A proteins in SAHF, or other hallmarks of the senescenceprogram, such as expression of SA ß-gal activity andsenescence-associated cell cycle exit.

Based on these findings and others reported previously (57,86), we propose the following stepwise model, comprised of dependentand independent steps, for the formation of SAHF (Fig. 10).Initially, histone chaperone protein HIRA and HP1 (HP1 ${alpha}$ , -ß,and - ${gamma}$ ) are recruited to PML nuclear bodies. Our previous kineticanalysis of SAHF formation showed that HIRA and HP1 proteinsenter PML nuclear bodies prior to any detectable chromosomecondensation or any other molecular marker of SAHF (86). SinceHP1ß ${Delta}$ N does not affect the localization of HIRA toPML bodies but does block recruitment of endogenous HP1 proteinsto PML bodies and does not go to PML bodies itself (data notshown), we conclude that HIRA enters PML bodies independentlyof HP1 proteins.

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FIG. 10. A stepwise model for the formation of SAHF in senescent human cells. This model indicates the key steps in SAHF formation, after initiation by senescence triggers. Dashed lines are steps which are currently poorly defined (see text for details). Abbreviations: HMT, histone methyltransferase; Me, methylated lysine 9 histone H3; Ac, acetylated histone. HMGA proteins are shown in mature SAHF (56), but their point of entry is not known.

The role served by the translocation of HIRA and HP1 proteinsto PML bodies is not known. However, PML bodies have been suggestedas sites of assembly of macromolecular complexes and also assites of protein modification (24, 31, 61). Significantly, incells approaching senescence, HP1 ${gamma}$ becomes phosphorylated onserine 93. Interestingly, we have not detected analogous phosphorylationof HP1 ${alpha}$ and -ß. The nonphosphorylatable mutant HP1 ${gamma}$ (S93A)efficiently enters PML bodies but does not efficiently localizeto SAHF. Thus, HP1 ${gamma}$ might be phosphorylated inside PML bodiesand phosphorylation might target HP1 ${gamma}$ to SAHF (86). Alternatively,HP1 ${gamma}$ might be phosphorylated after the protein exits PML bodiesen route to SAHF.

In the earliest discernible change in chromatin structure itself,individual chromosomes condense to form single SAHF. Previously,we showed that chromatin condensation depends on the histonechaperone ASF1a and is driven by a complex of ASF1a and itsbinding partner HIRA (86). Here we have extended this to showthat chromosome condensation requires an interaction of ASF1awith histone H3 and HIRA. Since the HIRA/ASF1a complex servesas a chaperone for deposition of the histone H3/H4 complex intochromatin (65, 76), it seems likely that chromosome condensationby HIRA and ASF1a depends on their chaperone activity. Conceivably,chromosome condensation depends, in part, on increased nucleosomedensity due to HIRA/ASF1a-mediated nucleosome deposition. Thisis consistent with many previous reports that transcriptionallyactive chromatin is depleted of nucleosomes. This is true atboth a genome-wide and a local chromatin level (2, 4, 6-8, 15,44, 52, 87). Moreover, a previous study reported that the facultativeheterochromatin of the inactive X chromosome has a higher nucleosomedensity than most other regions of the nucleus (62). Together,this suggests that whole chromosome condensation and gene silencingmay result from increased nucleosome density throughout thechromosome.

The HIRA/ASF1a chaperone complex preferentially utilizes histoneH3.3 as a deposition substrate (46, 76). Significantly, histoneH3.3 accumulates in fibroblasts approaching senescence and innondividing differentiated cells, in some cases to about 90%of the total histone H3, presumably with the majority beingin inactive chromatin (11, 13, 30, 41, 59, 64, 66, 80, 83).Unfortunately, because histone H3.3 and canonical H3.1 differonly by five amino acids, they cannot presently be differentiatedimmunologically and there is no straightforward way to ask whetherendogenous histone H3.3 is specifically enriched in SAHF. Theidea that SAHF might contain histone H3.3 may initially seemunlikely, because deposition of histone H3.3 is typically linkedto transcription activation (5, 49, 52, 69, 81), whereas SAHFis a form of transcriptionally silent facultative heterochromatin(56, 57, 86). However, the apparent inconsistency in this ideais merely an extension of an existing paradox. Specifically,HIRA and its orthologs in other species are typically involvedin gene silencing and formation of heterochromatin (9, 29, 39,40, 63, 70-72, 74), whereas HIRA's favored deposition substrate,histone H3.3, is linked to transcriptional activation (5, 49,52, 69, 81). However, to our knowledge, histone H3.3 per sehas not been shown to directly cause or contribute to transcriptionactivation, and a proportion of histone H3.3 does carry posttranslationalmarks characteristic of transcriptionally silent chromatin (32,47, 49). Therefore, histone H3.3 is unlikely to be exclusivelylinked to transcription activation. Instead, deposition of histoneH3.3 may be associated with any major remodeling of chromatin,perhaps as a way to "reset" histone modifications. To expressthis idea, Ooi and coworkers have suggested that histone H3.3is a chromatin "repair" variant (58). Concordant with this proposal,after egg fertilization in flies, dHIRA activity is requiredfor the replacement of protamines by histone H3.3-containingnucleosomes in decondensing sperm chromatin (46). By this view,the HIRA/ASF1a complex might drive formation of SAHF by depositionof histone H3.3-containing nucleosomes.

In line with the idea that chromosome condensation to form SAHFresults primarily from increased nucleosome density, chromosomecondensation into SAHF does not require the accumulation ofH3K9Me or the deposition of heterochromatic proteins HP1 andmacroH2A. Our previous kinetic analysis showed that chromatincondensation occurs prior to the accumulation of H3K9Me andthe deposition of HP1 and the histone variant macroH2A in chromatin(86). Here we have shown that chromosome condensation, triggeredby an activated Ras oncogene or ectopic expression of ASF1a,efficiently occurs in the absence of high levels of stably boundHP1 proteins. Together, these results eliminate the possibilitythat H3K9Me, HP1, or macroH2A drives chromosome condensation.The finding that facultative heterochromatin can form in theabsence of stably bound HP1 proteins is consistent with studiesof facultative heterochromatin in nucleated vertebrate erythrocytes,which ordinarily forms without HP1 proteins (26). In sum, theHIRA/ASF1a complex appears to drive chromosome condensationby acting upstream of characteristic heterochromatin modificationsand associated proteins, most likely by contributing to nucleosomeassembly through the deposition of histone H3/H4 complexes.

The final steps of SAHF formation consist of recruitment ofmacroH2A and HP1 proteins to chromatin. These two steps arenot separable, based on a temporal analysis alone (86). However,we have shown here that the recruitment of macroH2A occurs inthe absence of stably bound HP1 proteins. At this time, we cannotexclude the possibility that recruitment of HP1 proteins tochromatin depends on prior loading of histone macroH2A. However,since we know of no evidence in support of this idea, we proposethat HP1 and macroH2A proteins are independently loaded ontochromatin at approximately the same time. We find that phosphorylationof HP1 ${gamma}$ on S93 is required for its efficient recruitment to heterochromatin.Interestingly, another study found that HP1 ${gamma}$ phosphorylated onthis residue is localized to euchromatin in immortal and transformedcells (45) (it should be noted that these authors numbered theprocessed form of HP1 ${gamma}$ and so referred to the same residue asS83). Thus, phosphorylation of this site might target HP1 ${gamma}$ todifferent chromatin sites depending on the physiological context.

Remarkably, loading of abundant HP1 proteins onto chromatinis not required for two hallmarks of the senescent phenotype:expression of SA ß-gal and senescence-associated cellcycle exit. We obviously cannot rule out the possibility thatthe residual chromatin-bound HP1 proteins are sufficient tomediate HP1 functions that are required for these senescencephenotypes. However, these results raise the possibility thatHP1 proteins do not contribute to the acute onset of the senescentphenotype. Instead, HP1 proteins might be required for the long-termmaintenance of SAHF and the senescent state. Alternatively,HP1 proteins might secure the senescent state in the face ofgenetic alterations or cellular perturbations that compromiseother aspects of the senescence program. These ideas remainto be tested.

In contrast to our results with HP1 proteins, Narita and coworkersfound through shRNA knock-down experiments that the HMGA proteinHMGA2 is required for the formation of SAHF (56). This mightsuggest that HMGA proteins are incorporated into SAHF quiteearly during SAHF assembly, perhaps at the time of chromosomecondensation. However, until this is directly demonstrated,we have omitted HMGA's point of entry into SAHF from our model.

Although Fig. 10 provides a framework model for the formationof SAHF, many other questions remain. For example, we do notknow the triggers responsible for localization of HIRA and HP1proteins to PML bodies. We do not know the specific reason forHIRA's localization to PML bodies and the spatial and mechanisticrelationships between its localization to PML bodies and theformation of SAHF. Finally, we do not know the identity of thekinase responsible for the phosphorylation of HP1 ${gamma}$ , the histonemethyltransferase that methylates lysine 9 of histone H3 tocreate H3K9Me, or the factors required for the deposition ofmacroH2A into SAHF. Studies to answer these questions are ongoing.Meanwhile, the model proposed in Fig. 10 provides a valuableconceptual framework for thinking about these questions, aswell as summarizing a large body of existing knowledge.

ACKNOWLEDGMENTS

We thank Ken Zaret and Takashi Sekiya for purified recombinanthistone H3.1, John Pehrson for macroH2A antibodies, Bill Hahnand Bob Weinberg for pBABE-RasV12, and all members of the Adamslaboratory for critical discussions.

This study was supported by NIH grant GM062281 and Leukemiaand Lymphoma Society grant 1520-04 to P.D.A. and an AFAR grantto R.Z.

FOOTNOTES

* Corresponding author. Mailing address: W446, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111. Phone: (215) 728-7108. Fax: (215) 728-3616. E-mail: peter.adams{at}fccc.edu.

Published ahead of print on 22 January 2007.

Supplemental material for this article may be found at http://mcb.asm.org/.

REFERENCES

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