Isoform-specific Intermolecular Disulfide Bond Formation of Heterochromatin Protein 1 (HP1)*

Three mammalian isoforms of heterochromatin protein 1 (HP1), α, β, and γ, play diverse roles in gene regulation. Despite their structural similarity, the diverse functions of these isoforms imply that they are additionally regulated by post-translational modifications. Here, we have identified intermolecular disulfide bond formation of HP1 cysteines in an isoform-specific manner. Cysteine 133 in HP1α and cysteine 177 in HP1γ were involved in intermolecular homodimerization. Although both HP1α and HP1γ contain reactive cysteine residues, only HP1γ readily and reversibly formed disulfide homodimers under oxidative conditions. Oxidatively dimerized HP1γ strongly and transiently interacted with TIF1β, a universal transcriptional co-repressor. Under oxidative conditions, HP1γ dimerized and held TIF1β in a chromatin component and inhibited its repression ability. Our results highlight a novel, isoform-specific role for HP1 as a sensor of the cellular redox state.

Heterochromatin protein 1 (HP1) was originally characterized as an abundant protein that binds pericentric heterochromatin (1). HP1 acts as a scaffold-like molecule, which is composed of two conserved domains as follows: the chromodomain (CD) 2 and the chromoshadow domain (CSD). The variable hinge region separates these two domains (2). The CD recognizes methylated lysine 9 of histone H3 (H3K9), which recruits HP1 to specific sites within the genome (3)(4)(5). The CSD promotes HP1 homodimer formation and provides a surface for interaction with a variety of other chromatin proteins (6,7). Although genetic experiments previously revealed that HP1 works as a repressor of gene activation by propagation of a heterochromatin structure, emerging evidence has elucidated its diverse functions other than gene silencing (8). Some of these functions are regulated in an isoform-specific manner (9).
In vertebrates, three isoforms of HP1 exist as follows: ␣, ␤, and ␥, all of which share highly conserved domains. Tethering any HP1 isoform upstream of a promoter equally triggers gene silencing concomitant with local chromatin condensation and an increase in H3K9 methylation (10 -12), indicating their common silencing ability. However, nonredundant functions (13,14), different binding affinities to other proteins (15)(16)(17) and different localizations in tissues (18,19), of these three HP1 isoforms imply that ␣, ␤, and ␥ are functionally diverse. Furthermore, recent evidence clarified apparently opposite functions of HP1 isoforms, e.g. a role in transcriptional activation or in transcriptional elongation (20,21). One mechanism that could account for such functional diversity of HP1 isoforms is post-translational modification, which could cause conformational changes in the molecule. In fact, reversible modifications of HP1 (e.g. phosphorylation) can modulate its function in response to various stimuli or cellular environments, suggesting an active role for HP1 beyond its known function as a marker of heterochromatin (17,22). However, the precise modulatory mechanism across three HP1 isoforms that leads to functional differences remains to be elucidated.
Here, we identified isoform-specific disulfide bond formation as a novel post-translational modification of HP1. We analyzed the biochemical and functional characteristics of this oxidative modification. These data may offer a new insight into a novel role for HP1 during the cellular response to oxidative stress.
The supernatant was collected as the cytosolic fraction. Extraction buffer (20 mM HEPES, pH 7.9, 1.5 mM MgCl 2 , 0.42 M NaCl, 0.2 mM EDTA, 25% glycerol) was added to the pellet, and ultrasonic agitation was performed (30-s sonication with 30-s interval, 4 -6 times at 0°C; Bioruptor, CosmoBio). The suspension was incubated for 15 min at 4°C and centrifuged at 20,000 ϫ g for 10 min. The supernatant was collected as the nuclear extract.
Column Chromatography-For anion exchange, whole cells were lysed with buffer A (20 mM Tris, pH 8.0, 5% acetonitrile) containing 5 mM EDTA and 1% Nonidet P-40 and incubated at 4°C for 15 min. The lysate was centrifuged at 20,000 ϫ g for 5 min, and the supernatant was filtered and loaded onto an anion-exchange column (Q-Sepharose High Performance, GE Healthcare) pre-equilibrated with buffer A. After unbound samples were washed, protein was eluted with a linear gradient (0 -100%) of buffer B (buffer A with 1.0 M NaCl). For reversephase HPLC, purified protein samples and nuclear extracts were prepared with 0.3% trifluoroacetic acid (TFA) and 20% acetonitrile and applied to a phenyl reverse-phase column (4.6 ϫ 250 mm; Nakalai Tesque). Bound proteins were eluted by a segmented linear gradient of increasing concentrations of buffer B (acetonitrile and 0.1% TFA) in buffer A (0.1% TFA) at a flow rate of 0.5 ml/min. Buffer B was increased at a rate of 1.0%/fraction (fast gradient) or 0.2%/fraction (slow gradient). Collected fractions were dried by a centrifugal evaporator and reconstituted with SDS sample buffer with or without 2.5% 2-mercaptoethanol (reducing or nonreducing conditions, respectively).
Triton Extraction-Triton extraction was carried out as described previously with modification (23). Cells were lysed with a hypotonic lysis buffer with 0.5% Nonidet P-40 and centrifuged at 20,000 ϫ g for 5 min (as described above). The pellet was lysed in extraction buffer with 0.2% Triton X-100, incubated on ice for 30 min, and centrifuged at 20,000 ϫ g for 5 min. The supernatant was kept as a Triton-soluble fraction. The remaining pellet was lysed in SDS sample buffer (250 mM Tris, 5% SDS, and 5% glycerol) with or without 2.5% 2-mercaptoethanol (reducing or nonreducing conditions, respectively), and ultrasonic agitation was performed as described above. After centrifugation at 20,000 ϫ g for 5 min, the supernatant was kept as a Triton-insoluble fraction.
RNAi Knockdowns and Generation of HEK293T Stable Cells-Lentiviral particles derived from the pLKO.1-puro-containing shRNA sequence were purchased from the Mission shRNA library (Sigma). The oligonucleotide sequences of the shRNA were as follows: shRNA-6, CGACGTGTAGTGAATGGG-AAA; and shRNA-7, GCGTTTCTTAACTCTCAGAAA. Lentiviral particles were used to transduce human umbilical vein endothelial cells (HUVECs) or HEK293T cells in the presence of 8 g/ml Polybrene. To generate a HEK293T stable cell line, the infected cells were selected with 1 g/ml puromycin. The stable cells in which HP1␥ was almost completely depleted were next transfected with pEF-DEST51 HP1␥-FLAG WT or a C177S mutant (cloned from murine cDNA and resistant to shRNA), and the stable cells were selected with 5 g/ml blasticidin.
GAL4-luciferase Reporter Assay-pC3-ERHBD-GAL4 or pC3-ERHBD-GAL4-KAP1 (TIF1␤) with pGL4.31-PSV40-GAL4UAS were transfected using Lipofectamine 2000 into subconfluent HEK293T stable cells that were passaged 1 day before transfection. After 24 h, 0.04% ethanol or 4-OHT (500 nM) was added to the culture medium. Forty eight h after transfection, luciferase activity was measured by a luminometer (Lumat LB9507). Intranuclear mRNA levels of luciferase were measured as follows. Twenty four h after transfection, 4-OHT (500 nM) was added to the culture medium. Forty eight h after transfection, cells were lysed with a hypotonic lysis buffer with 0.5% Nonidet P-40 and centrifuged at 20,000 ϫ g for 5 min (as described above). From the nuclear pellet, total RNA was isolated using RNA-Bee (Cosmo Bio). Total RNA was treated with DNase (Turbo DNA-free, Applied Biosystems) and was reverse-transcribed using a high capacity cDNA reverse transcription kit (Applied Biosystems). Luciferase mRNA levels were measured by real time quantitative PCR (SYBR Green ER, Invitrogen). Firefly luciferase cDNA was amplified using the following primers: 5Ј-TACCCACTCGAAGACGGGAC-3Ј and 5Ј-ACTCGGCGTAGGTAATGTCCACCTC-3Ј. Human 18 S ribosomal RNA was measured using the following primers: 5Ј-GTAACCCGTTGAACCCCATT-3Ј and 5Ј-CCATCCAA-TCGGTAGTAGCG-3Ј. The relative levels of luciferase mRNA were normalized to the mRNA levels of 18 S ribosomal RNA.

HP1␣ Forms Dimers via Disulfide Bonds through Cysteine
133-During purification of HP1␣ in our previous work (24), we found that endogenous HP1␣ separates into two peaks by fractionation using reverse-phase HPLC. To confirm this finding, we fractionated whole cell lysates from HEK293T cells using two-step column chromatography (Fig. 1A). Endogenous HP1␣ was eluted at a salt concentration ranging from 0.3 to 0.35 M on a Q-Sepharose anion-exchange column (Fig. 1D, top panel). We applied this single peak to a reverse-phase column. After elution with a fast gradient, HP1␣ was still detected as a single peak (Fig. 1D, 2nd panel). However, when eluted with a slow gradient, HP1␣ separated into two peaks representing a hydrophilic and a hydrophobic form (Fig. 1D, 3rd panel). Two other HP1␣ antibodies against different epitopes also detected both bands (data not shown), suggesting that these were biochemically different forms of HP1␣. Even after direct fractionation of the nuclear extract, which includes the bulk of HP1␣ protein (Fig. 1B), endogenous HP1␣ showed a similar bimodal distribution (Fig. 1D, 4th panel). In other primary cells (HUVECs, neonatal rat cardiomyocytes, and rat cardiac fibroblasts), similar bimodal peaks were observed (supplemental Fig. S1). In contrast, recombinant HP1␣ expressed in Escherichia coli (Fig. 1C) exhibited only one peak with elution characteristics similar to those of the hydrophilic peak under the same separating condition used for the endogenous protein (Fig. 1D, bottom panel). These data suggest that two different forms of HP1␣ endogenously exist in multiple cell types and that the late-eluted hydrophobic species may be a post-translationally modified form.
To further elucidate the molecular characteristics of these two forms of HP1␣, we used recombinant FLAG-tagged HP1␣ (HP1␣-FLAG). As with endogenous HP1␣, HP1␣-FLAG existed mainly as a nuclear protein (Fig. 1E) and exhibited the FIGURE 1. Endogenous HP1␣ shows a bimodal distribution after protein purification by reverse-phase HPLC. The late-eluted fraction of HP1␣ is oxidatively modified to form a disulfide bond. A, schematic representation of HP1␣ purification from cell lysates using sequential column chromatography. B, equal quantities of cytosolic and nuclear fractions from HEK293T cells were resolved by SDS-PAGE and probed with the indicated antibodies. C, GST-HP1␣ expressed in E. coli was purified and cleaved by Factor Xa (upper panel) and detected with anti-HP1␣ antibody (lower panel). D, HEK293T cell lysate was fractionated by a Q-Sepharose HP anion-exchange column. Eluted fractions were resolved by reducing SDS-PAGE and probed with anti-HP1␣ antibody (top panel). The x axis at the upper edge indicates salt concentration. HP1␣ fractions eluted from the anion-exchange column were next applied to a phenyl reverse-phase column. The fractions were eluted by a fast gradient (buffer B, 1.0% increase of acetonitrile concentration/fraction, 2nd panel from the top) or by a slow gradient (buffer B, 0.2%/fraction, 3rd panel from the top). Nuclear extraction from HEK293T cells (4th panel from the top) or HP1␣ purified from E. coli (bottom panel) was fractionated with the same slow gradient. The eluted fractions were resolved by reducing SDS-PAGE and probed with anti-HP1␣ antibody. E, equal quantities of cytosolic and nuclear fractions from HEK293T cells expressing HP1␣-FLAG were resolved by SDS-PAGE and probed with the indicated antibodies. F, nuclear extract from HEK293T cells expressing HP1␣-FLAG was directly applied to a reverse-phase column, and the eluted fractions were resolved by reducing SDS-PAGE and probed with the indicated antibodies. G, diagrams of the representative deletion mutant or point mutant of the HP1␣ protein during stepwise mutation analysis (left column). Nuclear extractions from HEK293T cells expressing each mutant protein were fractionated by reverse-phase HPLC, resolved by SDS-PAGE, and probed with anti-FLAG antibody (right column). H, endogenous HP1␣ was purified from the HEK293T cell lysate as shown in A. The fractions eluted from the reverse-phase column were resolved by SDS-PAGE under nonreducing conditions and probed with anti-HP1␣ antibody. I, nuclear extract from HEK293T cells was incubated with 2 mM DTT or 2.5% 2-mercaptoethanol (ME) for 30 min at 4°C and then applied to a reverse-phase column. The eluted fractions were resolved by SDS-PAGE and probed with anti-HP1␣ antibody. D and F-I, the x axis at the lower edge indicates fraction numbers. same bimodal distribution after reverse-phase HPLC (Fig. 1F). Thus, we concluded that HP1␣-FLAG undergoes the same modification as endogenous HP1␣, validating the use of the tagged protein for further analysis. Initially, we attempted to detect the specific modification directly by matrix-assisted laser desorption/ionization and time-of-flight mass spectrometry (MALDI-TOF/MS) (supplemental Fig. S2, A-C). Although we detected peptide masses from both fractions corresponding to ϳ75% of the entire HP1␣ sequence (supplemental Fig. S2B), we did not detect any distinct features in the mass spectra under two different digestion conditions (trypsin or Asp-N) (supplemental Fig. S2C). We next tried to detect a modified residue by making multiple, stepwise mutations throughout the entire HP1␣ molecule. We hypothesized that HP1␣-FLAG lacking the modified residue would fractionate into a single peak by reverse-phase HPLC. First, we thoroughly screened the CD and hinge region, both of which are reported to be posttranslationally modified (17,22). However, we could not determine any specific amino acid residue from the mutational analysis (supplemental Fig. S2D). Second, we screened the CSD (supplemental Fig. S2E) and found that a deletion mutant lacking residues 119 -150 (⌬119 -150) was eluted as a single peak. We further narrowed down the deleted sequence 119 -150 and finally found that a mutant in which cysteine 133 (Cys-133) was replaced by alanine (C133A) was eluted as a single peak (Fig.  1G). These data suggest that the single cysteine 133 residue is responsible for the separation of the hydrophobic fraction of HP1␣.
Among post-translational modifications of cysteine, oxidation is a common feature. The thiol side chain can be oxidized to sulfenic acid (-SOH), sulfenyl amide (-SN), a disulfide bond (-SS-) or an irreversibly oxidized form (25). We examined the electrophoresis pattern of the two separated fractions of HP1␣ under nonreducing conditions and found that the hydrophobic form of HP1␣ shifted to a molecular weight twice its size, indicating that this HP1␣ formed a homodimer (Fig. 1H). In contrast, the mobility of the hydrophilic HP1␣ was unchanged. Because this dimer was nondissociable both under the strong acidic conditions of the reverse-phase HPLC and under the denaturing conditions during SDS-PAGE, it seemed to be linked by a covalent bond. Pretreatment with reducing agents, such as 2 mM DTT or 2.5% 2-mercaptoethanol, completely abolished the hydrophobic fraction (Fig. 1I). Taken together, these data suggest that endogenous HP1␣ dimerizes by intermolecular disulfide bond formation via Cys-133.
HP1␣ and HP1␥ Both Possess an Isoform-specific Cysteine Residue for Disulfide Bond Formation-The sequence identity among the three HP1 isoforms is remarkably high ( Fig. 2A), with up to 80% homology in the CSD. However, Cys-133 is specific to HP1␣ and is replaced by a serine in HP1␤ and HP1␥ (highlighted in red in Fig. 2A). Therefore, we evaluated whether this oxidative modification was specific for HP1␣. Endogenous HP1␤ was fractionated as a single peak by reverse-phase HPLC. However, endogenous HP1␥ was isolated as two separate peaks (Fig. 2B). Both the hydrophilic and the hydrophobic fractions of HP1␥ were eluted independently of those of HP1␣ suggesting that these two isoforms did not interact with each other during reverse-phase HPLC fractionation. Similar to HP1␣, the hydro-phobic form of HP1␥ also dimerized (Fig. 2C). HP1␤ contains only two cysteines, both of which are conserved among the isoforms (Cys-59 and Cys-160 of HP1␣; highlighted in blue in Fig. 2A). HP1␥ has three cysteines, and one of the cysteines, Cys-177, is an isoform-specific residue located in the C terminus of the CSD. This residue is replaced by tyrosine in HP1␣ and HP1␤ (highlighted in red in Fig. 2A). Mutational analysis of these cysteine residues revealed that only isoform-specific Cys-133 of HP1␣ and Cys-177 of HP1␥ were involved in dimerization (Fig. 2D). Mutating the corresponding residues of HP1␤, Ser-129 (matched to Cys-133 of HP1␣) or Tyr-173 (matched to Cys-177 of HP1␥), to cysteines created the late-eluted hydrophobic form (Fig. 2E). These hydrophobic forms of HP1␤ dimerized similarly with HP1␣ and HP1␥ (Fig. 2F). The other two HP1␤ mutants, S141C and S162C, did not form disulfide bonds. Together, these data suggest that even though their overall structures are highly conserved, endogenous HP1␣ and HP1␥ possess isoform-specific cysteine residues involved in the intermolecular disulfide bond formation. These two positions of the disulfide-linked cysteines are structurally sensitive to oxidation within the CSD.
HP1␥ Is More Sensitive to Oxidation than HP1␣ in Vitro-We tested whether the differences in the positions of the modified cysteine residues between HP1␣ and HP1␥ influenced their sensitivity to oxidation in vitro. Under mild oxidative conditions, only a low level of dimerized HP1␣ was detected even after a long exposure to air oxidation (Fig. 3A, left panels). In contrast, under the same conditions, HP1␥ was easily oxidized to form disulfide bonds (Fig. 3A, right panels). Treatment with DTT reversed the disulfide formation of HP1␥. These data indicate that HP1␥ is more sensitive to oxidation and more readily forms a disulfide dimer in vitro.
Using purified and oxidized HP1␥-FLAG, the intermolecular disulfide bond was confirmed by MALDI-TOF/MS analysis. The late-eluted dimerized fraction of HP1␥-FLAG was resolved by nonreducing SDS-PAGE, and the excised band was divided into two samples. One sample was reduced, carbamidomethylated with iodoacetamide, and digested by trypsin. The other sample was directly digested without pretreatment. The expected digested peptide, including Cys-177, consisted of the C terminus of HP1␥ and lysine residue within the linker peptide (Fig. 3B). The mass spectrum peak of 3084.32, which was detected only in the nonreduced sample, corresponded to the estimated mass of the dimeric peptide connected by a disulfide bond via Cys-177 (3084.35) (Fig. 3C, upper panel). In contrast, the peak at 1600.68, which was detected only in the reduced sample, corresponded to the estimated mass of the monomeric peptide, including carbamidomethylated Cys-177 (1600.71) (Fig. 3C, lower panel). No other significant mass spectral peaks from the intermolecular disulfide bond were detected.
HP1␥, but Not HP1␣, Readily Forms Disulfide Bonds under in Vivo Oxidative Conditions-We assessed whether this oxidative modification was promoted under in vivo oxidative conditions using a pro-oxidant agent, 2-methyl-1,4-naphthoquinone (menadione), which caused oxidative stress in cells (Fig. 4A) (26). Menadione treatment caused a dose-and time-dependent increase in the disulfide bond formation of HP1␥ in COS7 cells (Fig. 4B, left two panels). The disulfide dimerization of HP1␥ FIGURE 2. Both HP1␣ and HP1␥ possess isoform-specific cysteine residues that are oxidatively modified to form disulfide bonds. A, amino acid sequence alignment among mouse HP1 isoforms. Crosswise two-headed arrows indicate the N-terminal CD and C-terminal CSD. The bold blue arrow and bold white line along the CSD indicate ␤-sheet and ␣-helix, respectively. Blue highlights represent the following: two cysteine residues conserved among the HP1 family (Cys-59 and Cys-160; HP1␣). Red highlights represent the following: position of the cysteine residue specific to HP1␣ (Cys-133) or HP1␥ (Cys-177). The arrowhead indicates the position of the mutated HP1␤ serine residue (shown in E). B, nuclear extract from HEK293T cells was directly applied to a reverse-phase column, and the eluted fractions were resolved by SDS-PAGE and probed with anti-HP1␣, -␤, or -␥ antibodies. The immunoblotting procedure was performed by consecutive stripping and reprobing with each antibody of the same membrane. The upper band of fraction 20 in the bottom panel (arrowhead) indicates the residual signal from hydrophilic HP1␣. C, hydrophobic fractions of HP1␥ purified from HEK293T cells (as shown in Fig. 1A) were resolved by SDS-PAGE under reducing or nonreducing conditions and probed with anti-HP1␥ antibody. D, nuclear extract from HEK293T cells expressing each HP1␣-FLAG (top three panels) or HP1␥-FLAG (bottom three panels) with a cysteine-to-serine mutation was fractionated by reverse-phase HPLC, resolved by SDS-PAGE, and probed with anti-FLAG antibody. E, nuclear extract from HEK293T cells expressing HP1␤-FLAG with each serine-to-cysteine or tyrosine-to-cysteine mutation was fractionated by reverse-phase HPLC, resolved by SDS-PAGE, and probed with anti-FLAG antibody. F, late-eluted hydrophobic fraction of the HP1␤-FLAG mutant (S129C or Y173C) was resolved by reducing or nonreducing SDS-PAGE and probed with anti-FLAG antibody. B-E, the x axis at the lower edge indicates fraction numbers. was rapidly formed within minutes and was only formed via Cys-177 (Fig. 4C). The I165E mutation, which inhibits both noncovalent ␣-helix dimer formation and proper nuclear localization (6 -7), decreased, but not completely, the amount of disulfide dimers of HP1␥ (supplemental Fig. S3A). These data suggest that the oxidative dimerization of HP1␥ requires the proper localization and formation of constitutive, noncovalent dimers.
In contrast to HP1␥, an increase in dimerized HP1␣ was not observed under the same in vivo oxidative conditions (Fig. 4B,  right panel). The dimerized forms of HP1␣ and HP1␥ under basal conditions were almost undetectable without using the large scale purification shown in Fig. 1 because of their relatively low abundance before oxidant treatment. Menadione treatment promoted HP1␥ dimerization in various cells, but the extent of dimerization varied among cell types (supplemental Fig. S3B), suggesting that the reactivity of HP1␥ to reactive oxygen species stimulation varied according to cell type. In each cell, an increase in dimerized HP1␣ was not observed (data not shown). These results demonstrate that there is a clear difference in oxidation sensitivity among HP1 family members. Although both HP1␣ and HP1␥ have oxidationsensitive cysteines in their sequences, HP1␥ perceives oxidative conditions and is able to more readily form a disulfide dimer than HP1␣.
In HEK293T cells, the dimerized HP1␥ was subsequently reduced to the monomer form after removal of the oxidant (Fig.  4D, upper panel), but HP1␥ remained dimerized when continuously exposed to the oxidants (Fig. 4D, lower panel), suggesting that this oxidative modification was reversible. H 2 O 2 , known as an endogenous source of reactive oxygen species, also promoted dimerization of HP1␥ (Fig. 4E). This effect of H 2 O 2 was relatively weak in HEK293T cells when compared with the treatment of menadione. However, the same concentration of H 2 O 2 substantially increased the amount of dimerized HP1␥ in HUVECs (Fig. 4E, lower panel). Therefore, we further examined the molecular characteristics of the disulfide dimerization of HP1␥ using HUVECs.

Under Oxidative Conditions, HP1␥ Strongly and Transiently Interacts with TIF1␤ and Holds It in a Chromatin Component-
The CSD of HP1, which includes Cys-177 at its C terminus, creates a binding surface for other proteins (27). Therefore, disulfide modification of HP1␥ may affect the interactions between HP1 and HP1-binding proteins. Because many candidate effectors that bind to HP1 exist (8), we screened the interacting proteins of HP1␥ under oxidative conditions using metabolically radiolabeled HUVECs expressing recombinant HP1␥-FLAG transduced with adenovirus. Among the co-immunoprecipitated proteins, one protein band was detected after treatment with H 2 O 2 (Fig. 5A, arrowhead). The bound protein was purified and analyzed by MALDI-TOF/MS. The amino acid sequence of the digested peptides corresponded to TIF1␤ (also known as TRIM28 or KAP1), which is a universal co-repressor of gene transcription and is a well known interacting partner of HP1 (28 -31). Co-immunoprecipitation analysis showed that endogenous HP1␥ strongly interacted with TIF1␤ in a dose-dependent manner after H 2 O 2 treatment (Fig. 5B). TIF1␤ did not interact with HP1␥ with a C177S mutation under oxidative conditions, suggesting that the disulfide bond formation of HP1␥ enhanced the interaction of these proteins (Fig.  5C). When the oxidant was removed, TIF1␤ dissociated again from HP1␥, suggesting that this enhanced endogenous interac-  OCTOBER 8, 2010 • VOLUME 285 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 31343 tion was transient (Fig. 5D). Structurally, disulfide dimerization via Cys-177 is formed at the C terminus of the CSD, just adjacent to the binding interface for the PXVXL motif, which is a well characterized binding sequence in HP1-interacting proteins, including TIF1␤ (Fig. 5E) (7).

Isoform-specific Oxidative Modification of HP1
We next examined the localization changes of these proteins before and after oxidant treatment. No remarkable change in localization was detected by immunostaining (data not shown). However, biochemical analysis using Triton extraction verified the TIF1␤ translocation. HP1␥ existed mainly in the Tritoninsoluble chromatin component, whereas TIF1␤ was distrib-uted both in the soluble and the insoluble components (Fig. 5F). Under oxidative conditions, HP1␥ dimerized and was maintained in the insoluble components. Concomitant with HP1␥ dimerization, the insoluble component of TIF1␤ transiently increased. The knockdown of endogenous HP1␥ combined with the replacement by a C177S mutant of HP1␥ inhibited the translocation of TIF1␤, suggesting that HP1␥ held TIF1␤ on chromatin only when oxidized via Cys-177 (Fig. 5G). These data suggest that the intracellular redox state is transduced to the conformational and localization change of the repressor complex via oxidative modification of HP1␥. Dimerized HP1␥ under Oxidative Conditions Inhibits the Repression Ability of TIF1␤-To clarify whether the repression ability of TIF1␤ was promoted or inhibited when trapped by HP1␥ under oxidative conditions, we used a GAL4-based transcriptional reporter assay. In HEK293T cells, menadione treatment promoted the disulfide dimerization of HP1␥ and the interaction between HP1␥ and TIF1␤ more prominently than H 2 O 2 treatment (supplemental Fig. S3B and Figs. 4E and 6A). Therefore, we used menadione treatment for further analysis in HEK293T cells. We generated HEK293T cells stably expressing shRNA for HP1␥ and shRNA-resistant recombinant FLAGtagged HP1␥ WT or C177S mutant. In these cells, endogenous HP1␥ was almost completely depleted and was replaced by the dimerizable or undimerizable recombinant proteins (Fig. 6B). To evaluate the transcriptional repression ability of TIF1␤, we transfected the plasmids encoding ERHBD-GAL4 as a control or ERHBD-GAL4-TIF1␤ fusion protein with the reporter plasmids in these cells (12,32). The transcriptional regulatory activity of the ERHBD fusion protein is post-translationally controlled by the addition of 4-OHT to the culture medium (12). In our experimental conditions where ERHBD-GAL4 and ERHBD-GAL4-TIF1␤ were equally expressed (Fig. 6C, left  panel), only ERHBD-GAL4-TIF1␤ repressed transcription of luciferase with 4-OHT (500 nM) in the HEK293T stable cells (Fig. 6C, right panel). The extent of repression was similar between the cells expressing either the HP1␥-FLAG WT or the C177S mutant under nonoxidative conditions. When the cells were treated with menadione, HP1␥-FLAG WT strongly interacted with ERHBD-GAL4-TIF1␤ as was similarly observed with endogenous proteins (Fig. 6D). To examine the transcrip- tional change of the luciferase gene under oxidative conditions, we measured the intranuclear mRNA levels of luciferase by quantitative PCR instead of luciferase protein enzymatic activity (Fig. 6E). We chose this end point because the oxidation of HP1␥ was too rapid to properly evaluate its effect on luciferase transcription by measuring luciferase protein enzymatic activity. Under these conditions, menadione treatment relieved the levels of luciferase transcription repressed by ERHBD-GAL4-TIF1␤ in the cells expressing HP1␥-FLAG WT but did not relieve the levels in the cells expressing the C177S mutant (Fig.  6F). These data suggest that dimerized HP1␥ under oxidative conditions inhibits the repression ability of TIF1␤.
It remained unclear whether the intranuclear redox-sensing mechanism through the oxidative modification of HP1␥ plays a role in the cellular response to extrinsic oxidative stress. Therefore, we assessed the effect of this modification on cell survival under oxidative conditions using HEK293T cells stably expressing shRNA against HP1␥ (supplemental Fig. S4A). Depletion of HP1␥ using shRNA uniformly decreased cell viability under oxidative conditions induced by menadione treatment (supplemental Fig. S4B). For a rescue experiment, these stable clones were transduced with an adenoviral vector encoding WT HP1␥-FLAG or C177S HP1␥-FLAG. Both HP1␥ vectors were cloned from murine cDNA and were resistant to shRNA against human HP1␥. Transduction of both adenoviral constructs at a multiplicity of infection of 20 resulted in nearly equal expression of recombinant HP1␥ with endogenous HP1␥ and yielded a similar disulfide dimerization pattern (supplemental Fig. S4C). Under these conditions, WT HP1␥-FLAG rescued cell viability after menadione treatment in each stable clone, but the C177S HP1␥-FLAG mutant did not rescue cell viability (supplemental Fig. S4D). These results suggest that HP1␥ disulfide dimerization plays a pivotal role in cell survival under oxidative conditions.

DISCUSSION
In this study, we identified isoform-specific disulfide bond formation, which is a novel post-translational modification of HP1, using a unique column chromatography method. Biochemical analysis revealed two isoform-specific reactive cysteine residues, cysteine 133 in HP1␣ and cysteine 177 in HP1␥. In particular, HP1␥ readily and reversibly formed disulfide dimers under oxidative conditions. Dimerized HP1␥ strongly interacted with TIF1␤ and held it in a chromatin component. The GAL4 tethering repression assay revealed that the tight interaction of the repressor proteins had a reversing effect for transcriptional repression.
Several post-translational modifications of HP1 have been reported. Specifically, the linker region between the CSD and CD is highly amenable to post-translational modifications, especially phosphorylation that affects silencing activity or nuclear location of HP1 (17,(33)(34)(35). Also in the CD, Thr-51 of HP1␤ has been shown to be phosphorylated in response to DNA damage (22). More recently, a comprehensive proteomic analysis revealed that all HP1 isoforms are highly modified by phosphorylation, acetylation, methylation, and formylation both in the CD and in the CSD (36). Prior to this study, however, no oxidative modification of HP1 had been identified. Because oxidative modifications at cysteine residues would be easily disrupted under reducing conditions, such modifications may be detected only by the unique HPLC-based method used in this study and not by ordinary mass spectrometry analysis.
Both isoform-specific cysteines involved in forming disulfide bonds reside in a structurally flexible region of the CSD. Cys-133 of HP1␣ lies in the long loop between the ␤1 and ␤2 sheets, and Cys-177 of HP1␥ lies in the C-terminal region. Introducing cysteine residues into these flexible sites of HP1␤ conferred the ability to form disulfide bonds, suggesting that these sites have specific structures in the oxidative center. Although both cysteines were reactive, a distinct difference of sensitivity to oxidation existed. Each location of reactive cysteines and the surrounding structure might determine the sensitivity of HP1␣ and -␥ to oxidation. Under both in vitro and in vivo oxidative conditions, HP1␥ readily formed disulfide bonds. In contrast, only minimal disulfide formation of HP1␣ by oxidation was observed under our experimental conditions. The reactivity of HP1␣ under oxidation might be observed under different conditions. Nonetheless, the isoform specificity and functional importance of Cys-133 in HP1␣ has been reported previously (15).
HP1 has been reported to form dimers via the CSD, but these dimers are not mediated by disulfide bonds or other covalent bonds (6,37,38). Thus, HP1 dimerizes in at least two ways. The interface of the noncovalently linked dimer involves a symmetrical interaction on helix ␣2 of the CSD (27) and creates a nonpolar groove structure, which is a binding site for the PXVXL motif in HP1-interacting proteins, such as TIF1␤ (Fig. 5E) (7). Because reactive cysteine 177 in HP1␥ is located in the C terminus adjacent to the groove structure, disulfide bond formation at this site likely affects the binding affinity of HP1␥. Indeed, HP1␥ strongly and transiently interacted with TIF1␤ and promoted its translocation to a chromatin component stringently depending on the oxidative status of cysteine 177. This rapid reacting mechanism to transduce cellular redox state to a conformational change like a clear "on-off switch" suggests that HP1␥ is a functional redox sensor.
During the cellular response to oxidative stress, an increase in oxidants can trigger alterations in transcription levels through direct activation or by promoting a change in the subcellular localization of transcription factors by oxidizing reactive cysteine residues (25). Among these oxidative responses, the disulfide dimerization of HP1␥ demonstrated in this study appears to be one of the most rapid transcriptional regulatory mechanisms. TIF1␤ is a universal co-repressor for the Krüppelassociated box domain containing the zinc finger protein (KRAB-ZNF) family of transcription factors, and it is the major protein binding the CSD of HP1 (28 -31). TIF1␤ also works as a scaffold for the repressor complex, and its interaction with HP1 is essential for its repression activity (12, 39 -41). Recent findings have revealed that the binding of HP1 to TIF1␤ is essential for their coordinated function on the promoter of the endogenous genes (42). Therefore, the reversing effect for the repressive ability of TIF1␤ caused by HP1␥ disulfide dimerization might be required for a short period of adaptation against oxidative stress. Downstream genes regulated by these scaffold complexes remain to be clarified in the future analysis.
In conclusion, our study suggests that HP1 potentially acts as a rapid redox sensor, and it may connect the intracellular redox state with transcriptional regulation under various physiological conditions.