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To whom correspondence should be addressed: Dept. of Biological Science, Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555, Japan. Tel.: 81-96-342-3450; Fax: 81-96-342-3450
* This work was supported by a grant from the Ministry of Education, Culture, Sports, Science, and Technology and by a COE grant from MEXT Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S3. 1 A research fellow of the Japan Society for the Promotion of Science.
Small ubiquitin-related modifiers, SUMO-2/3 and SUMO-1, are involved in gene regulation and nuclear structures. However, little is known about the roles of SUMO, in heterochromatin formation of mammalian cells. Here we demonstrate that SUMOs directly interact with human MCAF1, which forms complexes with either the methyl-CpG-binding protein MBD1 or SETDB1, which trimethylates histone H3 at lysine 9 (H3-K9) in the presence of MCAF1. Modification of MBD1 with either SUMO-2/3 or SUMO-1 facilitated the interaction between MBD1 and MCAF1, suggesting that SUMOylation links the methylation of DNA and histones. In a cultured human cell line, SUMOs were localized in MBD1- and MCAF1-containing heterochromatin regions that were enriched in trimethyl-H3-K9 and the heterochromatin proteins HP1β and HP1γ. Specific knockdown of either SUMO-2/3 or SUMO-1 induced dissociation of MCAF1, trimethyl-H3-K9, and the HP1 proteins from the MBD1-containing heterochromatin foci, suggesting a requirement for SUMOs for heterochromatin assembly. These findings provide insights into the roles of SUMOylation in the regulation of heterochromatin formation and gene silencing.
The abbreviations used are: SUMO, small ubiquitin-related modifier; siRNA, small interfering RNAs; TRD, transcriptional repression domain; AM, ATFa-associated modulator; GST, glutathione S-transferase; MBP, maltose-binding protein; PBS, phosphate-buffered saline; EGFP, enhanced green fluorescent protein; E3, SUMO-protein isopeptide ligase; PML, promyelocytic leukemia.
3The abbreviations used are: SUMO, small ubiquitin-related modifier; siRNA, small interfering RNAs; TRD, transcriptional repression domain; AM, ATFa-associated modulator; GST, glutathione S-transferase; MBP, maltose-binding protein; PBS, phosphate-buffered saline; EGFP, enhanced green fluorescent protein; E3, SUMO-protein isopeptide ligase; PML, promyelocytic leukemia.
are covalently attached to their target proteins by a process referred to as SUMOylation. This process involves an enzymatic pathway, which is similar to the pathway employed in the ubiquitin conjugation cascade (
). SUMO-2 and -3 are more closely related to each other (95% amino acid identity) than to SUMO-1 (∼50% identity). It has become increasingly clear that a wide variety of cellular proteins can be modified by either SUMO-2/3, SUMO-1, or both, leading to alterations in many signaling pathways associated with their target proteins. Most, but not all, of the SUMOylation target proteins appear to be involved in maintenance of nuclear integrity, regulation of nuclear transport, or control of chromatin functions, such as transcription, replication, repair, recombination, and chromatin modification (
Currently, there are numerous examples of co-repressor proteins that can be SUMOylated and transcription factors whose activities are down-regulated by SUMOylation, implying the involvement of a SUMO modification system in the regulation of transcriptional repression and maintenance of silenced heterochromatin (
). Although the exact mechanisms by which SUMO modification contributes to such anti-activation activities in chromatin remain poorly defined, it has been speculated that SUMOylation mediates changes in gene expression and chromatin assembly, at least, by serving as a binding platform for recruiting other chromatin proteins that may transmit or amplify the SUMO signal and maintain the silenced state of chromatin (
), implying the possibility that histone-modifying enzymes may act as effectors for SUMO modification signals.
Covalent addition of a methyl group to the 5-position of cytosine (5mC) is the major modification of DNA in vertebrate genomes. This modification predominantly occurs within CpG dinucleotides and is involved in a wide range of biological phenomena, including genomic imprinting, X chromosome inactivation, and tissue-specific gene expressions (
). These proteins utilize transcriptional co-repressors or mediators to silence transcription and also modify the surrounding chromatin, thereby providing a link between DNA methylation and chromatin remodeling and modifications. Among these proteins, MBD1 is relatively well characterized and has been implicated in regulating chromatin structure and gene silencing through a currently unknown mechanism that probably involves histone modifications, such as deacetylation and H3-K9 methylation (
Considering the biological consequences of the effect of MBD1 on DNA methylation, the roles of an MBD1-interacting protein, designated MBD1-containing chromatin-associated factor 1 (MCAF1; also known as ATFa-associated modulator (AM)), are intriguing. Specifically, this protein has been reported to interact with MBD1 via the transcriptional repression domain (TRD) at the C-terminal region of MBD1 (
), suggesting that it acts as a recruiter for a wide range of proteins that can modulate gene regulation and chromatin formation. However, the mechanisms involved in regulating the assembly of macromolecular complexes containing MCAF1 and how such chromatin complexes contribute to the regulation of gene silencing and heterochromatin formation remain largely uncharacterized.
Here we report that MCAF1 interacts with SUMO-2/3 and SUMO-1, with a preference for SUMO-2/3, via short peptide sequences similar to the SUMO-binding motif. By using a human cultured cell line, we demonstrate SUMOylation of MBD1 both in vitro and in vivo, as well as facilitated anchorage of MCAF1 to SUMOylated MBD1. Furthermore, RNA interference experiments directed against SUMO-2/3 or SUMO-1 reveal that depletion of the SUMO pathways perturbs the assembly of MCAF1, trimethyl-H3-K9, HP1β, and HP1γ at MBD1-containing heterochromatin. Taken together, our results indicate that SUMOs function as epigenetic modulators for heterochromatin formation, at least in part by regulating the MCAF1-MBD1 interaction.
Plasmids—To generate pAS2-1 SUMO-3G-SUMO-3G, human SUMO-3 was amplified by PCR using the following oligonucleotides: forward primer, 5′-CATGCCATGGCCGACGAAAAGCCCAAG-3′ (an NcoI site is underlined); reverse primer, 5′-CTGCAGAACCAGCACAATGGTCCCGTCTGCTGTTGGAACAC-3′ (a BstXI site is underlined); forward primer, 5′-CTGCAGAACCATTGTGCTGGGCCGACGAAAAGCCCAAG-3′ (a BstXI site is underlined); and reverse primer, 5′-CGCGGATCCTCATCCCGTCTGCTGTTGGAACAC-3′ (a BamHI site is underlined). The generated PCR fragments were digested with NcoI, BstXI, and BamHI and cloned into pAS2-1 predigested with NcoI and BamHI. To generate pGEX4T-1 SUMO-1/2/3, SUMO sequences were PCR-amplified and subcloned into pGEX4T-1 (Amersham Biosciences). To generate pET30 SUMO-1/2/3, SUMO sequences were PCR-amplified and subcloned into pET30 (Novagen). To generate pcDNA3 DsRed MCAF1-(965-975), MCAF1-(965-975) was annealed using the following oligonucleotides: 5′-AATTCGGTGTCATTGATCTCACAATGGATGATGAAGAGTGAC-3′ and 5′-TCGAGTCACTCTTCATCATCCATTGTGAGATCAATGACACCG-3′, and subcloned into pcDNA3 DsRed monomer.
To construct plasmids expressing MCAF1-D968A and -L969A, site-directed mutagenesis was performed. MBD1-(373-605) was PCR-amplified and subcloned into pMAL-c2X (New England Biolabs) using the following oligonucleotides: forward primer, 5′-CGGAATTCCGTTGGCGCCAATGCCTGCAGTTT-3′ (an EcoRI site is underlined), and reverse primer: 5′-CCGTCTAGACTACTGCTTTCTAGCTCCAGGTTT-3′ (an XbaI site is underlined). MCAF1 and MBD1 expression plasmids were used as described previously (
Yeast Two-hybrid Screening—The yeast strain AH109 carrying pAS2-1 SUMO-3G-SUMO-3G was transformed with a mouse 11-day embryo cDNA library constructed in pACT2 (Clontech). Plasmids harboring cDNAs were recovered from histidine-positive colonies.
Cell Culture, Small Interfering RNAs (siRNAs), and Transient Transfection—HeLa and C-33A cells were cultured in Dulbecco's modified Eagle's medium nutrient mixture F-12 Ham (Sigma) supplemented with 5% fetal calf serum and antibiotics under 5% CO2. HeLa cells were transfected with plasmid DNAs using FuGENE 6 (Roche Applied Science). For siRNA experiments, C-33A cells were transfected with 20 nm siRNA duplex oligoribonucleotides using Oligofectamine (Invitrogen) and harvested at 60 h post-transfection. The siRNA duplexes used were designed to target the mRNAs encoding human SUMOs. The sequences were as follows: SUMO-1 number 1, 5′-GAAUCAUACUGUCAAAGACAGGGUG-3′ (Stealth™ siRNA; Invitrogen); SUMO-1 number 2, 5′-CACAUCUCAAGAAACUCAA-3′ (21-mer siRNA; Japan Bioservice); SUMO-2 number 1, 5′-GGCCUACUGCGAGAGGCAGGGCUUG-3′ (Stealth™ siRNA; Invitrogen); SUMO-2 number 2, 5′-UGAGGCAGAUCAGAUUCAG (21-mer siRNA; Japan Bioservice); SUMO-3 number 1, 5′-AGCCUAUUGUGAACGACAGGGAUUG-3′ (Stealth™ siRNA; Invitrogen); SUMO-3 number 2: 5′-GAUUAAGAGGCAUACACCA (21-mer siRNA; Japan Bioservice). The siRNAs for GL3 were described previously (
Bacterial SUMOylation, Protein Expression, and Purification—SUMO-modified MBP-MBD1-(373-605) was obtained using an Escherichia coli SUMOylation system. E. coli strain BL21(DE3) co-transformed with pMAL-MBD1-(373-605) and pT-E1E2S3 (or pT-E1E2S1) protein expression vectors (
) was used to express MBP-tagged and SUMO-modified MBD1-(373-605). Expression of recombinant proteins was induced with 0.2 mm isopropyl β-d-galactopyranoside at 25 °C for 18 h. Purification of GST fusion proteins, MBP fusion proteins, and His6 fusion proteins was carried out as described previously (
GST Pulldown Assay—The standard conditions used were as follows: bacterially expressed GST and GST fusion proteins (3 μg) were immobilized on glutathione-Sepharose beads (Amersham Biosciences) and incubated with His6-tagged proteins (3 μg) or E. coli lysates in a buffer (500 μl) consisting of 20 mm HEPES, pH 7.9, 20 mm KCl, 1.5 mm MgCl2, 0.01 mm ZnCl2, 10% glycerol, 0.1% Triton X-100, and 0.1 mm dithiothreitol for 1 h at 4 °C. For the binding assay described in Fig. 5A, the pulldown assay was performed in a buffer (500 μl) consisting of 25 mm HEPES, pH 7.5, 150 mm NaCl, 0.05% Nonidet P-40, and 1mm dithiothreitol (
Immunoprecipitation—C-33A and HeLa cells were treated with phosphate-buffered saline (PBS) containing 0.1% Nonidet P-40 and 20 mmN-ethylmaleimide on ice for 10 min, rinsed with ice-cold PBS, and lysed on ice with RIPA buffer (50 mm HEPES, pH 8.0, 150 mm NaCl, 1% Nonidet P-40, 0.1% sodium deoxycholate, 20 mmN-ethylmaleimide, 0.1 mm dithiothreitol, and protease inhibitors). After sonication and centrifugation for 15 min, nuclear cell lysates were incubated with specific antibodies or control IgG for 30 min at 4 °C. Next, 30 μl of protein G-agarose beads (Amersham Biosciences) was added, and the samples were incubated for a further 1 h. The beads were extensively washed with RIPA buffer as the binding buffer. The proteins associated with the beads were separated by SDS-PAGE and subjected to immunoblot analysis.
Indirect Immunofluorescence Assay—For immunofluorescence labeling, cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature and then permeabilized with 0.2% Triton X-100 in PBS for 5 min. Following three rinses with PBS, the cells were sequentially incubated with a specific primary antibody, followed by an appropriate secondary antibody. The secondary antibodies used were as follows: Alexa-488-conjugated donkey anti-rat IgG (Molecular Probes), Cy3-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch), Cy3-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch), and Cy5-conjugated donkey anti-goat IgG (Jackson ImmunoResearch). After three further washes with PBS, the cells were mounted under coverslips and analyzed using an Olympus IX71 microscope and the Lumina Vision software (Mitani Co.).
MCAF1 Binds to SUMO-1 and SUMO-2/3 with a Preference for SUMO-2/3—To identify possible chromatin proteins that may specifically recognize the SUMO moiety and be recruited to sites of SUMOylation, we performed a yeast two-hybrid screening using the Gal4-SUMO-3G-SUMO-3G fragment, which was incapable of conjugation, as bait. After multiple rounds of screening of a mouse 11-day embryo cDNA library, we isolated a clone encoding amino acids 787-1306 of the ATFa-associated factor AM, a mouse homolog of human MCAF1 (Fig. 1A).
As a step toward understanding the role of MCAF1 binding to SUMO, we first performed in vitro GST pulldown assays to investigate whether the binding was direct and whether the binding affinities varied among the SUMO paralogs. When beads bound to GST-SUMO-1, GST-SUMO-2, GST-SUMO-3, or GST alone were incubated with a lysate from C-33A human cervical cancer cells, MCAF1 associated more efficiently with GST-SUMO-2/3-beads than with GST-SUMO-1-beads (Fig. 1B). MCAF1 binding to SUMO-2 and SUMO-1 was direct, because bacterially expressed recombinant full-length His6-MCAF1 protein was retained on GST-SUMO-conjugated beads (Fig. 1C).
MCAF1 Contains a Conserved Amino Acid Sequence Similar to the SUMO-binding Motif—Mouse and human MCAF1 proteins have been demonstrated to belong to an evolutionarily conserved family of MCAF/AM proteins (
). A comparison of MCAF family members revealed two highly conserved domains in both mouse and human MCAF1 proteins, referred to as domain 1 and domain 2. To determine the region responsible for binding to SUMOs, we generated a series of deletion mutants of human MCAF1 and performed GST pulldown assays. The results revealed that 11 amino acid residues (amino acids 965-975) of MCAF1 (MCAF1-(965-975)) located between domain 1 and domain 2 were sufficient for a specific interaction with His6-SUMO-3 (Fig. 1A). A semi-quantitative binding analysis revealed that the affinity of MCAF1-(965-975) for His6-SUMO-2/3 was ∼5-fold higher than that for His6-SUMO-1 (Fig. 1D).
A data base search for MCAF1-(965-975) using the basic local alignment search tool algorithm BLAST detected several peptide sequences encoded in mouse, rat, dog, and chicken MCAF1 (supplemental Fig. 1A), suggesting evolutionary conservation of this amino acid sequence. We also found that the N-terminal half of this sequence partially overlapped with a recently described hydrophobic amino acid cluster in the SUMO-binding motif present in PIAS1, PIASx, SAE2, PML IV, RanBP2/Nup358, and thymine DNA glycosylase (Fig. 1E) (
). It should be mentioned that our alanine scanning analysis revealed that valine at residue 966, isoleucine at 967, aspartic acid at 968, leucine at 969, and threonine at 970 in MCAF1-(965-975) were essential for the binding (supplemental Fig. 1B). In addition, we found that peptides in which Asp at 973 or glutamic acid at 975 was substituted for asparagine, glutamine, or alanine, respectively, showed dramatically reduced affinities for SUMO-2/3 (supplemental Fig. 1B) as well as SUMO-1 (data not shown), suggesting an important role of this acidic amino acid cluster for the binding. Although several previously described SUMO-interacting peptides, including PIAS1, PIASx, SAE2 and PML IV, also contain an acidic cluster at their C terminus (Fig. 1E) (
), the roles of the negatively charged residues in the SUMO binding have not been investigated in detail.
The SUMO-MCAF1 Interaction Occurs in Vivo—To examine the importance of the amino acid residues in MCAF1-(965-975) for the binding to SUMO-2/3, two MCAF1 point mutants in which Asp-968 and Leu-969 were substituted for alanine were generated and designated His6-MCAF1-D968A and His6-MCAF1-L969A, respectively. GST pulldown assays revealed that neither of the mutants showed stable binding to GST-SUMO-2/3 (Fig. 2A), despite the observed association between wild-type His6-MCAF1 and GST-SUMO-2/3, indicating that Asp-968 and Leu-969 are critical for the interaction with SUMO-2/3 in vitro. Importantly, when the MCAF1 mutants were fused to EGFP (EGFP-MCAF1-D968A and EGFP-MCAF1-L969A) and co-expressed with Myc-SUMO-3G in HeLa cells, Myc-SUMO-3G was barely enriched in EGFP foci in cells expressing either the EGFP-MCAF1-D968A mutant (Fig. 2B, lower panel) or EGFP-MCAF1-L969A mutant (data not shown), whereas a substantial amount of Myc-SUMO-3G accumulated in EGFP foci in cells expressing wild-type EGFP-MCAF1 (Fig. 2B, upper panel). Given that Myc-SUMO-3G is a SUMO-3 mutant incapable of conjugation, these data provide evidence that MCAF1 interacts noncovalently with SUMO-2/3 in vivo, and further confirm that Asp-968 and Leu-969 are necessary components for the binding to SUMO-2/3. Similar results were obtained when Myc-SUMO-1G was used (data not shown).
Endogenous SUMO-2/3, SUMO-1, and MCAF1 Are Co-localized—Thus far, our results had implied that MCAF1 acts as a SUMO-binding protein. Therefore, we next attempted to assess the physiological relevance of the endogenous SUMO-MCAF1 interaction using mammalian cultured cells. As shown in Fig. 3A, indirect immunofluorescence experiments revealed a large number of C-33A cells containing discrete nuclear foci in which SUMO-2/3 and MCAF1 were co-localized, although some SUMO-2/3 and MCAF1 may also be distributed throughout the nucleoplasm, except for the nucleolus. Both the sizes and numbers of the SUMO-2/3-MCAF1 foci varied among individual cells with an average of 2-6 clear and large foci per cell. Double staining with anti-SUMO-2/3 and anti-SUMO-1 antibodies (Fig. 3B, upper panel) or anti-MCAF1 and anti-SUMO-1 antibodies (data not shown) revealed that the merged signals of the nuclear foci were almost indistinguishable, suggesting enrichment of SUMO-1 in SUMO-2/3-MCAF1 foci as well. Therefore, these data indicate the relevance of not only SUMO-2/3-MCAF1 but also SUMO-1-MCAF1 interactions in vivo. Although it is possible that a larger amount of SUMO-2/3 may be enriched with MCAF1 via the preferential association of SUMO-2/3 with MCAF1, our present indirect immunofluorescence technique could not quantitatively compare the levels of SUMO-2/3 versus SUMO-1 accumulation in MCAF1 foci.
SUMOs and MCAF1 Are Enriched in Heterochromatin Proteins—Because MCAF1 was enriched in heterochromatin foci that usually contain HP1β, HP1γ, trimethyl-H3-K9, and MBD1 (
), we tested whether SUMO foci overlapped with these heterochromatin proteins in C-33A cells. Staining with anti-SUMO-2/3 (Fig. 3B) or anti-SUMO-1 (data not shown) antibodies demonstrated that the SUMOs were co-localized with heterochromatin marker proteins. In contrast, other nonheterochromatic nuclear proteins, including the RNA splicing factor SC35 (Fig. 3B), nuclear scaffold protein hnRNP-U/SAF-A (data not shown), and PML bodies (data not shown) did not show significantly merged signals with SUMO-MCAF1 foci. In particular, the transcriptionally active chromatin marker trimethyl-H3-K4, facultative heterochromatin marker trimethyl-H3-K27, and HP1 family protein HP1α did not shown any significantly merged signals with SUMO-MCAF1 foci (Fig. 3B). Taken together, these observations suggest that SUMO-MCAF1 foci represent MBD1-containing constitutive heterochromatic regions in C-33A cells.
SUMOylated Forms of MCAF1 Are Barely Detectable—Because MCAF1 and SUMOs interacted in vitro and co-localized in vivo, one could argue that MCAF1 itself could be an efficient substrate for SUMOylation. To elucidate whether MCAF1 was SUMOylated in C-33A cells, we performed an immunoprecipitation analysis of endogenous MCAF1 (supplemental Fig. 2). When the proteins immunoprecipitated with the anti-MCAF1 antibody were probed with either anti-MCAF1, anti-SUMO-2/3, or anti-SUMO-1 antibodies, none of the antibodies detected any SUMOylated bands, which were expected to migrate more slowly than the 240-kDa band of nonmodified full-length MCAF1 (supplemental Fig. 2, arrowhead). Because more than 95% of the total cellular pool of MCAF1 could be extracted under our experimental conditions (data not shown), these results indicate that either most of the endogenous MCAF1 is modified very poorly, if at all, by SUMOs, or SUMOylated MCAF1 is unstable in C-33A cells.
MBD1 Is an Efficient Substrate for SUMOylation—We therefore hypothesized that there may be SUMOylated proteins in MBD1-containing heterochromatin and that SUMOylation of such proteins may provide sufficient anchoring of MCAF1 to heterochromatin regions. A straightforward test of this idea was to determine the SUMOylated proteins residing in heterochromatin and to investigate whether SUMOylation of such proteins provided a sufficient binding platform for MCAF1. For these purposes, we undertook a “candidate screening” approach to identify proteins that could be SUMOylated and should be present in heterochromatin.
Among the proteins tested, the methyl-CpG-binding protein MBD1 was identified as a good candidate. As shown in Fig. 4, A and B, the bacterial SUMOylation system (
) revealed that a GST protein fused to the full-length form of MBD1 (GST-MBD1) was efficiently SUMOylated, further demonstrating that the C-terminal region of MBD1 (MBD1-(373-605)), which contained the TRD, was responsible for SUMOylation. Moreover, we revealed SUMOylation of MBD1 using an ectopic expression system in mammalian cultured cells (Fig. 4C). When we expressed FLAG-tagged MBD1 in HeLa cells and performed an immunoprecipitation analysis with an anti-FLAG antibody, multiple high molecular mass bands were detected by the anti-SUMO-2/3 antibodies, suggesting that FLAG-MBD1 was efficiently modified by endogenous SUMO-2/3 (Fig. 4C, lanes 2 and 5). Multiple SUMOylated bands were also detected when FLAG-MBD1 was transfected along with Myc-SUMO-3 (Fig. 4C, lanes 3 and 6). Of note, FLAG-MBD1 proteins modified by Myc-SUMO-3 migrated slightly more slowly than FLAG-MBD1 proteins modified by endogenous SUMO-2/3, due to the addition of Myc tag moieties. We also found that ectopically expressed FLAG-MBD1 was efficiently modified by either endogenous SUMO-1 or ectopically expressed Myc-SUMO-1 (data not shown). Taken together, these results demonstrate that MBD1 is efficiently modified by either SUMO-2/3, SUMO-1, or both in vitro as well as in vivo.
Endogenous MBD1 Is SUMOylated—To formally confirm that MBD1 is indeed a physiological SUMOylation substrate, we immunoprecipitated endogenous MBD1 and investigated the existence of the SUMOylated form of MBD1. As shown in Fig. 4D, in addition to the 85- and 90-kDa bands corresponding to nonmodified forms of the endogenous spliced variants of MBD1 (
), an anti-MBD1 antibody precipitated multiple high molecular mass bands, particularly around 140 and 180 kDa (Fig. 4D, white circles). Furthermore, these bands could be superimposed with bands detected by the anti-SUMO-2/3 and anti-SUMO-1 antibodies, demonstrating SUMOylation of endogenous MBD1. It should be noted that a single molecule of SUMO is expected to migrate at ∼15 kDa. Therefore, the band shifts from 85 to 90 kDa (nonmodified forms) to 140-180 kDa (modified forms) suggested that 4-6 molecules of either SUMO-2/3, SUMO-1, or both were appended to a single molecule of MBD1. Taken together, these results indicate that a significant proportion of endogenous MBD1 is modified by either SUMO-2/3, SUMO-1, or both in vivo.
SUMO Modification of MBD1 Enhances the Association between MBD1 and MCAF1—Next, we performed GST pulldown assays to test whether SUMOylation of MBD1 increased its affinity for MCAF1 compared with the affinity of the nonmodified form of MBD1 for MCAF1. When recombinant MBP fused to MBD1-(373-605) was modified with SUMO-3 in the bacterial SUMOylation system (
), the total bacterial lysate contained a mixture of SUMO-3-modified MBP-MBD1-(373-605), in which different numbers of SUMO-3 were appended to the MBD1-(373-605) moiety of the MBP fusion protein (Fig. 5A, lane 7). Upon incubation of GST-MCAF1 or GST-MCAF1-(965-975) with a total lysate containing the mixture of SUMOylated MBD1-(373-605), followed by a GST pulldown assay, we found that SUMO-3-modified MBP-MBD1-(373-605) proteins, rather than the nonmodified form of MBP-MBD1-(373-605), were efficiently enriched in the fraction precipitated with GST-MCAF1 beads (Fig. 5A, lane 2) or GST-MCAF1-(965-975) beads (lane 4). Comparisons between the 80-kDa band of the nonmodified form of MBD1-(373-605) (Fig. 5A, white arrows) and the 120-(Fig. 5A, black arrowheads) and 160-kDa (Fig. 5A, white arrowheads) bands of SUMO-3-modified MBD1-(373-605) revealed enrichment of p120 and p160, respectively. It should be noted that, given that the free form of SUMO-3 was expected to be ∼15-kDa, p120 and p160 may represent MBD1-(373-605) containing two and five molecules of SUMO-3, respectively.
By using GST pulldown assays, we also tested the binding of GST-MCAF1 to SUMO-1-conjugated forms of MBP-MBD1-(373-605). As shown in Fig. 5A (lanes 8-14), both GST-MCAF1 and GST-MCAF1-(965-975) were able to bind to SUMO-1-modified MBP-MBD1-(373-605). However, their interactions appeared somewhat weaker than the interactions of GST-MCAF1/MCAF1-(965-975) with SUMO-3 modified MBP-MBD1-(373-605) (Fig. 5, A and B). Collectively, these results suggest that the SUMOylated form of MBD1 provides a more stable association with MCAF1 than the nonmodified form of MBD1.
Depletion of Either the SUMO-2/3 or SUMO-1 Pathway Perturbs the Assembly of MCAF1, Trimethyl-H3-K9, and HP1 Foci—Our finding that SUMOylation of MBD1 facilitates its association with MCAF1 supports the idea that MCAF1 is enriched in heterochromatin regions, at least in part, via anchoring to SUMO-modified MBD1. Therefore, we used RNA interference directed against SUMO-2/3 to investigate whether SUMO-2/3 is critical for the assembly of MCAF1 at MBD1-containing heterochromatin regions in vivo. To demonstrate the specificity of the RNA interference technique, we used two different siRNA duplexes for SUMO-1 (SUMO-1 numbers 1 and 2 siRNAs) and two independent combinations of SUMO-2 and -3 siRNA duplexes (SUMO-2/3 numbers 1 and 2 siRNAs). For control experiments, an siRNA against firefly luciferase GL3 was used. In each siRNA experiment, judging from immunofluorescence analysis using anti-SUMO-2/3 or anti-SUMO-1 antibodies, ∼60% of the cells showed no detectable levels of either SUMO-1 or SUMO-2/3 at 60 h post-transfection with either SUMO-2/3 or SUMO-1 siRNAs, respectively, whereas no reduction in the signals was observed in the control experiments (Fig. 6, A-E; and data not shown).
A comparison of the SUMO knockdown cells and control cells revealed no significant differences in terms of the localization of MBD1 or the number of MBD1 foci (data not shown), implying that depletion of either the SUMO-2/3 or SUMO-1 pathway may not completely impair the ability of MBD1 to localize at specific DNA regions, possibly via interactions between the methyl-CpG-binding domain of MBD1 and methylated DNA. In contrast, 52 and 61% of SUMO-2/3 and SUMO-1 knockdown cells, respectively, showed no significant accumulation of MCAF1 at MBD1-containing foci (Fig. 6, A and F). Moreover, 75 and 67% of SUMO-2/3 and SUMO-1 knockdown cells, respectively, showed decreased signals for trimethyl-H3-K9 around the MBD1-containing regions (Fig. 6, B and F). In agreement with these observations, 57 and 43% of SUMO-2/3 and SUMO-1 knockdown cells, respectively, showed HP1β delocalization from the MBD1 foci (Fig. 6, C and F), implying that the formation of MBD1-containing heterochromatin was perturbed. We further tested the subcellular localizations of other HP1 proteins, HP1γ and HP1α, and found delocalization of HP1γ, but not HP1α, in SUMO-2/3 and SUMO-1 siRNA-treated cells (Fig. 6, D and E, and data not shown). Taken together, we conclude that the SUMO-2/3 and SUMO-1 pathways are involved in the anchorage of MCAF1 and are also critical for methylation of histone H3-K9 and targeting of HP1β and HP1γ to MBD1-containing heterochromatin.
Perturbation of the SUMO Pathway by Ectopic Expression of MCAF1-(965-975) Induces Delocalization of MCAF1 and HP1β—The idea of facilitated anchorage of MCAF1 to MBD1-containing heterochromatin by SUMOylation was further tested by using cells that ectopically overexpressed MCAF1-(965-975). When a monomeric red fluorescent protein fused to MCAF1-(965-975) (DsRed-MCAF1-(965-975)) was transiently overexpressed in C-33A cells, the signals for both endogenous SUMO-2/3 (Fig. 7A) and SUMO-1 (data not shown) were remarkably reduced, suggesting that overproduction of monomeric DsRed-MCAF1-(965-975) effectively perturbed both SUMO pathways, possibly via sequestration of the SUMOs and/or competition with pre-existing SUMO-binding proteins. Significantly, in these DsRed-MCAF1-(965-975)-expressing cells, the numbers of cells in which both MCAF1 and HP1β were delocalized from MBD1-containing foci increased (Fig. 7, B and C), reminiscent of the depletion of the SUMO pathway by siRNAs described above. Because overexpression of monomeric DsRed alone had no apparent effect on the localizations of MCAF1 and HP1β at MBD1 foci (Fig. 7, A-C, upper panels), these results indicate the importance of the SUMO-binding region, MCAF1-(965-975), for regulating the anchorage of MCAF1 to MBD1-containing heterochromatin and further support the idea that SUMO-MCAF1 interactions are required for the proper assembly of MBD1-containing heterochromatin.
MCAF1 Is a SUMO-binding Protein—In this report, we have identified a previously unknown SUMO-binding region in MCAF1 (MCAF1-(965-975)) that facilitates its interaction with SUMOs. Significantly, we have revealed that MCAF1-(965-975) shows similarity to a previously characterized SUMO-binding motif (
). Moreover, we found that MCAF1 preferentially interacts with SUMO-2/3 rather than SUMO-1 in vitro. These results imply a previously unappreciated function of MCAF1 as a modulator for SUMO modification signals with the potential to preferentially transmit signals derived from SUMO-2/3-modified proteins. In addition, we found that following multisite modification and/or poly-chain formation of SUMOs on MBD1-(373-605), MCAF1 appeared to further increase its affinity for MBD1. This finding further supports the idea that MCAF1 functions as a modulator of SUMO signaling and suggests that MCAF1 may be able to amplify SUMO signals to downstream events. Thus, our results define MCAF1 as a novel class of SUMO regulators and predict the existence of a previously undescribed SUMO-MCAF1-based regulatory network.
MBD1 Is a SUMOylation Substrate—In this study, we have shown for the first time that the methyl-CpG-binding protein MBD1 is SUMOylated in mammalian cells, suggesting direct linkage of the SUMO modification pathway with a wide variety of important epigenetic cellular phenomena regulated by DNA methylation, including gene silencing and heterochromatin formation. It is feasible that other methyl DNA-binding proteins, besides MBD1, that reside in heterochromatin may also be SUMOylated. We are currently investigating whether other methyl DNA-binding proteins, including other MBD family proteins (
), are SUMOylated using the bacterial SUMOylation system.
It is currently unclear how SUMOylation of MBD1 is regulated and whether an E3 ligase that up-regulates SUMOylation of MBD1 is present. Because previous studies have shown direct interactions of MBD1 with MCAF1 (
), we first suspected that MCAF1 may act as a SUMO E3 ligase toward MBD1. However, neither binding of an E2 enzyme (Ubc9) to MCAF1 nor the ability of MCAF1 to enhance SUMOylation of MBD1 in vitro was observed (supplemental Fig. 3, A and B). In addition, although many of the SUMO E3 ligases reported to date have the ability to become auto-SUMOylated (
), MCAF1 appears to be inefficiently auto-SUMOylated. Thus, there is still poor evidence to support a role for MCAF1 as a SUMO E3 ligase. It will be interesting in the near future to identify the SUMO E3 ligases for MBD1 and to elucidate how such E3 ligases regulate the SUMOylation of MBD1 during cell cycle progression and cellular differentiation.
The SUMO Modification Pathway Cross-talks with the DNA and Histone Methylation Pathways—We have shown that SUMOylation of MBD1 provides sufficient anchoring of MCAF1 at heterochromatin regions in C-33A cells and demonstrated that depletion of either the SUMO-2/3 or SUMO-1 pathway results in delocalization of trimethyl-H3-K9, HP1β, and HP1γ from MBD1 foci, suggesting the possibility that destabilization of the interaction between MCAF1 and MBD1 perturbs the histone methylation pathway and the proper assembly of heterochromatin proteins at MBD1-containing DNA regions.
The molecular mechanism for how the SUMO pathway is linked to histone methylation and heterochromatin formation in mammalian cells currently remains to be fully elucidated, although two possibilities appear feasible. First, augmented recruitment of MCAF1 via an interaction with SUMOylated MBD1 may contribute to the maintenance of a stable assembly of H3-K9 methyltransferases at MBD1-containing heterochromatin regions, thereby stabilizing the association of HP1 with MBD1-containing heterochromatin. In this study, we have not identified any such putative H3-K9 methyltransferases that can be recruited by MCAF1 complexed with SUMOylated MBD1. However, we suggest that SETDB1 is a likely candidate for this scenario, because previous reports have described that MCAF1 is a regulatory subunit of the SETDB1 histone H3-K9 methyltransferase complex (
). Second, anchorage of MCAF1 to SUMOylated MBD1 may enhance the recruitment of chromatin remodeling activity and/or histone chaperone activity that somehow preferentially incorporate trimethyl-H3-K9. The chromatin assembly factor CAF1, which interacts with MBD1, represents a likely candidate for this scenario (
), although neither facilitated association of CAF1 with MCAF1-MBD1 complexes nor accumulation of CAF1 at SUMO-MCAF1 foci in C-33A cells have yet been demonstrated.
Regardless of the mechanism, our results imply a direct link between the SUMO pathway and the methylation of DNA and histone methylation. In addition, the dramatic effects on the nuclear localizations of several heterochromatin proteins, including trimethyl-H3-K9 and HP1 proteins, in either SUMO-2/3- or SUMO-1-depleted cells reveal highly dynamic features of the SUMO modification pathway in the context of heterochromatin formation. It is intriguing that HP1α, a well described constitutive heterochromatin protein in mammalian cells (
), seems to exhibit a different role in terms of the regulation of the SUMO-enriched heterochromatin regions in C-33A cells compared with the two other members of the HP1 protein family examined. Although these results imply that the various HP1 members have only overlapping functions with respect to the regulation of SUMO-enriched heterochromatin, the physiological relevance of this phenomenon remains to be elucidated in future studies.
In conclusion, our results have provided direct evidence that modification of MBD1 by either SUMO-2/3, SUMO-1, or both and facilitated anchorage of MCAF1 to SUMOylated MBD1 participate in the formation of heterochromatin in C-33A cells. Considering the increasing numbers of SUMO substrates involved in chromatin modification, remodeling, and epigenetic control, our findings lay the foundation for future exploration of currently undiscovered SUMO signaling events that regulate gene silencing and heterochromatin formation in both physiological and pathological situations.
We thank Dr. N. Saitoh and Dr. S. Watanabe for critically reading this manuscript and all our laboratory staff members for their helpful discussions.
This work is dedicated to the late S. Mizuno, who was an excellent mentor to H. S. during his graduate studies at Tohoku University, Sendai, Japan, and the late A. P. Wolffe, who was a good advisor to H. S. during his postdoctoral training at the National Institutes of Health, Bethesda. Their enthusiasm and curiosity were the motivation behind this study to find a link between the SUMO modification pathway and the mechanisms behind chromatin dynamics and epigenetic control.