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Human BAHD1 Promotes Heterochromatic Gene Silencing

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Human BAHD1 Promotes Heterochromatic Gene Silencing Human BAHD1 promotes heterochromatic gene silencing He´ le` ne Biernea,1, To Nam Thama, Eric Batscheb, Anne Dumayc, Morwenna Leguillouc, Sophie Kerne´ is-Golsteynd, Be´ atrice Regnaulte, Jacob S. Seelerf, Christian Muchardtb, Jean Feunteunc, and Pascale Cossarta,1 aInstitut Pasteur, Unite´des Interactions Bacte´ries Cellules, Inserm U604, and INRA USC2020, Paris, F-75015, France; bInstitut Pasteur, Unite´deRe´ gulation Epige´ne´ tique, INSERM Avenir, CNRS URA 2578, Paris, F-75015, France; cLaboratoire Ge´nomes et Cancers, FRE CNRS 2939, Villejuif, F-94805, France; dInstitut Pasteur, Plate-forme de Microscopie Electronique, Paris, F-75015, France; eInstitut Pasteur, Plate-forme Puces a`ADN, Paris, F-75015, France; and fInstitut Pasteur, Unite´Organisation Nucle´aire et Oncoge´ne` se, INSERM U579, Paris, F-75015, France Edited by Jasper Rine, University of California, Berkeley, CA, and approved June 30, 2009 (received for review February 6, 2009) Gene silencing via heterochromatin formation plays a major role in proaches, including two-hybrid screen, knockdown, and gain-of- cell differentiation and maintenance of homeostasis. Here we report function experiments. the identification and characterization of a novel heterochromatini- zation factor in vertebrates, bromo adjacent homology domain– Results containing protein 1 (BAHD1). This nuclear protein interacts with HP1, BAHD1 Is a Nuclear Protein. A single gene encodes BAHD1 in MBD1, HDAC5, and several transcription factors. Through electron vertebrates, and no ortholog is found in invertebrates or plants, and immunofluorescence microscopy studies, we show that BAHD1 suggesting that BAHD1 is involved in vertebrate-specific functions. overexpression directs HP1 to specific nuclear sites and promotes the In addition to its C-terminal BAH domain, the BAHD1 protein formation of large heterochromatic domains, which lack acetyl his- contains a N-terminal proline-rich region and a central nuclear tone H4 and are enriched in H3 trimethylated at lysine 27 (H3K27me3). localization signal (Fig. 1A). Immunofluorescence labeling with an Furthermore, ectopically expressed BAHD1 colocalizes with the het- antibody generated against the last 17 residues of the protein erochromatic inactive X chromosome (Xi). The BAH domain is required indicated that BAHD1 localizes to interphase nuclei in human cells for BAHD1 colocalization with H3K27me3, but not with the Xi chro- (Fig. 1A). But the staining was relatively weak, in agreement with CELL BIOLOGY mosome. As highlighted by whole genome microarray analysis of the reportedly low BAHD1 gene expression in a broad range of BAHD1 knockdown cells, BAHD1 represses several proliferation and human tissues (11, 12). Consequently, we cloned BAHD1 cDNA survival genes, in particular the insulin-like growth factor II gene into an expression vector. One day after transfection, BAHD1 (IGF2). When overexpressed, BAHD1 specifically binds the CpG-rich P3 tagged with the V5 epitope localized exclusively to the nucleus, promoter of IGF2, which increases MBD1 and HDAC5 targeting at this whereas V5-tagged cyan fluorescent protein (V5-CFP), used as a locus. This region contains DNA-binding sequences for the transcrip- control, localized to both the nucleus and the cytoplasm (Fig. 1A), tion factor SP1, with which BAHD1 coimmunoprecipitates. Collec- tively, these findings provide evidence that BAHD1 acts as a silencer confirming that BAHD1 is a nuclear protein. by recruiting at specific promoters a set of proteins that coordinate heterochromatin assembly. BAHD1 Interacts With Chromatin Repressors and Transcription Fac- tors. Given that BAH domain–containing proteins, such as the BAH domain ͉ epigenetics ͉ heterochromatin ͉ HP1 ͉ polycomb yeast Sir3 and mammalian MTA proteins, are components of multiprotein silencing complexes (10, 13), we hypothesized that BAHD1 also belongs to a chromatin-modifying complex, and ene repression in eukaryotes in response to developmental searched for its partners using a large-scale yeast two-hybrid screen. or environmental signals is a complex multistep process G This screen identified a series of nuclear BAHD1 interactors requiring the concerted action of many cellular factors, including DNA-binding transcription factors and cofactors. It also involves [supporting information (SI) Table S1], several of which are well- chromatin components and chromatin-binding/-modifying pro- characterized chromatin-associated repressors, including the 3 iso- ␣ ␤ ␥ teins, which are implicated in the establishment and maintenance forms of heterochromatin protein 1 (HP1 , , and ), the methyl- of a condensed chromatin state, also referred to as facultative CpG–binding protein MBD1, the histone H3 methyltransferase heterochromatin (1). Deregulation of these chromatin regula- KMT1E, the histone deacetylase HDAC5, and the ATP-dependent tory factors and aberrant chromatin modifications play impor- chromatin remodeling enzyme CHD1. This screen also revealed tant roles in various human diseases, including cancers, devel- several transcription and RNA-processing factors. We validated the opmental abnormalities, and neurologic disorders (2–6). ability of BAHD1 to associate with at least 2 of its partners, HP1␣ Moreover, several recent reports have linked histone modifica- and MBD1, through pull-down assays. In contrast to GST alone, tions and infectious diseases (7). During a screen for human GST-HP1␣ or GST-MBD1 purified proteins added to nuclear proteins that interact with microbial factors and that might play extracts of HEK293 cells expressing V5-BAHD1 or the control a role in bacterial pathogenicity, we identified a putative chro- V5-CFP were able to pull down V5-BAHD1, but not the control matin-associated protein, bromo adjacent homology domain– containing protein 1 (BAHD1), which remains uncharacterized. One study correlated repression of the BAHD1 gene to poor Author contributions: H.B. designed research; H.B., T.N.T., A.D., M.L., S.K.-G., and B.R. performed research; H.B., M.L., J.S.S., C.M., and J.F. contributed new reagents/analytic prognosis in lung cancer (8), raising the possibility that BAHD1 tools; H.B., E.B., B.R., J.F., and P.C. analyzed data; and H.B. and P.C. wrote the paper. might act as a tumor suppressor. This connection between The authors declare no conflict of interest. BAHD1 and pathological processes prompted us to characterize This article is a PNAS Direct Submission. the function of this protein. Data deposition: The data reported in this paper have been deposited in the Gene BAHD1 is so named because it contains a C-terminal BAH Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE16097). domain, which is found in several chromatin-binding proteins 1To whom correspondence may be addressed. E-mail: [email protected] or involved in transcriptional repression (9), including the yeast Sir3 [email protected]. protein (10). We investigated whether BAHD1 could be involved This article contains supporting information online at www.pnas.org/cgi/content/full/ in heterochromatin formation using a combination of ap- 0901259106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0901259106 PNAS Early Edition ͉ 1of6 Downloaded by guest on October 2, 2021 V5-CFP (Fig. 1B), demonstrating that BAHD1 associates with HP1␣ and MBD1. BAHD1 Represses Basal Transcription. The chromatin-associated fac- tors HP1␣ and MBD1 play key roles in the formation of repressive chromatin by binding methylated histone H3 and methylated DNA, respectively (14–16). Consequently, we expected that BAHD1 would induce transcriptional repression. To test this hypothesis, BAHD1 was fused to the DNA-binding domain (DBD) of the GAL4 protein and coexpressed in HEK293 cells with reporters containing GAL4 binding sites linked to either TK or MMTV promoters upstream of the luciferase gene. GAL4-BAHD1 inhib- ited luciferase activity in a dose-dependent manner, with a repres- sive effect comparable to that of GAL4-HP1␣ and GAL4-MBD1 (Fig. S1). These results demonstrate that BAHD1 acts as a repres- sor when targeted to a promoter. Because BAHD1 can interact with heterochromatin factors, it may promote gene silencing through an heterochromatinization process. Increasing BAHD1 Expression Levels in Cells Triggers Heterochromatin Formation. An important feature of heterochromatin is its ability to spread from a specific nucleation site (14). We reasoned that if BAHD1 promotes the assembly of a heterochromatinization ma- chinery at genomic microdomains, then increasing its amount in cells should stimulate the spread of heterochromatin throughout large genomic regions, a phenomenon visible by microscopy tech- niques. To address this question, we expressed YFP-BAHD1 in human C3SV40 fibroblasts for 24 h or 48 h. YFP-BAHD1 localized in the nucleus as dispersed foci that increase in size with increasing YFP-BAHD1 expression to form larger structures (Fig. 1C). Im- portantly, these structures were visible in living cells as patches of phase-dense material (Movie S1), which did not result from un- specific aggregation, because they were not observed on expression of either YFP or another tagged nuclear protein, such as GFP-PML (data not shown). To examine whether YFP-BAHD1–associated foci were heterochromatin areas, we first examined whether they correlated with histone hypoacetylation, a specific feature of het- erochromatin (1). Accordingly, acetyl-histone H4 was largely ex- cluded from YFP-BAHD1 foci (Fig. 1D). We then confirmed by electron microscopy that these nuclear structures
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