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Isolation and Characterization of Mammalian HDAC10, a Novel Histone Deacetylase*

  • Hung-Ying Kao
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  • Chih-Hao Lee
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  • Andrei Komarov
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  • Chris C. Han
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  • Ronald M. Evans
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  • Author Footnotes
    * This work was supported by a start-up fund (to H. Y. K.) and National Institutes of Health Grant HD27183 (to R. M. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EBI Data Bank with accession number(s) AF407272 (HDAC10α) and AF407273(HDAC10β).
    § Recipient of the James T. Pardee-Carl A. Gerstacker Assistant Professor of Cancer Research Faculty, and Chair in Cancer Research at CWRU Cancer Center. To whom correspondence may be addressed: Dept. of Biochemistry, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106. Tel.: 216-844-7572; Fax: 216-368-3419; E-mail: [email protected]
    ‖ Investigator of the Howard Hughes Medical Institute at the Salk Institute for Biological Studies, and March of Dimes Chair in Molecular and Developmental Biology. To whom correspondence may be addressed: The Howard Hughes Medical Institute, Gene Expression Laboratory, The Salk Institute for Biological Studies, 10010 Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-453-4100, Ext. 1585; Fax: 858-455-1349; E-mail: [email protected]
      Acetylation of histone core particles plays an important role in modulating chromatin structure and gene expression. The acetylation status of the histone tails is determined by two opposing enzymatic activities, histone acetyltransferases and histone deacetylases (HDACs). Here we describe the isolation and characterization of HDAC10, a novel class II histone deacetylase. Molecular cloning and Northern blot analyses reveal that the HDAC10 transcript is widely expressed and subjected to alternative splicing. HDAC10 is both nuclear and cytoplasmic, a feature reminiscent of HDACs 4, 5, and 7. Distinct from other family members, HDAC10 harbors an amino-terminal catalytic domain and a carboxyl pseudo-repeat that shares significant homology with its catalytic domain. Mutational analysis reveals that transcriptional repression by HDAC10 requires its intrinsic histone deacetylase activity. Taken together, HDAC10 represents a distinct HDAC that may play a role in transcription regulation.
      HAT
      histone acetyltransferase
      HDAC
      histone deacetylase
      SMRT
      silencing mediator for retinoid and thyroid hormone receptor
      EST
      expressed sequence tag
      DAPI
      4,6-diamidino-2-phenylidole
      NES
      nuclear export sequence
      DOTAP
      N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate
      YFP
      yellow fluorescent protein
      ORF
      open reading frame
      HEK
      human embryonic kidney
      The ability to modify chromatin structure, either locally or globally, is a critical feature of signal transduction pathways and gene regulation. Accordingly, the amino-terminal tails of histones are targets for several modifications including methylation, phosphorylation, and acetylation (
      • El-Osta A.
      • Wolffe A.P.
      ,
      • Roth S.Y.
      • Denu J.M.
      • Allis C.D.
      ). Recent studies suggest that the amino-terminal tails of histones play a critical role in modulating chromatin structure and hence, regulation of gene expression (
      • Cheung P.
      • Allis C.D.
      • Sassone-Corsi P.
      ,
      • Hansen J.C.
      • Tse C.
      • Wolffe A.P.
      ,
      • Strahl B.D.
      • Allis C.D.
      ,
      • Turner B.M.
      ,
      • Luger K.
      • Mader A.W.
      • Richmond R.K.
      • Sargent D.F.
      • Richmond T.J.
      ). Acetylation of histone tails is thought to create an open chromatin structure, thereby promoting the accessibility of transcription factors (
      • Luger K.
      • Mader A.W.
      • Richmond R.K.
      • Sargent D.F.
      • Richmond T.J.
      ). Consistent with this view, histone acetylation at specific lysines is associated with transcriptional activation, whereas deacetylated histones are found in transcriptionally silent regions. The acetylation status of the histone tails is determined by the interplay between histone acetyltransferases (HATs)1 andhistone deacetylases (HDACs). The identification of HATs and HDACs represents a critical step toward our understanding of transcription regulation.
      To date, three classes of HDACs exist that can be distinguished by size, catalytic domain, subcellular localization, and mode of action. Mammalian class I HDACs including HDACs 1, 2, 3, and 8 resemble the yeast global transcriptional regulator Rpd3. Class II HDACs (HDACs 4–7) are similar to yeast Hda1. HDACs 4, 5, and 7 harbor a highly conserved carboxyl-terminal catalytic domain and constitute a subfamily, whereas HDAC6 is distinct in that it contains a duplicated catalytic domain (
      • Grozinger C.M.
      • Hassig C.A.
      • Schreiber S.L.
      ,
      • Verdel A.
      • Khochbin S.
      ). The prototype of class III HDACs is the yeast Sir2 protein whose enzymatic reaction appears to be unique and requires the cofactor, NAD+ (
      • Guarente L.
      ). The enzymatic activity of class I and class II HDACs can be efficiently blocked by the treatment of trichostatin A, whereas that of class III HDACs is trichostatin A-insensitive (
      • Guarente L.
      ).
      Both class I and class II HDACs function, in part, through direct or indirect association with transcriptional corepressors, such as SMRT, nuclear receptor corepressor, and mSin3A (
      • Alland L.
      • Muhle R.
      • Hou H.
      • Potes J.
      • Chin L.
      • Schreiber-Agus N.
      • DePinho R.A.
      ,
      • Heinzel T.
      • Lavinsky R.M.
      • Mullen T.M.
      • Soderstrom M.
      • Laherty C.D.
      • Torchia J.
      • Yang W.M.
      • Brard G.
      • Ngo S.D.
      • Davie J.R.
      • Seto E.
      • Eisenman R.N.
      • Rose D.W.
      • Glass C.K.
      • Rosenfeld M.G.
      ,
      • Nagy L.
      • Kao H.Y.
      • Chakravarti D.
      • Lin R.J.
      • Hassig C.A.
      • Ayer D.E.
      • Schreiber S.L.
      • Evans R.M.
      ,
      • Hassig C.A.
      • Fleischer T.C.
      • Billin A.N.
      • Schreiber S.L.
      • Ayer D.E.
      ,
      • Laherty C.D.
      • Yang W.M.
      • Sun J.M.
      • Davie J.R.
      • Seto E.
      • Eisenman R.N.
      ,
      • Huang E.Y.
      • Zhang J.
      • Miska E.A.
      • Guenther M.G.
      • Kouzarides T.
      • Lazar M.A.
      ,
      • Kao H.Y.
      • Downes M.
      • Ordentlich P.
      • Evans R.M.
      ). In several instances, HDACs have been shown to directly interact with sequence-specific DNA-binding transcription factors and repress their activity (
      • Ng H.H.
      • Bird A.
      ). The class I HDACs are nuclear proteins that are generally small in size ranging from 40 to 55 kDa and are expressed ubiquitously. In contrast, class II HDACs are larger in size ranging from 100 to 130 kDa and can shuttle between the nucleus and cytoplasm (
      • Wang A.H.
      • Bertos N.R.
      • Vezmar M.
      • Pelletier N.
      • Crosato M.
      • Heng H.H.
      • Th'ng J.
      • Han J.
      • Yang X.J.
      ,
      • Lu J.
      • McKinsey T.A.
      • Zhang C.L.
      • Olson E.N.
      ,
      • Lu J.
      • McKinsey T.A.
      • Nicol R.L.
      • Olson E.N.
      ). The nuclear export of class II HDACs depends on highly conserved serine residues among class II HDACs and their association with 14-3-3 proteins (
      • Grozinger C.M.
      • Schreiber S.L.
      ,
      • McKinsey T.A.
      • Zhang C.L.
      • Olson E.N.
      ,
      • McKinsey T.A.
      • Zhang C.L.
      • Lu J.
      • Olson E.N.
      ,
      • Wang A.H.
      • Kruhlak M.J.
      • Wu J.
      • Bertos N.R.
      • Vezmar M.
      • Posner B.I.
      • Bazett-Jones D.P.
      • Yang X.J.
      ,
      • Zhao X.
      • Ito A.
      • Kane C.D.
      • Liao T.S.
      • Bolger T.A.
      • Lemrow S.M.
      • Means A.R.
      • Yao T.P.
      ). Furthermore, the expression patterns of HDACs 4, 5, and 7 are tissue-specific with heart, lung, and skeletal muscle showing the greatest abundance of mRNA (
      • Grozinger C.M.
      • Hassig C.A.
      • Schreiber S.L.
      ,
      • Verdel A.
      • Khochbin S.
      ,
      • Kao H.Y.
      • Downes M.
      • Ordentlich P.
      • Evans R.M.
      ,
      • Fischle W.
      • Emiliani S.
      • Hendzel M.J.
      • Nagase T.
      • Nomura N.
      • Voelter W.
      • Verdin E.
      ,
      • Fischle W.
      • Dequiedt F.
      • Fillion M.
      • Hendzel M.J.
      • Voelter W.
      • Verdin E.
      ). Indeed, class II HDACs have been shown to regulate the activity of myocyte enhancer factor 2, a protein family involved in muscle differentiation as well as heart development (
      • Wang A.H.
      • Bertos N.R.
      • Vezmar M.
      • Pelletier N.
      • Crosato M.
      • Heng H.H.
      • Th'ng J.
      • Han J.
      • Yang X.J.
      ,
      • Lu J.
      • McKinsey T.A.
      • Nicol R.L.
      • Olson E.N.
      ,
      • Lemercier C.
      • Verdel A.
      • Galloo B.
      • Curtet S.
      • Brocard M.P.
      • Khochbin S.
      ,
      • Miska E.A.
      • Karlsson C.
      • Langley E.
      • Nielsen S.J.
      • Pines J.
      • Kouzarides T.
      ,
      • Youn H.D.
      • Grozinger C.M.
      • Liu J.O.
      ,
      • Bodmer R.
      • Venkatesh T.V.
      ). These three HDACs also interact with Pox virus and zinc finger domain-containing factors, such as BCL6 and PLZF, and may mediate their repression activity (
      • Khochbin S.
      • Kao H.Y.
      ).
      Here we describe the isolation of HDAC10 and characterization of its biochemical properties. Molecular cloning and Northern blot analyses reveal that human HDAC10 is subjected to alternative splicing, generating at least two spliced isoforms, HDAC10αand HDAC10β. We find that the tissue distribution ofHDAC10 is distinct from that of other HDACs and shows by fluorescence microscopy that HDAC10α, similar to HDACs 4, 5, and 7, can be nuclear or cytoplasmic. HDAC10α harbors intrinsic HDAC activity, which is essential for its associated repression activity. Taken together, we conclude that HDAC10 is a novel histone deacetylase, which plays a role in transcriptional regulation.

      EXPERIMENTAL PROCEDURES

       Cell Cultures

      HEK293, NIH-3T3, and A549 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum along with penicillin-streptomycin antibiotics.

       Isolation of HDAC10 cDNAs and Plasmid Construction

      Human HDAC10 was isolated by PCR reactions using 5′-primer (5′-CATGAATTCGGTACCATGGGGACCGCGCTTGTGTACC-3′) and 3′-primer (5′CATTCTAGAGTCGACTCAAGCCACCAGGTGAGGATGGC-3′) according to the putative HDAC10 sequence deposited in GenBank (GenBankTM accession number CAB63048). A cDNA library derived from human hepatocellular carcinoma cell line HepG cells was used as a template for PCR amplication (Bioline). PCR products were digested with EcoRI/SalI and ligated with EcoRI/SalI-linearized pVP16 (Promega). The DNA sequence was confirmed by the dideoxy-sequencing method. Full-length HDAC10α was isolated from pVP16-HDAC10 byEcoRI/SalI digestion and subcloned into CMX-FLAG, CMX-Gal4, and CMX-1Y to create CMX-FLAG-HDAC10α, CMX-Gal4-HDAC10α, and CMX-1Y-HDAC10α. Truncation, deletion, and mutation constructs were generated by PCR and subcloned into vectors, CMX-Gal4 (a mammalian expression vector harboring the yeast Gal4 DNA-binding domain) (
      • Kao H.Y.
      • Downes M.
      • Ordentlich P.
      • Evans R.M.
      ) and CMX-1Y, a yellow fluorescenceprotein (YFP) expression vector (
      • Kao H.Y.
      • Downes M.
      • Ordentlich P.
      • Evans R.M.
      ). Site-directed mutagenesis was carried out with the QuickChange mutagenesis kit according to instructions by the manufacturer (Stratagene). The resulting mutants were confirmed by sequencing. Sequence analyses were performed using the DNASTAR program and the BLAST program from NCBI. Mouse HDAC10 EST clone (GenBankTM accession number AI323102) was requested from Genome System, Inc. Sequence analyses and the prediction of open reading frames were carried out using the DNASTAR program. CMX-HA-HDAC5, CMX-HA-HDAC7, and CMX-FLAG-HDAC1 have been reported previously (
      • Downes M.
      • Ordentlich P.
      • Kao H.Y.
      • Alvarez J.G.
      • Evans R.M.
      ). HDAC4 was generated by a PCR reaction using pBJ5-HDAC4-FLAG, a gift from Dr. Schreiber, as a template and cloned into a HA-tagged vector, CMX-1H, to create CMX-HA-HDAC4.

       RNA Isolation and Northern Blot Analyses

      Mouse tissues were from 3-month-old C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME), and RNA was isolated using TRIzol reagent (Invitrogen). All samples were separated on 1% agarose gels. Hybridization was carried out at 42 °C with 50% formamide. Human tissue and cancer cell blots were purchased from CLONTECH. Northern blots were probed with a full-length HDAC10 cDNA. AnEcoRI fragment containing 1.6-kb mouse HDAC10cDNA was used as a probe to analyze the mouse tissue blot. Northern blot analyses were performed using hybridization and washing protocols according to a previously published report (
      • Sambrook J.
      • Maniatis T.
      • Fritsch E.F.
      Molecular Cloning: A Laboratory Manual.
      ).

       Green Fluorescence Microscopy

      NIH-3T3 cells were plated onto 2-well chamber slides (Nunc) and transfected using Lipofectin (Invitrogen). After 36 h, cells were washed in 1× phosphate-buffered saline and fixed in 3% paraformaldehyde. Cells were washed three times with 1× phosphate-buffered saline. DAPI was applied to the samples after the final wash to visualize nuclei. Images were visualized using a LEICA fluorescence microscope equipped with a camera.

       Transient Transfection

      A549 cells (60–70% confluence, 48-well plate) were cotransfected with 16.6–66.6 ng of pCMX-GAL4 and pCMX-GAL4-HDAC10 constructs, 100 ng of pMH100-TK-luciferase, and 100 ng of pCMX-LacZ in 200 μl of Dulbecco's modified Eagle's medium containing 10% fetal bovine serum by the DOTAP-mediated procedure (
      • Kao H.Y.
      • Downes M.
      • Ordentlich P.
      • Evans R.M.
      ). After 24 h, the medium was replaced. Cells were harvested and assayed for luciferase activity 36–48 h after transfection. The luciferase activity was normalized to the level of β-galactosidase activity. Each transfection was performed in triplicate and repeated at least three times.

       Coimmunoprecipitation

      Coimmunoprecipitation was carried out according to a published protocol (
      • Kao H.Y.
      • Downes M.
      • Ordentlich P.
      • Evans R.M.
      ). 10-cm plates of HEK293 cells were transfected with 10 μg of the appropriate plasmids using Targefect F1 (Targeting Systems, San Diego, CA). Cells were harvested 48 h later and lysed in NET-N buffer (50 mm Tris, pH 8.0, 150 mm NaCl, 10% glycerol, 0.5% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, and protease inhibitors mixture (Roche Molecular Biochemicals)) for 15 min at 4 °C, scraped, and centrifuged for 15 min at 13,000 rpm. The supernatant was kept as whole cell extract. After preclearing by incubation with protein A/G-agarose (Santa Cruz Biotechnologies), immunoprecipitations were carried out using either HA-agarose (Santa Cruz Biotechnologies) or M2-agarose (Sigma) and were allowed to proceed for 2 h at 4 °C. Beads were washed 3–4 times in lysis buffer without Triton X-100 for histone deacetylase assays and in 1× phosphate-buffered saline with 0.1% Nonidet P-40 for coimmunoprecipitations. For coimmunoprecipitations, samples were heat denatured in SDS loading buffer, separated on SDS-PAGE gels, transferred to a nitrocellulose membrane, and probed with appropriate antibodies.

       Histone Deacetylase Assays

      Histone deacetylase assays were performed according to a described protocol (
      • Kao H.Y.
      • Downes M.
      • Ordentlich P.
      • Evans R.M.
      ). Briefly, 60,000 cpm of 3H-histones was incubated with FLAG antibody immunoprecipitates for 2 h at 37 °C. Reactions were stopped by the addition of acetic acid/HCl to a final concentration of 0.12 mol/0.72 mol and extracted with two volumes of ethyl acetate. Samples were centrifuged, and supernatants were analyzed in a scintillation counter. Each reaction contained approximately one-third of lysates harvested from 10-cm plates of cells.

      RESULTS

       Isolation of Human and Mouse cDNAs Encoding HDAC10

      To identify novel histone deacetylases, we carried out BLAST analyses against the histone deacetylase domain of mouse HDAC7. This analysis led to the identification of a putative human HDAC (GenBankTM accession number CAB63048) encoding a predicted 673 amino acid protein with an amino-terminal catalytic domain. Full-length cDNA was isolated from a cDNA library derived from human hepatocellular carcinoma cell line HepG cells by PCR reactions. PCR products were isolated, subcloned, and sequenced. Three clones representing two spliced isoforms, HDAC10α(GenBankTM accession number AF407272) andHDAC10β (GenBankTM accession number AF407273) were isolated. HDAC10α encodes a protein of 669 amino acids, and HDAC10β encodes a protein of 649 amino acids. Sequence comparison indicated that both forms lacked 4 amino acids encoded by GenBankTM accession number CAB63048 (amino acids 338–341) (Fig. 1A). In addition, HDAC10β is missing 20 amino acids (amino acids 253–272) found in HDAC10α. Because the missing 20 amino acids fall within a region highly conserved among HDACs and HDAC10β appears to be catalytically inactive, HDAC10α (referred to as HDAC10 hereafter) was used for characterization in this report.
      Figure thumbnail gr1a
      Figure 1HDAC10 is a new member of class II HDACs.A, amino acid sequence alignment between human HDAC10α (GenBankTM accession numberAF407272), human HDAC6, and Drosophila HDAC6. Conserved residues are shaded. The missing 20 amino acids (253) in HDAC10β are indicated by broken lines. The partially duplicated regions (rep-1 and rep-2) of HDAC10α are flanked byarrows. Putative nuclear receptor interacting motifs areunderlined. Putative nuclear exportsequences (NES) are indicated by dotted lines on top of the sequences. B, sequence alignment between partially duplicated regions, rep-1 and rep-2. The identical amino acids between repeats 1 and 2 are shown. C, schematic representation of class II HDACs. Histone deacetylase domain is shown in gray. The degree of identity and similarity between HDAC10 (100%) and individual class II HDACs are shown. D, sequence alignment between human HDAC10α and an alternatively spliced isoform of mouse HDAC10. The mouse HDAC10 EST clone does not encode a full-length HDAC10 protein. It encodes seven short conceptual open reading frames (marked ORF1–7). Four of these ORFs including ORFs 1, 3, 6, and 7 share a high degree of homology with human HDAC10α.
      Figure thumbnail gr1b
      Figure 1HDAC10 is a new member of class II HDACs.A, amino acid sequence alignment between human HDAC10α (GenBankTM accession numberAF407272), human HDAC6, and Drosophila HDAC6. Conserved residues are shaded. The missing 20 amino acids (253) in HDAC10β are indicated by broken lines. The partially duplicated regions (rep-1 and rep-2) of HDAC10α are flanked byarrows. Putative nuclear receptor interacting motifs areunderlined. Putative nuclear exportsequences (NES) are indicated by dotted lines on top of the sequences. B, sequence alignment between partially duplicated regions, rep-1 and rep-2. The identical amino acids between repeats 1 and 2 are shown. C, schematic representation of class II HDACs. Histone deacetylase domain is shown in gray. The degree of identity and similarity between HDAC10 (100%) and individual class II HDACs are shown. D, sequence alignment between human HDAC10α and an alternatively spliced isoform of mouse HDAC10. The mouse HDAC10 EST clone does not encode a full-length HDAC10 protein. It encodes seven short conceptual open reading frames (marked ORF1–7). Four of these ORFs including ORFs 1, 3, 6, and 7 share a high degree of homology with human HDAC10α.
      HDAC10 is leucine-rich and contains multiple copies of a (I/V/L)XX(I/V/L)(I/V/L) motif resembling the nuclear receptor-interacting motif present in coactivators and corepressors (
      • Heery D.M.
      • Kalkhoven E.
      • Hoare S.
      • Parker M.G.
      ,
      • Darimont B.D.
      • Wagner R.L.
      • Apriletti J.W.
      • Stallcup M.R.
      • Kushner P.J.
      • Baxter J.D.
      • Fletterick R.J.
      • Yamamoto K.R.
      ,
      • McInerney E.M.
      • Rose D.W.
      • Flynn S.E.
      • Westin S.
      • Mullen T.M.
      • Krones A.
      • Inostroza J.
      • Torchia J.
      • Nolte R.T.
      • Assa-Munt N.
      • Milburn M.V.
      • Glass C.K.
      • Rosenfeld M.G.
      ,
      • Nolte R.T.
      • Wisely G.B.
      • Westin S.
      • Cobb J.E.
      • Lambert M.H.
      • Kurokawa R.
      • Rosenfeld M.G.
      • Willson T.M.
      • Glass C.K.
      • Milburn M.V.
      ,
      • Perissi V.
      • Staszewski L.M.
      • McInerney E.M.
      • Kurokawa R.
      • Krones A.
      • Rose D.W.
      • Lambert M.H.
      • Milburn M.V.
      • Glass C.K.
      • Rosenfeld M.G.
      ,
      • Nagy L.
      • Kao H.-Y.
      • Love J.D.
      • Li C.
      • Banayo E.
      • Gooch J.T.
      • Krishna V.
      • Chatterjee K.
      • Evans R.M.
      • Schwabe J.W.R.
      ,
      • Shiau A.K.
      • Barstad D.
      • Loria P.M.
      • Cheng L.
      • Kushner P.J.
      • Agard D.A.
      • Greene G.L.
      ). Furthermore, four copies of putative nuclearexport sequences (NESs), similar to an NES motif LX2–3LX2–3LXL (
      • Fornerod M.
      • Ohno M.
      • Yoshida M.
      • Mattaj I.W.
      ), are present in HDAC10. The carboxyl-terminal region of HDAC10 shares significant homology with the amino-terminal catalytic domain (Fig. 1B), suggesting that HDAC10 contains a partially duplicated catalytic domain. This observation is interesting because HDAC6 also contains two catalytic domains. Indeed, BLAST analyses indicated that HDAC10 is a new member of the class II HDACs (Fig.1C).
      BLAST searches of mouse ESTs against the amino acid sequence of humanHDAC10 led to the identification of a mouseHDAC10 EST clone (GenBankTM accession numberAI323102). Interestingly, sequence analyses of this entire mouse EST insert suggests that it represents an additional isoform ofHDAC10 (Fig. 1D), which does not encode a full-length HDAC10. Instead, this EST clone contains seven short open reading frames (ORFs), four of which share extensive sequence homology with human HDAC10. These results strongly suggest that both human and mouse HDAC10 are subjected to alternative splicing that is conserved during evolution.

       Tissue Distribution of HDAC10

      Unlike class I HDACs, the expression of class II members appears to be tissue-specific (
      • Grozinger C.M.
      • Hassig C.A.
      • Schreiber S.L.
      ,
      • Verdel A.
      • Khochbin S.
      ,
      • Kao H.Y.
      • Downes M.
      • Ordentlich P.
      • Evans R.M.
      ,
      • Fischle W.
      • Emiliani S.
      • Hendzel M.J.
      • Nagase T.
      • Nomura N.
      • Voelter W.
      • Verdin E.
      ,
      • Fischle W.
      • Dequiedt F.
      • Fillion M.
      • Hendzel M.J.
      • Voelter W.
      • Verdin E.
      ). Northern blot analyses revealed human HDAC10 to be highly expressed in the liver, spleen, and kidney (Fig.2A) with two transcripts of 2.8 and 3.5 kb observed in many tissues. HDAC10 was also expressed in most of the cancer cell lines examined (Fig. 2B). The mouse homologue was similarly expressed (Fig. 2C), showing at least two transcripts.
      Figure thumbnail gr2
      Figure 2Tissue distribution of HDAC10.A, human HDAC10 is highly expressed in liver, spleen, and kidney. The human tissue blot was probed with a 32P-labeled full-length HDAC10 cDNA fragment. Transcripts of 2.8 and 3.5 kb were detected in most tissues. In some tissues, such as skeletal muscle, a larger transcript was also observed. B, a cancer cell line blot was examined together with a human tissue blot. Cancer cell lines, including HL60, S3, K-562, Raji, and SW480, predominantly express a single transcript. C, tissue distribution of mouse HDAC10. The mouse blots were probed using an EcoRI fragment from the mouse HDAC10 EST clone as described under “Experimental Procedures.”

       HDAC10 Is Localized in the Nucleus and Cytoplasm

      Whereas class I HDACs are primarily nuclear, class II HDACs have been found to shuttle between the nucleus and cytoplasm. The fact that HDAC10 contains four copies of putative NESs suggests that HDAC10 may shuttle between the nucleus and cytoplasm (Fig.3A). To examine the subcellular localization of HDAC10, we generated a construct encoding HDAC10 fused to the carboxyl terminus of YFP. YFP-HDAC10 was expressed in NIH-3T3 cells, and its subcellular localization was examined under fluorescence microscopy. Interestingly, HDAC10 was localized in both the nucleus and cytoplasm (Fig. 3B, left panel). This observation is reminiscent of the subcellular localization of HDACs 4, 5, and 7. Furthermore, the amino-terminal catalytic domain (1) (middle panel) and the carboxyl-terminal region (339) (right panel) of HDAC10 were also localized in both the nucleus and cytoplasm.
      Figure thumbnail gr3
      Figure 3HDAC10 is both nuclear and cytoplasmic in NIH-3T3 cells. A, putative NESs in HDAC10α. The conserved leucine residues are underlined. B, expression constructs of YFP-HDAC10 were transiently transfected into NIH-3T3 cells. 36 h later, the subcellular localization of HDAC10 was examined by fluorescence microscopy. Left panel, full-length HDAC10; middle panel, HDAC10 (1); right panel, HDAC10 (339). Top panels are YFP-HDAC10.Bottom panels are DAPI staining. C, HDAC10 does not associate with HDACs 4, 5, or 7. FLAG-HDAC10 and HA-HDACs 4, 5, and 7 were expressed either alone or together in HEK293 cells. After 48 h, whole cell extracts were prepared and fractionated on SDS-PAGE followed by Western blot analyses with either anti-FLAG (lanes 15–21) or anti-HA (lanes 22–28) antibodies. Alternatively, whole cell extracts were incubated with anti-FLAG antibody-conjugated beads, and bound fractions were washed and resolved on SDS-PAGE followed by Western blot analyses probed with anti-FLAG (lanes 1–7) or anti-HA (lanes 8–14) antibodies.
      The similar subcellular localization of HDAC10 prompted us to examine its potential association with HDACs 4, 5, or 7. FLAG-HDAC10 and HA-HDACs 4, 5, or 7 were either singly expressed or coexpressed in HEK293 cells, and immunoprecipitation experiments were carried out using anti-FLAG antibody-conjugated agarose beads. As shown in Fig.3C, whereas FLAG-HDAC10 was efficiently precipitated by anti-FLAG antibody-conjugated beads (lanes 4–7), HDACs 4, 5, or 7 were not coprecipitated (
      • Alland L.
      • Muhle R.
      • Hou H.
      • Potes J.
      • Chin L.
      • Schreiber-Agus N.
      • DePinho R.A.
      ,
      • Heinzel T.
      • Lavinsky R.M.
      • Mullen T.M.
      • Soderstrom M.
      • Laherty C.D.
      • Torchia J.
      • Yang W.M.
      • Brard G.
      • Ngo S.D.
      • Davie J.R.
      • Seto E.
      • Eisenman R.N.
      • Rose D.W.
      • Glass C.K.
      • Rosenfeld M.G.
      ,
      • Nagy L.
      • Kao H.Y.
      • Chakravarti D.
      • Lin R.J.
      • Hassig C.A.
      • Ayer D.E.
      • Schreiber S.L.
      • Evans R.M.
      ,
      • Hassig C.A.
      • Fleischer T.C.
      • Billin A.N.
      • Schreiber S.L.
      • Ayer D.E.
      ). As controls, Western blots of whole cell extracts showed good expression of all HDACs tested (lanes 15–28). Based on these results, we conclude that HDAC10 does not associate with HDACs 4, 5, or 7.

       Deacetylase Activity

      To determine whether HDAC10 harbors intrinsic histone deacetylase activity, FLAG-HDAC1 and FLAG-HDAC10 were transiently transfected into HEK293 cells. Whole cell extracts were prepared and immunoprecipitated with FLAG antibody-conjugated agarose beads. Western blot analyses were conducted to confirm the presence of FLAG-HDAC1 and HDAC10 in whole cell extracts (Fig.4A, lanes 2 and3) and in the immunoprecipitates (lanes 4 and5). As a control, a mock transfection with the vector alone was processed in parallel with the HDAC1 and HDAC10 samples (lanes 1 and 4). The immunoprecipitates were incubated with purified 3H-acetate-labeled histones, and the amount of released 3H-acetate was measured by scintillation counting. Whereas the control extracts contained background levels of HDAC activity, the HDAC10 immunocomplex exhibited a 9-fold higher activity than the control (Fig. 4B, lanes 1 and 3). The FLAG-HDAC1 immunocomplex exhibited even higher HDAC activity. We concluded that HDAC10 harbors intrinsic HDAC activity (lane 2).
      Figure thumbnail gr4
      Figure 4HDAC10 deacetylates histones in vitro.A, Western blots of HEK293 cells transfected with FLAG-HDAC1 or FLAG-HDAC10. Lanes 1–3, whole cell extracts; lanes 4–6, FLAG antibody immunoprecipitates. B, HDAC10 immunocomplex deacetylates histones in vitro. The amount of released 3H-labeled acetate is shown on the topof the bar. Lane 1, control; lane 2, FLAG-HDAC1; lane 3, FLAG-HDAC10. Note that HDAC1 has only one copy of the FLAG tag, whereas HDAC10 has three copies of the FLAG sequence.

       Transcriptional Repression

      Both class I and class II HDACs are able to repress basal transcription in transient transfection assays, presumably through their associated HDAC activities. HDAC10 was fused to the DNA-binding domain of Gal4 to determine repressor activity. A reporter plasmid containing four copies of the Gal4 binding site, cloned upstream of the thymidine kinase promoter and directing the expression of luciferase, was used to monitor the activity of Gal4-HDAC10 in this assay (Fig.5A). Whereas the catalytic domain alone represses transcription (lane 5), a maximal repression activity requires full-length HDAC10 (lane 2). Deletion or truncation of the HDAC domain significantly reduced repression activity (lanes 3, 4, and6). Structural studies of a trichostatin A-bound histone deacetylase homologue indicated that residues corresponding to Asp-170, Asp-172, and His-195 in HDAC10 are highly conserved among all HDACs and play an important role in binding Zn2+, an essential cofactor for histone deacetylase activiy (
      • Finnin M.S.
      • Donigian J.R.
      • Cohen A.
      • Richon V.M.
      • Rifkind R.A.
      • Marks P.A.
      • Breslow R.
      • Pavletich N.P.
      ). Alanine substitution at these residues has been shown to significantly decrease the enzymatic and transcriptional repression activities in HDAC1, HDAC5, and HDAC7 (
      • Downes M.
      • Ordentlich P.
      • Kao H.Y.
      • Alvarez J.G.
      • Evans R.M.
      ,
      • Hassig C.A.
      • Tong J.K.
      • Fleischer T.C.
      • Owa T.
      • Grable P.G.
      • Ayer D.E.
      • Schreiber S.L.
      ). Alanine substitutions at the corresponding positions in HDAC10 show that only residual activity associated with the carboxyl terminus of the enzyme (Fig. 5, lanes 6–9), Taken together, these results strongly suggest that HDAC10 repression activity is dependent on both catalytic and carboxyl-terminal function.
      Figure thumbnail gr5
      Figure 5HDAC10 harbors potent repression activity.A, schematic representation of the description of the transient transfection assays. A reporter plasmid containing four copies of Gal4 binding sites cloned upstream of thymidine kinase promoter and directing the expression of luciferase was used to monitor the trans-activation capacity of Gal4-HDAC10 in transient transfection assays. B, HDAC10 represses basal transcription in transient transfection assays. Fold repression activity is shown on the top of eachbar. Lane 1, control Gal4 vector alone;lane 2, Gal4-HDAC10 (full-length); lane 3, Gal4-HDAC10 (243); lane 4, Gal4-HDAC10 (1);lane 5, Gal4-HDAC10 (1); lane 6, Gal4-HDAC10 (339); lane 7, Gal4-HDAC10 (D170A); lane 8, Gal4-HDAC10 (D172A); lane 9, Gal4-HDAC10 (H195A).

      DISCUSSION

      In this study, we described the isolation of HDAC10, a novel mammalian class II histone deacetylase with transcriptional repressor activity. Human HDAC10 encodes at least two and possibly more differentially spliced isoforms. The largest HDAC10isoform encodes a protein of 669 amino acids and contains an amino-terminal histone deacetylase catalytic domain. Similar to other class II HDACs, HDAC10 can localize to either the nucleus or cytoplasm.
      Northern blot analyses indicated that the expression of HDAC10 may be ubiquitous with the highest expression in the liver, spleen, and kidney. Intriguingly, many cancer cell lines predominantly express a 2.8-kb transcript. Further investigation is underway to explore the significance of this observation. Sequence analyses of a mouse EST clone revealed a possible additional isoform of mammalian HDAC10 that lacks an intact deacetylase domain. This clone contains seven putative open reading frames and therefore may be a pseudogene. As with the human isoform, mouse HDAC10 is widely expressed in many tissues.
      Transcriptional repression by nuclear hormone receptors is mediated through their association with SMRT and nuclear receptor corepressor, which recruit histone deacetylase complexes containing both class I and class II HDACs (
      • Kao H.Y.
      • Downes M.
      • Ordentlich P.
      • Evans R.M.
      ). The association of nuclear hormone receptors with corepressors is mediated through the signature motif (I/L)XX(I/V)I, which is similar to the (I/V/L)XX(I/V/L)(I/V/L) motif in HDAC10. However, yeast two-hybrid assays detect no interaction between HDAC10 and the ligand binding domains of the retinoic acid receptor and thyroid hormone receptor (data not shown). Nonetheless, this does not rule out the possibility that HDAC10 may interact with other nuclear receptors.
      Fluorescence microscopy studies indicated that HDAC10 can be nuclear and/or cytoplasmic, a feature similar to that of HDACs 4, 5, and 7. Our sequence analyses suggested that two copies of putative NESs are present in the amino terminus (1) and carboxyl terminus (339) of HDAC10, respectively. This finding is inconsistent with our observation that these two fragments can also localize in the nucleus and cytoplasm. It has been suggested that HDACs function in the nucleus and that nuclear export is a simple yet effective way to inactivate HDAC (
      • Fischle W.
      • Dequiedt F.
      • Fillion M.
      • Hendzel M.J.
      • Voelter W.
      • Verdin E.
      ). However, the functional roles for HDACs in the cytoplasm can not be excluded.
      Using transient transfection assays, we showed that an HDAC10 fusion potently represses basal transcription from a Gal4-thymidine kinase promoter construct. The amino-terminal HDAC domain alone is able to repress transcription, although the carboxyl-terminal domain can repress approximately 3–4-fold. Alanine substitutions of several residues conserved in other HDACs (corresponding to amino acids Asp-170, Asp-172, and His-195 of HDAC10) eliminate the amino-terminal repression activity (
      • Downes M.
      • Ordentlich P.
      • Kao H.Y.
      • Alvarez J.G.
      • Evans R.M.
      ,
      • Hassig C.A.
      • Tong J.K.
      • Fleischer T.C.
      • Owa T.
      • Grable P.G.
      • Ayer D.E.
      • Schreiber S.L.
      ). These observations reveal that both the carboxyl and amino terminii are required for maximal repression.
      HDACs are often found as part of multi-component complexes. HDAC1 and HDAC2 copurify with mSin3A and Mi-2 (
      • Hassig C.A.
      • Fleischer T.C.
      • Billin A.N.
      • Schreiber S.L.
      • Ayer D.E.
      ,
      • Laherty C.D.
      • Yang W.M.
      • Sun J.M.
      • Davie J.R.
      • Seto E.
      • Eisenman R.N.
      ,
      • Zhang Y.
      • LeRoy G.
      • Seelig H.P.
      • Lane W.S.
      • Reinberg D.
      ,
      • Tong J.K.
      • Hassig C.A.
      • Schnitzler G.R.
      • Kingston R.E.
      • Schreiber S.L.
      ,
      • Xue Y.
      • Wong J.
      • Moreno G.T.
      • Young M.K.
      • Cote J.
      • Wang W.
      ), and HDAC3 associates with transcriptional corepressors and possibly with HDAC4 and HDAC5 (
      • Fischle W.
      • Dequiedt F.
      • Fillion M.
      • Hendzel M.J.
      • Voelter W.
      • Verdin E.
      ,
      • Guenther M.G.
      • Lane W.S.
      • Fischle W.
      • Verdin E.
      • Lazar M.A.
      • Shiekhattar R.
      ,
      • Underhill C.
      • Qutob M.S.
      • Yee S.P.
      • Torchia J.
      ,
      • Urnov F.D.
      • Yee J.
      • Sachs L.
      • Collingwood T.N.
      • Bauer A.
      • Beug H.
      • Shi Y.B.
      • Wolffe A.P.
      ,
      • Wen Y.D.
      • Perissi V.
      • Staszewski L.M.
      • Yang W.M.
      • Krones A.
      • Glass C.K.
      • Rosenfeld M.G.
      • Seto E.
      ). In an attempt to examine interactions between HDAC10 and other class II HDACs, we found no evidence of an association between HDAC10 and HDACs 4, 5, or 7. This observation suggests that HDAC10 has distinct protein association properties and thus may participate in new ways to control transcription.

      Acknowledgments

      We thank the sequencing facilities at the Salk Institute, Drs. David Samols, Cheng-Ming Chiang, and Ruth Yu for critical comments on the manuscript.

      REFERENCES

        • El-Osta A.
        • Wolffe A.P.
        Gene Expr. 2000; 9: 63-75
        • Roth S.Y.
        • Denu J.M.
        • Allis C.D.
        Annu. Rev. Biochem. 2001; 70: 81-120
        • Cheung P.
        • Allis C.D.
        • Sassone-Corsi P.
        Cell. 2000; 103: 263-271
        • Hansen J.C.
        • Tse C.
        • Wolffe A.P.
        Biochemistry. 1998; 37: 17637-17641
        • Strahl B.D.
        • Allis C.D.
        Nature. 2000; 403: 41-45
        • Turner B.M.
        Bioessays. 2000; 22: 836-845
        • Luger K.
        • Mader A.W.
        • Richmond R.K.
        • Sargent D.F.
        • Richmond T.J.
        Nature. 1997; 389: 251-260
        • Grozinger C.M.
        • Hassig C.A.
        • Schreiber S.L.
        Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4868-4873
        • Verdel A.
        • Khochbin S.
        J. Biol. Chem. 1999; 274: 2440-2445
        • Guarente L.
        Genes Dev. 2000; 14: 1021-1026
        • Alland L.
        • Muhle R.
        • Hou H.
        • Potes J.
        • Chin L.
        • Schreiber-Agus N.
        • DePinho R.A.
        Nature. 1997; 387: 49-55
        • Heinzel T.
        • Lavinsky R.M.
        • Mullen T.M.
        • Soderstrom M.
        • Laherty C.D.
        • Torchia J.
        • Yang W.M.
        • Brard G.
        • Ngo S.D.
        • Davie J.R.
        • Seto E.
        • Eisenman R.N.
        • Rose D.W.
        • Glass C.K.
        • Rosenfeld M.G.
        Nature. 1997; 387: 43-48
        • Nagy L.
        • Kao H.Y.
        • Chakravarti D.
        • Lin R.J.
        • Hassig C.A.
        • Ayer D.E.
        • Schreiber S.L.
        • Evans R.M.
        Cell. 1997; 89: 373-380
        • Hassig C.A.
        • Fleischer T.C.
        • Billin A.N.
        • Schreiber S.L.
        • Ayer D.E.
        Cell. 1997; 89: 341-347
        • Laherty C.D.
        • Yang W.M.
        • Sun J.M.
        • Davie J.R.
        • Seto E.
        • Eisenman R.N.
        Cell. 1997; 89: 349-356
        • Huang E.Y.
        • Zhang J.
        • Miska E.A.
        • Guenther M.G.
        • Kouzarides T.
        • Lazar M.A.
        Genes Dev. 2000; 14: 45-54
        • Kao H.Y.
        • Downes M.
        • Ordentlich P.
        • Evans R.M.
        Genes Dev. 2000; 14: 55-66
        • Ng H.H.
        • Bird A.
        Trends Biochem. Sci. 2000; 25: 121-126
        • Wang A.H.
        • Bertos N.R.
        • Vezmar M.
        • Pelletier N.
        • Crosato M.
        • Heng H.H.
        • Th'ng J.
        • Han J.
        • Yang X.J.
        Mol. Cell. Biol. 1999; 19: 7816-7827
        • Lu J.
        • McKinsey T.A.
        • Zhang C.L.
        • Olson E.N.
        Mol. Cell. 2000; 6: 233-244
        • Lu J.
        • McKinsey T.A.
        • Nicol R.L.
        • Olson E.N.
        Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4070-4075
        • Grozinger C.M.
        • Schreiber S.L.
        Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7835-7840
        • McKinsey T.A.
        • Zhang C.L.
        • Olson E.N.
        Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14400-14405
        • McKinsey T.A.
        • Zhang C.L.
        • Lu J.
        • Olson E.N.
        Nature. 2000; 408: 106-111
        • Wang A.H.
        • Kruhlak M.J.
        • Wu J.
        • Bertos N.R.
        • Vezmar M.
        • Posner B.I.
        • Bazett-Jones D.P.
        • Yang X.J.
        Mol. Cell. Biol. 2000; 20: 6904-6912
        • Zhao X.
        • Ito A.
        • Kane C.D.
        • Liao T.S.
        • Bolger T.A.
        • Lemrow S.M.
        • Means A.R.
        • Yao T.P.
        J. Biol. Chem. 2001; 276: 35042-35048
        • Fischle W.
        • Emiliani S.
        • Hendzel M.J.
        • Nagase T.
        • Nomura N.
        • Voelter W.
        • Verdin E.
        J. Biol. Chem. 1999; 274: 11713-11720
        • Fischle W.
        • Dequiedt F.
        • Fillion M.
        • Hendzel M.J.
        • Voelter W.
        • Verdin E.
        J. Biol. Chem. 2001; 276: 35826-35835
        • Lemercier C.
        • Verdel A.
        • Galloo B.
        • Curtet S.
        • Brocard M.P.
        • Khochbin S.
        J. Biol. Chem. 2000; 275: 15594-15599
        • Miska E.A.
        • Karlsson C.
        • Langley E.
        • Nielsen S.J.
        • Pines J.
        • Kouzarides T.
        EMBO J. 1999; 18: 5099-5107
        • Youn H.D.
        • Grozinger C.M.
        • Liu J.O.
        J. Biol. Chem. 2000; 275: 22563-22567
        • Bodmer R.
        • Venkatesh T.V.
        Dev. Genet. 1998; 22: 181-186
        • Khochbin S.
        • Kao H.Y.
        FEBS Lett. 2001; 494: 141-144
        • Downes M.
        • Ordentlich P.
        • Kao H.Y.
        • Alvarez J.G.
        • Evans R.M.
        Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10330-10335
        • Sambrook J.
        • Maniatis T.
        • Fritsch E.F.
        Molecular Cloning: A Laboratory Manual.
        2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 7.42-7.44
        • Heery D.M.
        • Kalkhoven E.
        • Hoare S.
        • Parker M.G.
        Nature. 1997; 387: 733-736
        • Darimont B.D.
        • Wagner R.L.
        • Apriletti J.W.
        • Stallcup M.R.
        • Kushner P.J.
        • Baxter J.D.
        • Fletterick R.J.
        • Yamamoto K.R.
        Genes Dev. 1998; 12: 3343-3356
        • McInerney E.M.
        • Rose D.W.
        • Flynn S.E.
        • Westin S.
        • Mullen T.M.
        • Krones A.
        • Inostroza J.
        • Torchia J.
        • Nolte R.T.
        • Assa-Munt N.
        • Milburn M.V.
        • Glass C.K.
        • Rosenfeld M.G.
        Genes Dev. 1998; 12: 3357-3368
        • Nolte R.T.
        • Wisely G.B.
        • Westin S.
        • Cobb J.E.
        • Lambert M.H.
        • Kurokawa R.
        • Rosenfeld M.G.
        • Willson T.M.
        • Glass C.K.
        • Milburn M.V.
        Nature. 1998; 395: 137-143
        • Perissi V.
        • Staszewski L.M.
        • McInerney E.M.
        • Kurokawa R.
        • Krones A.
        • Rose D.W.
        • Lambert M.H.
        • Milburn M.V.
        • Glass C.K.
        • Rosenfeld M.G.
        Genes Dev. 1999; 13: 3198-3208
        • Nagy L.
        • Kao H.-Y.
        • Love J.D.
        • Li C.
        • Banayo E.
        • Gooch J.T.
        • Krishna V.
        • Chatterjee K.
        • Evans R.M.
        • Schwabe J.W.R.
        Genes Dev. 1999; 13: 3209-3216
        • Shiau A.K.
        • Barstad D.
        • Loria P.M.
        • Cheng L.
        • Kushner P.J.
        • Agard D.A.
        • Greene G.L.
        Cell. 1998; 95: 927-937
        • Fornerod M.
        • Ohno M.
        • Yoshida M.
        • Mattaj I.W.
        Cell. 1997; 90: 1051-1060
        • Finnin M.S.
        • Donigian J.R.
        • Cohen A.
        • Richon V.M.
        • Rifkind R.A.
        • Marks P.A.
        • Breslow R.
        • Pavletich N.P.
        Nature. 1999; 401: 188-193
        • Hassig C.A.
        • Tong J.K.
        • Fleischer T.C.
        • Owa T.
        • Grable P.G.
        • Ayer D.E.
        • Schreiber S.L.
        Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3519-3524
        • Zhang Y.
        • LeRoy G.
        • Seelig H.P.
        • Lane W.S.
        • Reinberg D.
        Cell. 1998; 95: 279-289
        • Tong J.K.
        • Hassig C.A.
        • Schnitzler G.R.
        • Kingston R.E.
        • Schreiber S.L.
        Nature. 1998; 395: 917-921
        • Xue Y.
        • Wong J.
        • Moreno G.T.
        • Young M.K.
        • Cote J.
        • Wang W.
        Mol. Cell. 1998; 2: 851-861
        • Guenther M.G.
        • Lane W.S.
        • Fischle W.
        • Verdin E.
        • Lazar M.A.
        • Shiekhattar R.
        Genes Dev. 2000; 14: 1048-1057
        • Underhill C.
        • Qutob M.S.
        • Yee S.P.
        • Torchia J.
        J. Biol. Chem. 2000; 275: 40463-40470
        • Urnov F.D.
        • Yee J.
        • Sachs L.
        • Collingwood T.N.
        • Bauer A.
        • Beug H.
        • Shi Y.B.
        • Wolffe A.P.
        EMBO J. 2000; 19: 4074-4090
        • Wen Y.D.
        • Perissi V.
        • Staszewski L.M.
        • Yang W.M.
        • Krones A.
        • Glass C.K.
        • Rosenfeld M.G.
        • Seto E.
        Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7202-7207