Characterization of a human <em>RPD3</em> ortholog, HDAC3

^{Picower Institute for Medical Research, Manhasset, NY 11030; and}^{Gladstone Institute for Virology and Immunology, University of California, San Francisco, CA 94103}

^{To whom reprint requests should be addressed at: Gladstone Institute for Virology and Immunology, University of California, 365 Vermont Street, San Francisco CA 94103. e-mail:}ude.fscu.liamkciuq@nidreV_cirE.

Communicated by Anthony Cerami, The Kenneth S. Warren Laboratories, Tarrytown, NY

Received 1997 Nov 11; Accepted 1997 Dec 29.

Abstract

Histone acetylation levels in cells result from a dynamic equilibrium between competing histone acetylases and deacetylases. Changes in histone acetylation levels occur during both transcriptional activation and silencing. Cloning of the cDNA for a human histone deacetylase (HDAC1) has shown that it represents a human ortholog of the yeast transcriptional regulator RPD3. We have screened the expressed sequence tag database (National Center for Biotechnology Information) with the yeast RPD3 sequence and identified a human ortholog of RPD3, HDAC3. This cDNA encodes a protein of 428 amino acids with 58% sequence identity with HDAC1p. By using a specific polyclonal antiserum recognizing the C-terminal domain of HDAC3p and Western blotting, we detected a single ∼49-kDa band in several tumor cell lines. HDAC3p is expressed predominantly in the nuclear compartment. Immunoprecipitation experiments with either an antiserum against HDAC3p or an anti-FLAG antiserum and a flagged HDAC3 cDNA showed that HDAc3p exhibits deacetylase activity both on free histones and on purified nucleosomes. This deacetylase activity is inhibited by trichostatin, trapoxin, and butyrate in vitro to the same degree as the deacetylase activity associated to HDAC1p. These observations identify another member of a growing family of human HDAC genes.

Abstract

Posttranslational modifications of the N-terminal domain of histones, which include acetylation, methylation, phosphorylation, and ubiquitination, are emerging as an important step in the transcriptional regulation of eukaryotic genes. In particular, acetylation of specific lysine residues in the N-terminal portion of all histones consists of the transfer of an acetyl group from acetyl CoA onto the ɛ-amino group of lysine residues. Regulation of acetylation of the core histones is a dynamic process under the control of competing enzymes: histone acetyltransferases and histone deacetylases (HDACs) (1, 2).

Although a correlation between core histone acetylation and gene activity has been known for many years, the identification of the first histone acetyltransferase (3) as a homologue of the yeast transcriptional coactivator GCN5 has brought considerable support to the paradigm that histone acetylation/deacetylation plays an active role in transcriptional regulation. More recent studies have shown that other proteins previously identified as transcriptional regulators also possess histone acetyltransferase activity. These include p300 and CBP, coactivators that interacts with DNA binding factors (4, 5); P/CAF, a human homologue of yeast GCN5 that interacts with p300/CBP (6); and TAF_II250, a subunit of TFIID complex (7).

Independently, the target of action of the HDAC inhibitor trapoxin A was identified as a human ortholog of the yeast transcriptional regulator RPD3 (1). This gene, initially called HD1, is now called HDAC1, and its product exhibits HDAC activity (1). Another RPD3-related gene, initially called hRPD3 (8), now called HDAC2, encodes a protein interacting with YY1, a transcription factor that can act both as an activator or a repressor (9). HDAC1p and HDAC2p are part of two large repression complexes containing mSin3p, a mammalian homologue of the yeast corepressor SIN3 (9–11). Mammalian Sin3p was first identified as a protein interacting with Mad/Mxi proteins and required for their repressor function (12, 13). The HDAC1–Sin3 complex also plays a role of corepressor complex for N-Cor and SMRT, two silencing mediators for the unliganded retinoid and thyroid hormone receptors (14–16).

Herein, we report the identification and the characterization of a third human cDNA exhibiting homology to yeast RPD3. The protein encoded by this cDNA, HDAC3p, possesses intrinsic HDAC activity.

Acknowledgments

We thank David Spector (Cold Spring Harbor Laboratories) for performing the immunofluorescence microscopy analysis and Dr. Y. Nakamura (Kami-Ikebukuro Toshima-ku, Tokyo) and M.Yoshida (University of Tokyo, Tokyo) for reagents. We thank Chris Hassig (Harvard University) for discussions and for providing the protocol for in vitro acetylation of H4 peptide. C.V.L. is “Chercheur Qualifié” of the “Fonds National de la Recherche Scientifique” (Belgium). W.F. is supported by a fellowship from the Boehringer Ingelheim (Germany) and on a leave from the University of Tubingen (Germany). This work was supported in part by a grant from the National Institutes of Health of the United States Public Health Service (National Institute of General Medical Science/National Institute of Allergy and Infectious Diseases GM51671).

Acknowledgments

ABBREVIATIONS

HDAC	histone deacetylase
TSA	trichostatin A
TPX	trapoxin

ABBREVIATIONS

Note Added in Proof

While this manuscript was in preparation, Yang and coworkers (46) independently reported the identification of HDAC3.

Note Added in Proof

Footnotes

Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF039703","term_id":"2789655"}}AF039703).

Footnotes

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