Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation.
Journal: 1999/September - Genes and Development
ISSN: 0890-9369
PUBMED: 10444591
Abstract:
ATP-dependent nucleosome remodeling and core histone acetylation and deacetylation represent mechanisms to alter nucleosome structure. NuRD is a multisubunit complex containing nucleosome remodeling and histone deacetylase activities. The histone deacetylases HDAC1 and HDAC2 and the histone binding proteins RbAp48 and RbAp46 form a core complex shared between NuRD and Sin3-histone deacetylase complexes. The histone deacetylase activity of the core complex is severely compromised. A novel polypeptide highly related to the metastasis-associated protein 1, MTA2, and the methyl-CpG-binding domain-containing protein, MBD3, were found to be subunits of the NuRD complex. MTA2 modulates the enzymatic activity of the histone deacetylase core complex. MBD3 mediates the association of MTA2 with the core histone deacetylase complex. MBD3 does not directly bind methylated DNA but is highly related to MBD2, a polypeptide that binds to methylated DNA and has been reported to possess demethylase activity. MBD2 interacts with the NuRD complex and directs the complex to methylated DNA. NuRD may provide a means of gene silencing by DNA methylation.
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Genes Dev 13(15): 1924-1935

Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation

Howard Hughes Medical Institute (HHMI), Division of Nucleic Acids Enzymology, Department of Biochemistry, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854 USA; Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh EH9 3JR, UK; Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York 10021 USA
Present address: Lineberger Comprehensive Cancer Center, Deptartment of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7295 USA.
Corresponding author.
Received 1999 May 26; Accepted 1999 Jun 23.

Abstract

ATP-dependent nucleosome remodeling and core histone acetylation and deacetylation represent mechanisms to alter nucleosome structure. NuRD is a multisubunit complex containing nucleosome remodeling and histone deacetylase activities. The histone deacetylases HDAC1 and HDAC2 and the histone binding proteins RbAp48 and RbAp46 form a core complex shared between NuRD and Sin3-histone deacetylase complexes. The histone deacetylase activity of the core complex is severely compromised. A novel polypeptide highly related to the metastasis-associated protein 1, MTA2, and the methyl-CpG-binding domain-containing protein, MBD3, were found to be subunits of the NuRD complex. MTA2 modulates the enzymatic activity of the histone deacetylase core complex. MBD3 mediates the association of MTA2 with the core histone deacetylase complex. MBD3 does not directly bind methylated DNA but is highly related to MBD2, a polypeptide that binds to methylated DNA and has been reported to possess demethylase activity. MBD2 interacts with the NuRD complex and directs the complex to methylated DNA. NuRD may provide a means of gene silencing by DNA methylation.

Keywords: DNA methylation, histone deacetylase complex, nucleosome remodeling, gene silencing
Abstract

Packaging of DNA into chromatin allows the cell to store its genetic information efficiently and has an important role in regulating gene expression (Workman and Kingston 1998). Dynamic changes in chromatin structure can facilitate or prevent the access of the transcription machinery to nucleosomal DNA, leading to transcription regulation. Recent studies have revealed two mechanisms by which chromatin structure can be altered. One mechanism involves multisubunit protein complexes that use the energy derived from ATP hydrolysis to alter the structure of, or ‘remodel’, nucleosomes (for review, see Tsukiyama and Wu 1997; Kadonaga 1998; Varga-Weisz and Becker 1998; Travers 1999). The other mechanism involves covalent modification of nucleosomes, in particular acetylation of the core histone tails and methylation of DNA (for review, see Grunstein 1997; Kuo and Allis 1998; Struhl 1998; Ng and Bird 1999).

Since the discovery of histone acetylation by Allfrey et al. (1964), a general correlation between histone acetylation and gene activity has been established (Hebbes et al. 1988). The enzymes that catalyze histone acetylation and deacetylation have been identified (Brownell et al. 1996; Taunton et al. 1996). Several transcriptional coactivators have histone acetyltransferase (HAT) activity, whereas several transcriptional corepressors have histone deacetylase activity (for review, see Grunstein 1997; Kuo and Allis 1998; Struhl 1998). In addition, mutagenesis studies with Gcn5 and Rpd3, the prototypical histone acetyltransferase and deacetylase, respectively, confirmed the long-speculated role of histone acetylation and deacetylation in transcription regulation (Kadosh and Struhl 1998a; Kuo et al. 1998; Wang et al. 1998). Moreover, Rpd3/Sin3-dependent repression has been shown to be directly associated with the deacetylation of lysine 5 of histone H4 in the promoters of UME6-regulated genes (Kadosh and Struhl 1998b; Rundlett et al. 1998). However, how core histone acetylation/deacetylation leads to transcriptional activation/repression remains to be elucidated.

Methylation of cytosine at CpG dinucleotides is a common feature of many higher eukaryotic genomes. Many studies have established a general correlation between DNA methylation and gene inactivation (Razin and Riggs 1980). However, the underlying molecular mechanism remained unknown until recently. It was found that MeCP2, a protein that specifically binds to methylated DNA, copurifies with the Sin3A/HDAC corepressor complex and that the histone deacetylase inhibitor TSA relieves MeCP2-mediated transcriptional repression (Jones et al. 1998; Nan et al. 1998). Recently, four mammalian proteins containing regions homologous to the MeCP2 methyl-CpG-binding domain, MBD1-4, were identified by searching the expressed sequence tag (EST) databases (Hendrich and Bird 1998). Interestingly, MBD2 was claimed to be a DNA demethylase, and MBD4 was shown to be an endonuclease potentially involved in DNA mismatch repair (Bellacosa et al. 1999; Bhattacharya et al. 1999). The functions of MBD1 and MBD3 are unknown.

To develop a mechanistic understanding of how core histone acetylation regulates transcription, we have studied the histone deacetylases HDAC1 and HDAC2 (Taunton et al. 1996; Yang et al. 1996). Using a combination of conventional and affinity chromatography, we previously purified and characterized two HDAC1/HDAC2-containing histone deacetylase complexes, the Sin3A/HDAC complex, and the NuRD complex (Zhang et al. 1997, 1998a,b). The two protein complexes share four polypeptides: HDAC1, HDAC2, RbAp46, and RbAp48. In addition, each complex contains three unique polypeptides (Sin3A, SAP30, and SAP18 in the Sin3 complex, and Mi2, p70, and p32 in the NuRD complex). Interestingly, the NuRD complex also possesses nucleosome remodeling activity, most likely because of the presence of Mi2, a member of the SWI2/SNF2 helicase/ATPase family (Tong et al. 1998; Xue et al. 1998; Zhang et al. 1998a).

To gain insight into the function of the NuRD complex, we have identified its p70 and p32 subunits. We demonstrate that these polypeptides have an important role in modulating the histone deacetylase activity of NuRD. Furthermore, we provide evidence linking NuRD function to methylated DNA.

Different subunits of the NuRD complex were analyzed for histone deacetylase activity and for their ability to regulate the histone deacetylase activity of the HDAC/RbAp core complex. The activity was compared to that of the NuRD complex using equal Western blot units of HDACs. Assays were performed as described in Materials and Methods. The numbers shown represent an average of at least three independent assays.

Acknowledgments

We are grateful to Drs. E. Seto and S.C. Tsai for baculovirus expressing HDAC2, to S. Schreiber for baculovirus expressing HDAC1, to A. Verreault and B. Stillman for baculoviruses expressing RbAp46 and RbAp48, and to D. Gottschling for the plasmid encoding yeast Hat 1. We thank Dr. George Orphanides for critical reading of the manuscript. We also thank members of the Reinberg laboratory for stimulating discussions during the course of this work. Y.Z. is a recipient of a National Institutes of Health (NIH) fellowship (1F32GM19515-01). H.H.N. holds a Darwin Trust Scholarship. D.R. is supported by a grant from NIH (GM-48518) and from the HHMI. A.B. is supported by grants from the Wellcome Trust. P.T. is supported by grants from the National Science Foundation and the National Cancer Institute.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked ‘advertisement’ in accordance with 18 USC section 1734 solely to indicate this fact.

Acknowledgments

Footnotes

E-MAIL ude.jndmu@fdebnier; FAX (732) 235-5294.

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