Involvement of a Novel Zinc Finger Protein, MIZF, in Transcriptional Repression by Interacting with a Methyl-CpG-binding Protein, MBD2*

MBD2, a methyl-CpG-binding protein, is a component of the MeCP1 histone deacetylase (HDAC) complex and plays a critical role in DNA methylation-mediated transcriptional repression. To understand the molecular basis of the methylation-associated repression, we attempted to identify MBD2-interacting proteins by a yeast two-hybrid system. Using MBD2 as bait, we isolated a novel zinc finger protein, referred to as MIZF. A direct interaction between MBD2 and MIZF was confirmed by in vitro binding assays and immunoprecipitation experiments. Four of seven zinc fingers present in the C-terminal region of MIZF are required for binding with MBD2. The MIZF mRNA is expressed in all human tissues and cell lines examined. The subcellular localization of MIZF is distinct from that of MBD2, although both proteins co-localize in some areas of the nuclei; MIZF localizes diffusely in the nucleoplasmic region, whereas MBD2 preferentially localizes in major satellites. A reporter assay demonstrated that MIZF significantly abrogates transcriptional activities. This repression is attenuated by an HDAC inhibitor, trichostatin A, and is completely dependent on the interaction with MBD2. These results suggest that MIZF is abundantly present in cells and functions as a negative regulator of transcription by binding to MBD2 and recruiting HDAC-containing complexes.

[1]  L. Magnaghi-Jaulin,et al.  The three members of the pocket proteins family share the ability to repress E2F activity through recruitment of a histone deacetylase. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[2]  J. Mackay,et al.  Zinc fingers are sticking together. , 1998, Trends in biochemical sciences.

[3]  F. Ishikawa,et al.  MBD2‐MBD3 complex binds to hemi‐methylated DNA and forms a complex containing DNMT1 at the replication foci in late S phase , 2000, Genes to cells : devoted to molecular & cellular mechanisms.

[4]  J. Herman,et al.  Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer , 1999, Nature Genetics.

[5]  Wen‐Ming Yang,et al.  Transcriptional repression by YY1 is mediated by interaction with a mammalian homolog of the yeast global regulator RPD3. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[6]  J. Strouboulis,et al.  Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription , 1998, Nature Genetics.

[7]  A. Bird,et al.  MeCP2 Is a Transcriptional Repressor with Abundant Binding Sites in Genomic Chromatin , 1997, Cell.

[8]  Rudolf Jaenisch,et al.  Targeted mutation of the DNA methyltransferase gene results in embryonic lethality , 1992, Cell.

[9]  E. Ballestar,et al.  Mi-2 complex couples DNA methylation to chromatin remodelling and histone deacetylation , 1999, Nature Genetics.

[10]  A. Bird,et al.  Active Repression of Methylated Genes by the Chromosomal Protein MBD1 , 2000, Molecular and Cellular Biology.

[11]  K. Georgopoulos,et al.  The role of the Ikaros gene in lymphocyte development and homeostasis. , 1997, Annual review of immunology.

[12]  A. Bird,et al.  Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation. , 1999, Genes & development.

[13]  Identification of a mammalian protein that binds specifically to DNA containing methylated CpGs , 1989, Cell.

[14]  A. Razin,et al.  CpG methylation, chromatin structure and gene silencing—a three‐way connection , 1998, The EMBO journal.

[15]  M. Surani.  Imprinting and the Initiation of Gene Silencing in the Germ Line , 1998, Cell.

[16]  R. Evans,et al.  Zinc fingers: Gilt by association , 1988, Cell.

[17]  E. Craig,et al.  Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. , 1996, Genetics.

[18]  A. Bird,et al.  The thymine glycosylase MBD4 can bind to the product of deamination at methylated CpG sites , 1999, Nature.

[19]  H. Willard,et al.  Mammalian X-chromosome inactivation and the XIST gene. , 1992 .

[20]  Samuel H. Wilson,et al.  Human β-polymerase gene. Structure of the 5'-flanking region and active promoter , 1988 .

[21]  Paul Tempst,et al.  MBD2 is a transcriptional repressor belonging to the MeCP1 histone deacetylase complex , 1999, Nature Genetics.

[22]  R. Evans,et al.  The LAZ3(BCL-6) oncoprotein recruits a SMRT/mSIN3A/histone deacetylase containing complex to mediate transcriptional repression. , 1998, Nucleic acids research.

[23]  Colin A. Johnson,et al.  Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex , 1998, Nature.

[24]  M. Nakao,et al.  Tsutomu by Methyl-CpG Binding Protein MBD 1 Mechanism of Transcriptional Regulation , 2000 .

[25]  I. Sadowski,et al.  GAL4 fusion vectors for expression in yeast or mammalian cells. , 1992, Gene.

[26]  E. Li,et al.  Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases , 1998, Nature Genetics.

[27]  Y. Emori,et al.  Morphological Changes and Detachment of Adherent Cells Induced by p122, a GTPase-activating Protein for Rho* , 1999, The Journal of Biological Chemistry.

[28]  Rudolf Jaenisch,et al.  DNA hypomethylation leads to elevated mutation rates , 1998, Nature.

[29]  A. Bird,et al.  Identification and Characterization of a Family of Mammalian Methyl-CpG Binding Proteins , 1998, Molecular and Cellular Biology.

[30]  S. Cross,et al.  A component of the transcriptional represser MeCP1 shares a motif with DNA methyltransferase and HRX proteins , 1997, Nature Genetics.

[31]  S. Baylin,et al.  Methylation of the oestrogen receptor CpG island links ageing and neoplasia in human colon , 1994, Nature Genetics.

[32]  M. Bartolomei,et al.  Genomic imprinting in mammals. , 1997, Annual review of genetics.

[33]  Rudolf Jaenisch,et al.  Role for DNA methylation in genomic imprinting , 1993, Nature.

[34]  P. Jones,et al.  Inhibition of DNA methylation by 5-aza-2'-deoxycytidine suppresses the growth of human tumor cell lines. , 1998, Cancer research.

[35]  Timothy H. Bester.  Cloning of a mammalian DNA methyltransferase , 1988 .