DNA methylation inhibits transcription indirectly via a methyl-CpG binding protein

We have studied the mechanism by which DNA methylation inhibits transcription both in cell-free nuclear extracts and in the living cell. Repression of transcription in vitro for four different promoters was shown to be an indirect effect. The mediator of repression had properties indistinguishable from those of a methyl-CpG binding protein (MeCP-1) that has been previously identified. Use of differentially methylated promoters and methylated competitors in transient transfection assays suggested that indirect repression via MeCP-1 also occurs in the living cell. This was supported by the fact that MeCP-1-deficient cells showed much reduced repression of methylated genes.

[1]  A. Razin,et al.  Cloning, characterization, and expression in Escherichia coli of the gene coding for the CpG DNA methylase from Spiroplasma sp. strain MQ1(M.SssI). , 1990, Nucleic acids research.

[2]  A. Bird,et al.  Specific protection of methylated CpGs in mammalian nuclei , 1989, Cell.

[3]  C. Stocking,et al.  Functional analysis of a retroviral host-range mutant: altered long terminal repeat sequences allow expression in embryonal carcinoma cells. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[4]  J. Hurst,et al.  DNA methylation and the regulation of globin gene expression , 1983, Cell.

[5]  E. Murray,et al.  Site specific demethylation in the promoter of human gamma‐globin gene does not alleviate methylation mediated suppression. , 1987, The EMBO journal.

[6]  A. Graessmann,et al.  Chromatin structure is required to block transcription of the methylated herpes simplex virus thymidine kinase gene. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

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

[8]  A. Riggs,et al.  Sequence of the promoter region of the gene for human X-linked 3-phosphoglycerate kinase. , 1984, Gene.

[9]  H. Cedar,et al.  Expression of a cloned adenovirus gene is inhibited by in vitro methylation. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[10]  P. Yen,et al.  Transformation with DNA from 5-azacytidine-reactivated X chromosomes. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[11]  Howard Cedar,et al.  DNA methylation affects the formation of active chromatin , 1986, Cell.

[12]  J. Nevins,et al.  Role of an adenovirus E2 promoter binding factor in E1A-mediated coordinate gene control. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[13]  F. Watt,et al.  Cytosine methylation prevents binding to DNA of a HeLa cell transcription factor required for optimal expression of the adenovirus major late promoter. , 1988, Genes & development.

[14]  A. Feinberg,et al.  A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. , 1983, Analytical biochemistry.

[15]  M. Capecchi,et al.  Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells , 1987, Cell.

[16]  A. Riggs,et al.  DNA methylation and gene function. , 1980, Science.

[17]  P. Dobrzanski,et al.  Inactivation by sequence-specific methylations of adenovirus promoters in a cell-free transcription system , 1988, Journal of virology.

[18]  S. Iguchi-Ariga,et al.  CpG methylation of the cAMP-responsive enhancer/promoter sequence TGACGTCA abolishes specific factor binding as well as transcriptional activation. , 1989, Genes & development.

[19]  E. Linney,et al.  Mutation near the polyoma DNA replication origin permits productive infection of F9 embryonal carcinoma cells , 1981, Cell.

[20]  W. Schaffner,et al.  Sp1 transcription factor binds DNA and activates transcription even when the binding site is CpG methylated. , 1988, Genes & development.

[21]  A. Razin,et al.  Effect of in vitro DNA methylation on beta-globin gene expression. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[22]  O. Pongs,et al.  In vitro transcription with extracts of nuclei of Drosophila embryos. , 1985, Nucleic acids research.

[23]  T. Klimkait,et al.  Reactivation of the methylation-inactivated late E2A promoter of adenovirus type 2 by E1A (13 S) functions. , 1988, Journal of molecular biology.

[24]  H. Cedar,et al.  Effect of regional DNA methylation on gene expression. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[25]  D. Renz,et al.  DNA methylation of three 5' C-C-G-G 3' sites in the promoter and 5' region inactivate the E2a gene of adenovirus type 2. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[26]  Peter A. Jones,et al.  Cellular differentiation, cytidine analogs and DNA methylation , 1980, Cell.

[27]  T. Mohandas,et al.  Reactivation of an inactive human X chromosome: evidence for X inactivation by DNA methylation. , 1981, Science.

[28]  H. Cedar,et al.  Organization of 5-methylcytosine in chromosomal DNA. , 1978, Biochemistry.

[29]  J. Jiricny,et al.  Methylation of single CpG dinucleotides within a promoter element of the Herpes simplex virus tk gene reduces its transcription in vivo. , 1988, Gene.

[30]  G. Schaffner,et al.  Ribozyme, antisense RNA, and antisense DNA inhibition of U7 small nuclear ribonucleoprotein-mediated histone pre-mRNA processing in vitro , 1989, Molecular and cellular biology.

[31]  G. Schaffner,et al.  OVEC, a versatile system to study transcription in mammalian cells and cell-free extracts. , 1987, Nucleic acids research.

[32]  A. Razin,et al.  In vitro methylation of the hamster adenine phosphoribosyltransferase gene inhibits its expression in mouse L cells. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[33]  A. Schechter,et al.  Inducible transcription of five globin genes in K562 human leukemia cells. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[34]  D. Bednarik,et al.  Inactivation of the HIV LTR by DNA CpG methylation: evidence for a role in latency. , 1990, The EMBO journal.

[35]  A. van der Eb,et al.  A new technique for the assay of infectivity of human adenovirus 5 DNA. , 1973, Virology.

[36]  R. Hansen,et al.  Demethylation of specific sites in the 5' region of the inactive X-linked human phosphoglycerate kinase gene correlates with the appearance of nuclease sensitivity and gene expression , 1988, Molecular and cellular biology.

[37]  A. Bird,et al.  High levels of De Novo methylation and altered chromatin structure at CpG islands in cell lines , 1990, Cell.

[38]  R. Roeder,et al.  Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. , 1983, Nucleic acids research.