title: MeCP2 driven transcriptional repression in vitro: selectivity for methylated DNA, action at a distance and contacts with the basal transcription machinery.
abstract: The pathways for selective transcriptional repression of methylated DNA templates by the methyl-CpG-binding protein MeCP2 have been investigated using a purified in vitro transcription system that does not assemble chromatin. MeCP2 selectively inhibits transcription complex assembly on methylated DNA but does not destabilize a pre-assembled transcription complex. MeCP2 functions to repress transcription at a distance of >500 bp from the transcription start site. The transcription repression domain (TRD) of MeCP2 will repress transcription in vitro when fused to a heterologous Gal4 DNA-binding domain. The TRD associates with TFIIB. Exogenous TFIIB does not relieve transcriptional repression established by either intact MeCP2 or a Gal4-TRD fusion protein under these in vitro conditions, nor does the addition of histone deacetylase inhibitors. We find that the transcriptional repression established by both MeCP2 and the Gal4-TRD fusion protein in vitro also correlates with selective assembly of large nucleoprotein complexes. The formation of such complexes reflects a local concentration of DNA-bound transcriptional repressor that may stabilize a state of repression even in the presence of exogenous transcriptional machinery.
authors.affiliation: Laboratory of Molecular Embryology, National Institute of Child Heath and Human Development, NIH, Building 18T, Room 106, Bethesda, MD 20892-5431, USA.
authors.forename: N K
authors.initials: NK
authors.lastname: Kaludov
authors.forename: A P
authors.initials: AP
authors.lastname: Wolffe
mesh_terms: D002868:Chromosomal Proteins, Non-Histone; D004247:DNA / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D051783:Methyl-CpG-Binding Protein 2; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D035581:Transcription Factor TFIIB; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D051783:Methyl-CpG-Binding Protein 2; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D035581:Transcription Factor TFIIB; D014157:Transcription Factors; D004247:DNA; D005982:Glutathione Transferase