title: Direct association between PU.1 and MeCP2 that recruits mSin3A-HDAC complex for PU.1-mediated transcriptional repression.
abstract: PU.1, a member of the Ets family of transcription factors, is implicated in hematopoietic cell differentiation through its interactions with other transcriptional factors and cofactors. To identify a novel protein(s) binding to PU.1, we carried out affinity purification using a column of Glutathione-Sepharose beads bound to GST-PU.1 fusion protein and isolated several individual proteins using murine erythroleukemia (MEL) cell extracts. Sequence analysis of these proteins revealed that one was MeCP2 a methyl CpG binding protein. GST-pull-down assay and immunoprecipitation assay showed that PU.1 bound directly to MeCP2 via its Ets domain and MeCP2 bound to PU.1 via either its amino terminal domain or trans-repression domain. MeCP2 repressed transcriptional activity of PU.1 on a reporter construct with trimerized PU.1 binding sites. This downregulation was recovered in the presence of histone deacetylase inhibitor, trichostatin A (TSA). MeCP2 was integrated in PU.1-mSin3A-HDAC complex but not in PU.1-CBP complex. Chromatin immunoprecipitation (ChIP) assays showed that PU.1 and MeCP2 were collocated at the PU.1 binding site on the reporter construct and the PU.1 binding site of the intervening sequence 2 (IVS2) region in the intron of the beta-globin gene, which has been proposed to regulate expression of the gene, in undifferentiated MEL cells. The complex disappeared from the region during the course of erythroid differentiation of MEL cells. Our results suggest that MeCP2 acts as a corepressor of PU.1 probably due to facilitating complex formation with mSin3A and HDACs.
authors.affiliation: Department of Cell Genetics, Sasaki Institute, 2-2 Kanda Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan.
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Suzuki
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Yamada
authors.forename: Fumiko
authors.initials: F
authors.lastname: Kihara-Negishi
authors.forename: Takuya
authors.initials: T
authors.lastname: Sakurai
authors.forename: Tsuneyuki
authors.initials: T
authors.lastname: Oikawa
mesh_terms: D002454:Cell Differentiation; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins / Q000032:analysis / Q000737:chemistry / Q000502:physiology*; D006655:Histone Deacetylases / Q000378:metabolism*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism / Q000473:pathology; D051783:Methyl-CpG-Binding Protein 2; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000737:chemistry / Q000502:physiology*; D012097:Repressor Proteins / Q000378:metabolism* / Q000502:physiology*; D056264:Sin3 Histone Deacetylase and Corepressor Complex; D015534:Trans-Activators / Q000032:analysis / Q000737:chemistry / Q000502:physiology*; D000099232:Proto-Oncogene Protein Spi-1
chemical_list: D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D006655:Histone Deacetylases; D006877:Hydroxamic Acids; D051783:Methyl-CpG-Binding Protein 2; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins; D056264:Sin3 Histone Deacetylase and Corepressor Complex; D015534:Trans-Activators; D000099232:Proto-Oncogene Protein Spi-1; C497392:MECP2 protein, human; C099683:SIN3A transcription factor; C012589:trichostatin A