title: Cryptic MCAT enhancer regulation in fibroblasts and smooth muscle cells. Suppression of TEF-1 mediated activation by the single-stranded DNA-binding proteins, Pur alpha, Pur beta, and MSY1.
abstract: An asymmetric polypurine-polypyrimidine cis-element located in the 5' region of the mouse vascular smooth muscle alpha-actin gene serves as a binding site for multiple proteins with specific affinity for either single- or double-stranded DNA. Here, we test the hypothesis that single-stranded DNA-binding proteins are responsible for preventing a cryptic MCAT enhancer centered within this element from cooperating with a nearby serum response factor-interacting CArG motif to trans-activate the minimal promoter in fibroblasts and smooth muscle cells. DNA binding studies revealed that the core MCAT sequence mediates binding of transcription enhancer factor-1 to the double-stranded polypurine-polypyrimidine element while flanking nucleotides account for interaction of Pur alpha and Pur beta with the purine-rich strand and MSY1 with the complementary pyrimidine-rich strand. Mutations that selectively impaired high affinity single-stranded DNA binding by fibroblast or smooth muscle cell-derived Pur alpha, Pur beta, and MSY1 in vitro, released the cryptic MCAT enhancer from repression in transfected cells. Additional experiments indicated that Pur alpha, Pur beta, and MSY1 also interact specifically, albeit weakly, with double-stranded DNA and with transcription enhancer factor-1. These results are consistent with two plausible models of cryptic MCAT enhancer regulation by Pur alpha, Pur beta, and MSY1 involving either competitive single-stranded DNA binding or masking of MCAT-bound transcription enhancer factor-1.
authors.affiliation: Department of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, Minnesota 55905, USA.
authors.forename: Leslie E
authors.initials: LE
authors.lastname: Carlini
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Getz
authors.forename: Arthur R
authors.initials: AR
authors.lastname: Strauch
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Kelm
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D001483:Base Sequence; D019921:Biotinylation; D002460:Cell Line; D002478:Cells, Cultured; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D000939:Epitopes; D005347:Fibroblasts / Q000378:metabolism*; D017930:Genes, Reporter; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000166:cytology* / Q000378:metabolism; D009154:Mutation; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D011233:Precipitin Tests; D011485:Protein Binding; D011817:Rabbits; D051381:Rats; D000091262:TEA Domain Transcription Factors; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D004247:DNA; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D000939:Epitopes; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D000091262:TEA Domain Transcription Factors; D014157:Transcription Factors; C491579:PURB protein, human; C121485:Pura protein, mouse; C108770:Purb protein, mouse; C057182:TEAD1 protein, human; C541744:Ybx1 protein, mouse