title: An Sp1-NF-Y/progesterone receptor DNA binding-dependent mechanism regulates progesterone-induced transcriptional activation of the rabbit RUSH/SMARCA3 gene.
abstract: Steroids regulate alternative splicing of rabbit RUSH/SMARCA3, an SWI/SNF-related transcription factor. Transactivation was evaluated in 2057 bp of genomic sequence. Truncation analysis identified a minimal 252-bp region with strong basal promoter activity in transient transfection assays. The size of the 5'-untranslated region (233 bp) and the transcription start site were determined by primer extension analysis. The transcription start site mapped to a consensus initiator (Inr) element in a TATA-less region with a downstream promoter element (+29). These elements were authenticated by mutation/deletion analysis. The Inr/downstream promoter element combination is conserved in the putative core promoter (-35/+35) of the human ortholog, suggesting that transcription initiation is similarly conserved. Two Sp1 sites that are also conserved in the putative promoter of human SMARCA3 and a RUSH binding site (-616/-611) that is unique to the rabbit promoter repress basal transcription. These sites were variously authenticated by gel shift and chromatin immunoprecipitation assays. Analysis of the proximal promoter showed the -162/+90 region was required for progesterone responsiveness in transient transfection assays. Subsequent mutation/deletion analysis revealed a progesterone receptor half-site mediated induction by progesterone. An overlapping Y-box (in the reverse ATTGG orientation) repressed basal transcription and progesterone-induced transcriptional activation in the presence of the Sp1 sites. The specificity of progesterone receptor and transcription factor NF-Y binding were authenticated by gel shift assays. Chromatin immunoprecipitation assays confirmed the Y-box effects were mediated in a DNA binding-dependent fashion. This represents a unique regulatory scenario in which ligand-dependent transactivation by the progesterone receptor is subject to Sp1/NF-Y repression.
authors.affiliation: Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, USA.
authors.forename: Aveline
authors.initials: A
authors.lastname: Hewetson
authors.forename: Beverly S
authors.initials: BS
authors.lastname: Chilton
mesh_terms: D017398:Alternative Splicing / Q000187:drug effects; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D023081:CCAAT-Binding Factor / Q000378:metabolism*; D002460:Cell Line; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics*; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011374:Progesterone / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D011817:Rabbits; D011980:Receptors, Progesterone / Q000378:metabolism*; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection; D014599:Uterus / Q000187:drug effects / Q000378:metabolism
chemical_list: D023081:CCAAT-Binding Factor; D004268:DNA-Binding Proteins; C100023:HLTF protein, human; D011980:Receptors, Progesterone; C104270:SMARCA3 protein, Oryctolagus cuniculus; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D011374:Progesterone; D004247:DNA