title: The transcription factor CCAAT-binding factor CBF/NF-Y regulates the proximal promoter activity in the human alpha 1(XI) collagen gene (COL11A1).
abstract: We have characterized the proximal promoter region of the human COL11A1 gene. Transient transfection assays indicate that the segment from -199 to +1 is necessary for the activation of basal transcription. Electrophoretic mobility shift assays (EMSAs) demonstrated that the ATTGG sequence, within the -147 to -121 fragment, is critical to bind nuclear proteins in the proximal COL11A1 promoter. We demonstrated that the CCAAT binding factor (CBF/NF-Y) bound to this region using an interference assay with consensus oligonucleotides and a supershift assay with specific antibodies in an EMSA. In a chromatin immunoprecipitation assay and EMSA using DNA-affinity-purified proteins, CBF/NF-Y proteins directly bound this region in vitro and in vivo. We also showed that four tandem copies of the CBF/NF-Y-binding fragment produced higher transcriptional activity than one or two copies, whereas the absence of a CBF/NF-Y-binding fragment suppressed the COL11A1 promoter activity. Furthermore, overexpression of a dominant-negative CBF-B/NF-YA subunit significantly inhibited promoter activity in both transient and stable cells. These results indicate that the CBF/NF-Y proteins regulate the transcription of COL11A1 by directly binding to the ATTGG sequence in the proximal promoter region.
authors.affiliation: Department of Anatomy, Biology, and Medicine, Oita Medical University, Hasama-machi, Oita 879-5593, Japan.
authors.forename: Noritaka
authors.initials: N
authors.lastname: Matsuo
authors.forename: Wang
authors.initials: W
authors.lastname: Yu-Hua
authors.forename: Hideaki
authors.initials: H
authors.lastname: Sumiyoshi
authors.forename: Keiko
authors.initials: K
authors.lastname: Sakata-Takatani
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Nagato
authors.forename: Kumiko
authors.initials: K
authors.lastname: Sakai
authors.forename: Mami
authors.initials: M
authors.lastname: Sakurai
authors.forename: Hidekatsu
authors.initials: H
authors.lastname: Yoshioka
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D023081:CCAAT-Binding Factor / Q000502:physiology*; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D003094:Collagen / Q000235:genetics* / Q000378:metabolism*; D024161:Collagen Type XI / Q000235:genetics* / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D017353:Gene Deletion; D005786:Gene Expression Regulation; D005799:Genes, Dominant; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D023081:CCAAT-Binding Factor; D002843:Chromatin; D024161:Collagen Type XI; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C402244:nuclear factor Y; D003094:Collagen; D004247:DNA; D008156:Luciferases