The transcription factor CCAAT-binding factor CBF/NF-Y regulates the proximal promoter activity in the human alpha 1(XI) collagen gene (COL11A1).
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.
Matsuo
Noritaka
N
Department of Anatomy, Biology, and Medicine, Oita Medical University, Hasama-machi, Oita 879-5593, Japan.
Yu-Hua
Wang
W
Sumiyoshi
Hideaki
H
Sakata-Takatani
Keiko
K
Nagato
Hitoshi
H
Sakai
Kumiko
K
Sakurai
Mami
M
Yoshioka
Hidekatsu
H
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
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