The transcription factor CCAAT-binding factor CBF/NF-Y and two repressors regulate the core promoter of the human pro-alpha3(V) collagen gene (COL5A3).
To elucidate the mechanisms underlining alpha3(V) collagen chain expression, we performed an initial analysis of the structure and function of the core promoter of the human COL5A3 gene. The core promoter, which lacks a typical TATA motif and has a high GC content, was defined within the -129 bp immediately upstream from the major transcription start site by transient transfection experiments. In this region, we identified four DNA-protein complexes, named A, B, C, and D, by a combination of DNase I footprinting and electrophoretic mobility shift assays. Electrophoretic mobility shift assays using mutant oligonucleotide revealed that the complexes A, B, C, and D bind to -122 to -117, the -101 to -96, the -83 to -78, and the -68 to -57 bp, respectively. The competition assays using consensus oligonucleotides and supershift assays with specific antibodies showed that complex A consists of CBF/NF-Y. In a chromatin immunoprecipitation assay, CBF/NF-Y protein directly bound to this region, in vivo. Functional analysis showed that CBF/NF-Y activated the gene, whereas the proteins of complexes B and C repressed its activity. Furthermore, overexpression of a mutant form of the CBF-B/NF-YA subunit, which forms CBF/NF-Y with CBF-A/NF-YB and CBF-C/NF-YC subunits, inhibited promoter activity.
Department of Anatomy, Biology and Medicine, Faculty of Medicine, Oita University, 1-1 Hasama-machi, Oita 879-5593, Japan.
Hitoshi
H
Nagato
Noritaka
N
Matsuo
Hideaki
H
Sumiyoshi
Keiko
K
Sakata-Takatani
Masaru
M
Nasu
Hidekatsu
H
Yoshioka
D020816:Amino Acid Motifs; D001483:Base Sequence; D001667:Binding, Competitive; D023081:CCAAT-Binding Factor / Q000378:metabolism / Q000502:physiology*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D024062:Collagen Type V / Q000737:chemistry* / Q000235:genetics*; D004247:DNA / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D003851:Deoxyribonucleases / Q000378:metabolism; D017353:Gene Deletion; D006801:Humans; D047468:Immunoprecipitation; D019169:Jurkat Cells; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic; D014162:Transfection
D023081:CCAAT-Binding Factor; D002843:Chromatin; D024062:Collagen Type V; D017931:DNA Primers; D009841:Oligonucleotides; D014157:Transcription Factors; C402244:nuclear factor Y; D012313:RNA; D004247:DNA; D008156:Luciferases; D003851:Deoxyribonucleases