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The Transcription Factor CCAAT-binding Factor CBF/NF-Y and Two Repressors Regulate the Core Promoter of the Human Pro-α3(V) Collagen Gene (COL5A3)
To elucidate the mechanisms underlining α3(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 â129 bp imme...
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Published in: | The Journal of biological chemistry 2004-11, Vol.279 (45), p.46373 |
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Main Authors: | , , , , , |
Format: | Article |
Language: | English |
Online Access: | Get full text |
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Summary: | To elucidate the mechanisms underlining α3(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 â129 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 â122
to â117, the â101 to â96, the â83 to â78, and the â68 to â57 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. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.M406069200 |