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J. Biol. Chem., Vol. 279, Issue 45, 46373-46383, November 5, 2004

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The Transcription Factor CCAAT-binding Factor CBF/NF-Y and Two Repressors Regulate the Core Promoter of the Human Pro-3(V) Collagen Gene (COL5A3)^*

Hitoshi Nagato, Noritaka Matsuo, Hideaki Sumiyoshi, Keiko Sakata-Takatani, Masaru Nasu, and Hidekatsu Yoshioka¶

From the Department of Anatomy, Biology and Medicine, and Department of Immunology and Allergy, Faculty of Medicine, Oita University, 1-1 Hasama-machi, Oita 879-5593, Japan

To elucidate the mechanisms underlining 3(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.

Received for publication, June 1, 2004 , and in revised form, August 16, 2004.

^* This work was supported by Grants-in-Aid for Scientific Research 11470312 and 14370468 (to H. Y.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Table S1.

^¶ To whom correspondence should be addressed: Dept. of Anatomy, Biology and Medicine, Faculty of Medicine, Oita University, 1-1 Hasama-machi, Oita 879-5593, Japan. Tel.: 81-97-586-5670; Fax: 81-97-549-6302; E-mail: hidey{at}med.oita-u.ac.jp.

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