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J. Biol. Chem., Vol. 283, Issue 9, 5542-5553, February 29, 2008

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General Transcription Factor IIA- Increases Osteoblast-specific Osteocalcin Gene Expression via Activating Transcription Factor 4 and Runt-related Transcription Factor 2^*

Shibing Yu, Yu Jiang, Deborah L. Galson, Min Luo, Yumei Lai, Yi Lu, Hong-Jiao Ouyang, Jian Zhang, and Guozhi Xiao¹

From the Departments of Medicine and Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania 15240

ATF4 (activating transcription factor 4) is an osteoblast-enriched transcription factor that regulates terminal osteoblast differentiation and bone formation. ATF4 knock-out mice have reduced bone mass (severe osteoporosis) throughout life. Runx2 (runt-related transcription factor 2) is a runt domain-containing transcription factor that is essential for bone formation during embryogenesis and postnatal life. In this study, we identified general transcription factor IIA (TFIIA) as a Runx2-interacting factor in a yeast two-hybrid screen. Immunoprecipitation assays confirmed that TFIIA interacts with Runx2 in osteoblasts and when coexpressed in COS-7 cells or using purified glutathione S-transferase fusion proteins. Chromatin immunoprecipitation assay of MC3T3-E1 (clone MC-4) preosteoblast cells showed that in intact cells TFIIA is recruited to the region of the osteocalcin promoter previously shown to bind Runx2 and ATF4. A small region of Runx2 (amino acids 258–286) was found to be required for TFIIA binding. Although TFIIA interacts with Runx2, it does not activate Runx2. Instead, TFIIA binds to and activates ATF4. Furthermore, TFIIA together with ATF4 and Runx2 stimulates osteocalcin promoter activity and endogenous mRNA expression. Small interfering RNA silencing of TFIIA markedly reduces levels of endogenous ATF4 protein and Ocn mRNA in osteoblastic cells. Overexpression of TFIIA increases levels of ATF4 protein. Finally, TFIIA significantly prevents ATF4 degradation. This study shows that a general transcription factor, TFIIA, facilitates osteoblast-specific gene expression through interactions with two important bone transcription factors ATF4 and Runx2.

Received for publication, July 10, 2007 , and in revised form, December 31, 2007.

^* This work was supported by National Institutes of Health Grant DK072230 and Department of Defense Grant W81XWH-07-1-0160 (to G. X.). 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 supplemental Figs. S1 and S2.

¹ To whom correspondence should be addressed: Division of Hematology/Oncology, Dept of Medicine, University of Pittsburgh, Veterans Affairs Pittsburgh Healthcare System, Research and Development, 151-U, Rm. 2W-111, University Dr. C, Pittsburgh, PA 15240. Tel.: 412-688-6000 (Ext. 814459); Fax: 412-688-6960; E-mail: xiaog{at}upmc.edu.

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