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J. Biol. Chem., Vol. 282, Issue 1, 507-517, January 5, 2007

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The Homeobox Gene GAX Activates p21^WAF1/CIP1 Expression in Vascular Endothelial Cells through Direct Interaction with Upstream AT-rich Sequences^*

From the Division of Surgical Oncology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, The Cancer Institute of New Jersey, New Brunswick, New Jersey 088901

Tumors secrete pro-angiogenic factors to induce the ingrowth of blood vessels from the surrounding stroma, the end targets of which are vascular endothelial cells (ECs). The homeobox gene GAX inhibits angiogenesis and induces p21^WAF1/CIP1 expression in vascular ECs. To elucidate the mechanism through which GAX activates p21^WAF1/CIP1 expression, we constructed GAX cDNAs with deletions of the N-terminal domain, the homeodomain, or the C-terminal domain and then assessed these constructs for their ability to activate p21^WAF1/CIP1. There was an absolute requirement for the homeodomain, whereas deleting the C-terminal domain decreased but did not abolish transactivation of the p21^WAF1/CIP1 promoter by GAX. Deleting the N-terminal domain did abolish transactivation. Next, we performed chromatin immunoprecipitation and found, 15 kb upstream of the p21^WAF1/CIP1 ATG codon, an ATTA-containing GAX-binding site (designated A6) with a sequence similar to that of other homeodomain-binding sites. GAX was able to bind to A6 in a homeodomain-dependent manner and thereby activate the expression of a reporter gene coupled to this sequence, and this activation was abolished by mutating specific residues in this sequence. On the basis of the sequence of A6, we were then able to locate other ATTA-containing sequences that also bound GAX and activated transcription in reporter constructs. Finally, we found that the ability of these GAX deletions to induce G₀/G₁ arrest correlates with their ability to transactivate the p21^WAF1/CIP1 promoter. We conclude that GAX activates p21^WAF1/CIP1 through multiple upstream AT-rich sequences. Given the multiple biological activities of GAX in regulating EC function, identification of a putative GAX-binding site will allow the study of how GAX activates or represses other downstream targets to inhibit angiogenesis.

Received for publication, July 11, 2006 , and in revised form, October 5, 2006.

^* This work was supported by United States Department of Defense Grants DAMD17-02-1-0511 and DAMD17-03-1-0292 and NCI Grant 1 R01 CA111344 from the National Institutes of Health. 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.

¹ To whom correspondence should be addressed: Div. of Surgical Oncology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, The Cancer Institute of New Jersey, 195 Little Albany St., New Brunswick, NJ 08901. Tel.: 732-235-8524; Fax: 732-235-8098; E-mail: gorskidh{at}umdnj.edu.

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