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

Abstract

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.