The homeobox gene GAX activates p21 WAF1/CIP1 expression in vascular endothelial cells through direct interaction with upstream AT-rich sequences
The homeobox gene GAX activates p21 WAF1/CIP1 expression in vascular endothelial cells through direct interaction with upstream AT-rich sequences
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DOI：
10.1074/jbc.m606604200
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发表时间：
2007-01-05
影响因子：
4.8
通讯作者：
Gorski, David H.
中科院分区：
文献类型：
--
作者：
Chen, Yun;Leal, Alejandro D.;Gorski, David H.
肿瘤分泌促血管生成因子以诱导血管从周围基质向内生长，其最终靶点是血管内皮细胞（EC）。同源异型盒基因GAX抑制血管生成并诱导血管内皮细胞中p21（WAF 1/CIP 1）的表达。为了阐明GAX激活p（21 WAF 1/CIP 1）表达的机制，我们构建了缺失N-末端结构域、同源结构域或C-末端结构域的GAX cDNA，然后评估这些构建物激活p（21 WAF 1/CIP 1）的能力。有一个同源异型域的绝对要求，而删除C-末端结构域减少，但没有取消GAX的p21（WAF 1/CIP 1）启动子的反式激活。删除N-末端结构域确实废除了反式激活。接下来，我们进行了染色质免疫沉淀，并发现，类似于15 kb上游的p21（WAF 1/CIP 1）ATG密码子，ATTA含有GAX结合位点（指定为A6）的序列类似于其他同源结构域结合位点。GAX能够以同源结构域依赖的方式结合A6，从而激活与该序列偶联的报告基因的表达，并且通过突变该序列中的特定残基来消除这种激活。在A6序列的基础上，我们随后能够定位其它含有ATTA的序列，其也结合GAX并激活报告构建体中的转录。最后，我们发现这些GAX缺失诱导G（0）/G（1）停滞的能力与它们反式激活p21（WAF 1/CIP 1）启动子的能力相关。我们得出结论，GAX激活p21（WAF 1/CIP 1）通过多个上游AT丰富的序列。鉴于GAX在调节EC功能中的多种生物活性，鉴定推定的GAX结合位点将允许研究GAX如何激活或抑制其他下游靶点以抑制血管生成。
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 p(21WAF1/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 p(21WAF1/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, similar to 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 G(0)/G(1) 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.