Regulation of an endogenous locus using a panel of designed zinc finger proteins targeted to accessible chromatin regions - Activation of vascular endothelial growth factor A
Regulation of an endogenous locus using a panel of designed zinc finger proteins targeted to accessible chromatin regions - Activation of vascular endothelial growth factor A
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DOI：
10.1074/jbc.m011172200
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发表时间：
2001-04-06
影响因子：
4.8
通讯作者：
Wolffe, AP
中科院分区：
文献类型：
--
作者：
Liu, PQ;Rebar, EJ;Wolffe, AP
我们绘制了内源性血管内皮生长因子A （VEGF-A）染色体位点内DNase I可及性增强的保守区域。合成锌指蛋白（ZFP）转录因子被设计用于靶向DNA酶i超敏感区的DNA序列。当这些ZFPs融合到VP16或p65转录激活结构域时，能够激活VEGF-A基因的表达，通过mRNA积累和VEGF-A蛋白分泌来检测，其范围超过了缺氧胁迫诱导的范围。重要的是，单个工程ZFP转录因子诱导了具有明确生理功能的VEGF-A mRNA的多个剪接变体。我们提供的证据表明，ZFP转录因子融合到VP16和p65中，靶向转录起始位点上游或下游的两个不同的染色体位点>500碱基对，可以增强VEGF-A基因转录的激活。我们的策略提供了一种新的方法，可以在不改变内源性靶位点DNA序列的情况下，远距离剖析基因调控的要求。
We have mapped conserved regions of enhanced DNase I accessibility within the endogenous chromosomal locus of vascular endothelial growth factor A (VEGF-A). Synthetic zinc finger protein (ZFP) transcription factors were designed to target DNA sequences contained within the DNase I-hypersensitive regions. These ZFPs, when fused to either VP16 or p65 transcriptional activation domains, were able to activate expression of the VEGF-A gene as assayed by mRNA accumulation and VEGF-A protein secretion through a range exceeding that induced by hypoxic stress. Importantly, multiple splice variants of VEGF-A mRNA with defined physiological functions were induced by a single engineered ZFP transcription factor. We present evidence for an enhanced activation of VEGF-A gene transcription by ZFP transcription factors fused to VP16 and p65 targeted to two distinct chromosomal sites >500 base pairs upstream or downstream of the transcription start site. Our strategy provides a novel approach for dissecting the requirements for gene regulation at a distance without altering the DNA sequence of the endogenous target locus.