Molecular Biology Graduate Program, School of Dental Medicine University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.
PLoS One. 2013 Aug 21;8(8):e72358. doi: 10.1371/journal.pone.0072358. eCollection 2013.
The HIF1- and HIF2-mediated transcriptional responses play critical roles in solid tumor progression. Despite significant similarities, including their binding to promoters of both HIF1 and HIF2 target genes, HIF1 and HIF2 proteins activate unique subsets of target genes under hypoxia. The mechanism for HIF target gene specificity has remained unclear. Using siRNA or inhibitor, we previously reported that STAT3 or USF2 is specifically required for activation of endogenous HIF1 or HIF2 target genes. In this study, using reporter gene assays and chromatin immuno-precipitation, we find that STAT3 or USF2 exhibits specific binding to the promoters of HIF1 or HIF2 target genes respectively even when over-expressed. Functionally, HIF1α interacts with STAT3 to activate HIF1 target gene promoters in a HIF1α HLH/PAS and N-TAD dependent manner while HIF2α interacts with USF2 to activate HIF2 target gene promoters in a HIF2α N-TAD dependent manner. Physically, HIF1α HLH and PAS domains are required for its interaction with STAT3 while both N- and C-TADs of HIF2α are involved in physical interaction with USF2. Importantly, addition of functional USF2 binding sites into a HIF1 target gene promoter increases the basal activity of the promoter as well as its response to HIF2+USF2 activation while replacing HIF binding site with HBS from a HIF2 target gene does not change the specificity of the reporter gene. Importantly, RNA Pol II on HIF1 or HIF2 target genes is primarily associated with HIF1α or HIF2α in a STAT3 or USF2 dependent manner. Thus, we demonstrate here for the first time that HIF target gene specificity is achieved by HIF transcription partners that are required for HIF target gene activation, exhibit specific binding to the promoters of HIF1 or HIF2 target genes and selectively interact with HIF1α or HIF2α protein.
HIF1 和 HIF2 介导的转录反应在实体肿瘤进展中发挥关键作用。尽管它们具有显著的相似性，包括与 HIF1 和 HIF2 靶基因的启动子结合，但 HIF1 和 HIF2 蛋白在缺氧下激活独特的靶基因子集。HIF 靶基因特异性的机制尚不清楚。我们之前使用 siRNA 或抑制剂报告，STAT3 或 USF2 特异性地需要激活内源性 HIF1 或 HIF2 靶基因。在这项研究中，我们使用报告基因检测和染色质免疫沉淀，发现即使过表达，STAT3 或 USF2 也分别特异性地结合到 HIF1 或 HIF2 靶基因的启动子上。功能上，HIF1α 与 STAT3 相互作用，以 HIF1α HLH/PAS 和 N-TAD 依赖性方式激活 HIF1 靶基因启动子，而 HIF2α 与 USF2 相互作用，以 HIF2α N-TAD 依赖性方式激活 HIF2 靶基因启动子。在物理上，HIF1α HLH 和 PAS 结构域是其与 STAT3 相互作用所必需的，而 HIF2α 的 N-和 C-TAD 都参与与 USF2 的物理相互作用。重要的是，将功能性 USF2 结合位点添加到 HIF1 靶基因启动子中会增加启动子的基础活性及其对 HIF2+USF2 激活的反应，而用 HIF2 靶基因的 HBS 替换 HIF 结合位点不会改变报告基因的特异性。重要的是，RNA Pol II 主要与 STAT3 或 USF2 依赖性的 HIF1α 或 HIF2α 相关联在 HIF1 或 HIF2 靶基因上。因此，我们在这里首次证明，HIF 靶基因特异性是通过 HIF 转录伙伴实现的，这些伙伴是 HIF 靶基因激活所必需的，特异性地结合到 HIF1 或 HIF2 靶基因的启动子上，并选择性地与 HIF1α 或 HIF2α 蛋白相互作用。
