Copper Regulation of HIF-1 Transcription Activity [ChIP-seq]. Copper Regulation of HIF-1 Transcription Activity [ChIP-seq]

Copper (Cu) regulates hypoxia-inducible factor-1 (HIF-1) transcription activity by affecting the selectivity of HIF-1α targeting to the promoters of the affected genes. Here, we made an effort to provide a comprehensive understanding of Cu regulation of the selectivity of HIF-1α targeting across genome. We used tetraethylenepentamine (TEPA), a Cu selective chelator, to reduce Cu content in the cells. In hypoxia, we conducted chromatin immunoprecipitation combined with massively parallel DNA sequencing (ChIP-seq) to globally map the binding sites of HIF-1α, Pol Ⅱ (RNA polymeraseⅡ) and histone H3K27ac. We also performed RNA-sequencing (RNA-seq) in EA.hy926 cells under hypoxia (1% O2) with or without Cu depression to determine the profile of mRNA expression. Our analyses identified 3197 HIF-1α binding sites under hypoxia. Cu depression by TEPA reduced 1820 binding sites from the 3197, but induced additional 274 new binding sites. We analyzed these binding sites in the promoter and putative enhancer regions, coupled with their mRNA expression profiles, and found 281 Cu-dependent and 10 Cu-independent HIF-1α target genes. We found that the core bases “GGAA” and “TTCC” constituted the critical motifs for the binding sites of Cu-dependent genes. This study thus revealed that Cu, by affecting the binding of HIF-1α to the critical motifs in the promoter and putative enhancer regions of HIF-1 regulated genes, leads to the selectivity of HIF-1 regulated expression of Cu-dependent genes. Overall design: Analysis of DNA-binding profiling of HIF-1α, Pol Ⅱ and H3K27ac in TEPA treated and -untreated cells. .

铜（Cu）通过影响缺氧诱导因子-1α（HIF-1α）靶向受调控基因启动子的选择性，调控缺氧诱导因子-1（HIF-1）的转录活性。本研究旨在全面解析铜对全基因组范围内HIF-1α靶向选择性的调控机制。我们采用铜选择性螯合剂四乙烯五胺（tetraethylenepentamine, TEPA）降低细胞内铜含量。在缺氧条件下，我们通过染色质免疫共沉淀结合高通量测序（chromatin immunoprecipitation combined with massively parallel DNA sequencing, ChIP-seq），对HIF-1α、RNA聚合酶Ⅱ（RNA polymerase Ⅱ, Pol Ⅱ）以及组蛋白H3K27ac（histone H3K27ac）的结合位点进行全基因组定位。同时，我们在缺氧（1% O₂）条件下的EA.hy926细胞中开展了铜含量敲低与未敲低处理的RNA测序（RNA-sequencing, RNA-seq），以获取mRNA表达谱。数据分析显示，缺氧条件下共鉴定得到3197个HIF-1α结合位点。经TEPA处理实现铜含量敲低后，3197个结合位点中有1820个的结合信号显著减弱，同时新增274个结合位点。我们对这些位于启动子及推定增强子区域的结合位点及其对应的mRNA表达谱进行联合分析，最终鉴定得到281个铜依赖型及10个铜非依赖型HIF-1α靶基因。进一步分析发现，铜依赖型基因的HIF-1α结合位点的核心碱基序列为"GGAA"与"TTCC"。本研究揭示，铜通过影响HIF-1α与HIF-1调控基因启动子及推定增强子区域内关键基序的结合，实现了对铜依赖型基因HIF-1调控表达选择性的调控。整体实验设计：对经TEPA处理与未处理的细胞中HIF-1α、Pol Ⅱ及H3K27ac的DNA结合谱进行分析。