Genome-wide HIF-1 binding sites in HepG2 cells

Adaptation to hypoxia is mediated through a coordinated transcriptional response driven largely by Hypoxia-Inducible Factor 1 (HIF-1). The direct transcriptional targets of HIF-1 play important roles in facilitating both short-term and long-term adaptation to hypoxia. Alignment of the sequences encompassing all well-characterized HIF-1 binding sites has revealed a consensus core HRE motif of 5'-RCGTG-3' (R = A or G). Since the consensus HIF-1 binding motif is too promiscuous to accurately predict binding a priori, we used ChIP-chip to define HIF-1 chromatin binding on a genome-wide level. We integrated these results with gene expression profiling to interrogate mechanisms regulating hypoxia-induced gene expression, and to more comprehensively identify direct targets of HIF-1 transactivation. HepG2 cells were cultured under normoxic (ambient) or hypoxic (0.5%O2, 4 h) conditions. HIF-1 ChIP was performed with a HIF-1 polyclonal Ab. Triplicate ChIPed DNA and inputs were amplified and hybridized onto the Affymetrix Gene-Chip Human Tiling 2.0R Array Set. The MAT algorithm was used to identify peaks of probe intensity (‘‘hits’’).

细胞的低氧适应主要通过以缺氧诱导因子1（Hypoxia-Inducible Factor 1, HIF-1）为核心驱动的协同转录应答实现。HIF-1的直接转录靶标在介导细胞短期与长期低氧适应过程中均发挥重要作用。对所有已被充分表征的HIF-1结合位点所在序列进行比对分析后，可得到其共有核心缺氧反应元件（Hypoxia Response Element, HRE）基序：5'-RCGTG-3'（其中R代表腺嘌呤A或鸟嘌呤G）。由于该共有HIF-1结合基序的特异性过于宽泛，无法准确预先预测结合事件，因此我们采用染色质免疫沉淀芯片（Chromatin Immunoprecipitation-on-chip, ChIP-chip）技术，在全基因组范围内鉴定HIF-1的染色质结合位点。我们将上述实验结果与基因表达谱数据整合，以解析低氧诱导基因表达的调控机制，并更全面地鉴定HIF-1转录激活的直接靶标。本实验采用HepG2细胞，分别在常氧（环境空气）或低氧（体积分数0.5% O₂，培养4小时）条件下培养。采用HIF-1多克隆抗体完成HIF-1染色质免疫沉淀实验。将三份重复的ChIP富集DNA及输入对照DNA进行扩增后，与Affymetrix GeneChip人类平铺探针2.0R芯片组进行杂交。采用MAT算法识别探针信号强度的峰值（即“命中位点”）。