Genome-wide mapping of SIN3A binding sites by ChIP-seq in HUVEC exposed to hypoxia

Cells adapt to environmental changes, including fluctuations in oxygen levels, through the induction of specific gene expression programs. To identify genes regulated by hypoxia at the transcriptional level, we pulse-labeled HUVEC cells with 4-thiouridine and sequenced nascent transcripts. Then, we searched genome-wide binding profiles from the ENCODE project for factors that correlated with changes in transcription and identified binding of several components of the Sin3A co-repressor complex, including SIN3A, SAP30 and HDAC1/2, proximal to genes repressed by hypoxia. SIN3A interference revealed that it participates in the downregulation of 75% of the hypoxia-repressed genes in endothelial cells. Unexpectedly, it also blunted the induction of 47% of the upregulated genes, suggesting a role for this corepressor in gene induction. In agreement, ChIP-seq experiments showed that SIN3A preferentially localizes to the promoter region of actively transcribed genes and that SIN3A signal was enriched in hypoxia-repressed genes, prior exposure to the stimulus. Importantly, SINA3 occupancy was not altered by hypoxia in spite of changes in H3K27ac signal. In summary, our results reveal a prominent role for SIN3A in the transcriptional response to hypoxia and suggest a model where modulation of the associated histone deacetylase activity, rather than its recruitment, determines the transcriptional output. ChIP-Seq analysis of SIN3A binding in normoxia and hypoxia in HUVEC cells compared with their input as a control. The data is from a single biological replicate.

细胞可通过激活特定基因表达程序，适应包括氧水平波动在内的环境变化。为鉴定转录层面受低氧（hypoxia）调控的基因，我们以4-硫尿苷（4-thiouridine）对人脐静脉内皮细胞（Human Umbilical Vein Endothelial Cells, HUVEC）进行脉冲标记，并对新生转录本进行测序。随后，我们从ENCODE项目（ENCODE Project）的全基因组结合谱中筛选与转录变化相关的调控因子，并发现低氧抑制基因的邻近区域存在Sin3A共抑制复合物（Sin3A co-repressor complex）的多个组分结合，包括SIN3A、SAP30以及HDAC1/2。通过SIN3A基因干扰实验发现，该因子参与了内皮细胞中75%低氧抑制基因的下调过程。出乎意料的是，它同时削弱了47%上调基因的诱导表达，这表明该共抑制因子在基因诱导过程中也发挥作用。与此一致的是，染色质免疫共沉淀测序（Chromatin Immunoprecipitation sequencing, ChIP-seq）实验结果显示，SIN3A优先定位在活跃转录基因的启动子区域，且在低氧刺激前，低氧抑制基因区域的SIN3A信号即已富集。值得注意的是，尽管H3K27ac信号发生了变化，但低氧并未改变SIN3A的染色质结合占有率。综上，我们的研究结果揭示了SIN3A在低氧应答的转录调控过程中的重要作用，并提出了一个模型：决定转录输出的是相关组蛋白去乙酰化酶活性的调控，而非SIN3A的招募过程。本数据集包含以输入样本为对照的HUVEC细胞在常氧（normoxia）和低氧条件下SIN3A结合的ChIP-seq分析数据，且仅包含一次生物学重复。