Genome wide chromatin profile for Histone H1.4 (pan-H1.4) and Phosphorylated H1.4 (pS187-H1.4)

In this study, we use novel, highly specific custom antibodies against a linker histone variant and its modified form to conduct chromatin immunoprecipitation (ChIP) followed by Next Generation Sequencing (NGS) to investigate their binding profiles as a result of transcription inititation. Here we use an estrogen responsive breast cancer cell line, MCF7 to study the enrichment patterns of Histone H1.4 (pan-H1.4) and Phosphorylated H1.4 (pS187-H1.4) across the genome as a result of a short estrogen treatment (30 mins). We find that pS187-H1.4 is preferentially enriched at the promoters of active genes as opposed to the depletion of pan-H1.4 at the same site. Additionally, we use a short treatment of Flavopiridol- an inhibitory drug specific for Cyclin-dependent Kinase-9 (CDK9) which is responsible for the phosphorylation of H1.4 cells, to demonstrate the quenching of this signal at sites specificially activated as a result of the estrogen treatment. This demonstrates for the first time that pS187-H1.4 is associated with active genes and may even be involved in the regulation of gene activity. Overall design: Examination of Histone H1.4 and pS187H1.4 marks across the genome to evaluate their response to transcription activation

本研究针对一种连接组蛋白变体及其修饰形式，采用新型高特异性定制抗体开展染色质免疫共沉淀 (chromatin immunoprecipitation, ChIP) 实验，随后辅以下一代测序 (Next Generation Sequencing, NGS)，以探究二者在转录起始过程中的结合谱特征。本研究选用雌激素响应性乳腺癌细胞系MCF7，通过30分钟短期雌激素处理，分析组蛋白H1.4 (pan-H1.4)与磷酸化H1.4 (pS187-H1.4)在全基因组范围内的富集模式。研究发现，与泛H1.4在活性基因启动子区域出现的耗竭相反，pS187-H1.4优先富集于该类区域。此外，本研究使用针对细胞周期蛋白依赖性激酶9 (CDK9) 的特异性抑制药物黄酮哌啶醇进行短期处理——该激酶正是介导细胞内H1.4磷酸化的关键酶——证实雌激素处理激活的特定位点处的该信号会被淬灭。本研究首次证实，pS187-H1.4与活性基因存在关联，甚至可能参与基因活性的调控。整体实验设计：检测全基因组范围内的组蛋白H1.4与pS187-H1.4修饰标记，以评估二者对转录激活的响应情况。