Won Kyoung-Jae, Choi Inchan, LeRoy Gary, Zee Barry M, Sidoli Simone, Gonzales-Cope Michelle, Garcia Benjamin A
The Institute for Diabetes, Obesity, and Metabolism, Philadelphia, PA 19104 USA.
Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104 USA.
Epigenetics Chromatin. 2015 Apr 10;8:13. doi: 10.1186/s13072-015-0005-9. eCollection 2015.
Histone variants play further important roles in DNA packaging and controlling gene expression. However, our understanding about their composition and their functions is limited.
Integrating proteomic and genomic approaches, we performed a comprehensive analysis of the epigenetic landscapes containing the four histone variants H3.1, H3.3, H2A.Z, and macroH2A. These histones were FLAG-tagged in HeLa cells and purified using chromatin immunoprecipitation (ChIP). By adopting ChIP followed by mass spectrometry (ChIP-MS), we quantified histone post-translational modifications (PTMs) and histone variant nucleosomal ratios in highly purified mononucleosomes. Subsequent ChIP followed by next-generation sequencing (ChIP-seq) was used to map the genome-wide localization of the analyzed histone variants and define their chromatin domains. Finally, we included in our study large datasets contained in the ENCODE database. We newly identified a group of regulatory regions enriched in H3.1 and the histone variant associated with repressive marks macroH2A. Systematic analysis identified both symmetric and asymmetric patterns of histone variant occupancies at intergenic regulatory regions. Strikingly, these directional patterns were associated with RNA polymerase II (PolII). These asymmetric patterns correlated with the enhancer activities measured using global run-on sequencing (GRO-seq) data.
Our studies show that H2A.Z and H3.3 delineate the orientation of transcription at enhancers as observed at promoters. We also showed that enhancers with skewed histone variant patterns well facilitate enhancer activity. Collectively, our study indicates that histone variants are deposited at regulatory regions to assist gene regulation.
组蛋白变体在DNA包装和基因表达调控中发挥着更重要的作用。然而，我们对它们的组成和功能的了解有限。
整合蛋白质组学和基因组学方法，我们对包含四种组蛋白变体H3.1、H3.3、H2A.Z和macroH2A的表观遗传景观进行了全面分析。这些组蛋白在HeLa细胞中用FLAG标签标记，并使用染色质免疫沉淀（ChIP）进行纯化。通过采用ChIP后接质谱分析（ChIP-MS），我们对高度纯化的单核小体中的组蛋白翻译后修饰（PTM）和组蛋白变体核小体比率进行了定量。随后的ChIP后接下一代测序（ChIP-seq）用于绘制分析的组蛋白变体在全基因组的定位，并定义它们的染色质结构域。最后，我们在研究中纳入了ENCODE数据库中的大型数据集。我们新鉴定出一组富含H3.1和与抑制性标记macroH2A相关的组蛋白变体的调控区域。系统分析确定了基因间调控区域组蛋白变体占据的对称和不对称模式。引人注目的是，这些方向性模式与RNA聚合酶II（PolII）相关。这些不对称模式与使用全局运行测序（GRO-seq）数据测量的增强子活性相关。
我们的研究表明，H2A.Z和H3.3在增强子处划定了转录方向，正如在启动子处观察到的那样。我们还表明，具有偏斜组蛋白变体模式的增强子很好地促进了增强子活性。总体而言，我们的研究表明组蛋白变体沉积在调控区域以协助基因调控。
