Characterization of CpG sites that escape methylation on the inactive human X-chromosome

In many whole genome studies of gene expression or modified cytosines, data from probes localized to the X-chromosome are removed from analyses due to gender bias. Previously, we observed population differences in cytosine modifications between Caucasian and African lymphoblastoid cell lines (LCLs) on the autosomes using whole genome arrays to measure modified cytosines. DNA methylation plays a critical role in establishment and maintenance of X-chromosome inactivation in females. Therefore, we reasoned that by investigating cytosine modification patterns specifically on the X-chromosome, we could obtain valuable information about a chromosome that is often disregarded in genome-wide analyses. We investigated population differences in cytosine modification patterns along the X-chromosome between Caucasian and African LCLs and identified novel sites that escape methylation on the inactive X-chromosome (Xi) in females. We characterized the chromatin state of these loci by incorporating the extensive histone modification ChIP-seq data generated by ENCODE. To explore the relationship between DNA and histone modifications further, we hypothesized that BRD4, a protein that binds acetylated histones, could be preventing some sites from becoming de novo methylated. To test this, we treated 4 female LCLs with JQ1, a small molecule inhibitor of BRD4, but found that JQ1 treatment induced minor changes in cytosine modification levels, and the majority of sites escaping methylation on the Xi remained unmethylated. This suggests that other epigenetic mechanisms or transcription factors are likely playing a larger role in protecting these sites from de novo methylation on the Xi.在诸多针对基因表达或胞嘧啶修饰的全基因组研究中，由于存在性别偏倚，定位于X染色体的探针所产生的数据通常会被从分析流程中剔除。此前，我们借助全基因组芯片对胞嘧啶修饰进行检测，在常染色体层面观察到高加索人群与非洲人群的淋巴母细胞系（LCLs）之间存在胞嘧啶修饰的群体差异。DNA甲基化（DNA methylation）在雌性个体X染色体失活的建立与维持过程中发挥关键作用。因此我们推断，通过专门针对X染色体开展胞嘧啶修饰模式的研究，我们能够获取这类在全基因组分析中常被忽视的染色体的宝贵信息。我们对高加索与非洲人群来源的LCLs中X染色体的胞嘧啶修饰模式进行了群体差异分析，并鉴定出雌性个体中在失活X染色体（Xi）上免于甲基化的全新位点。我们通过整合ENCODE项目产生的海量组蛋白修饰染色质免疫共沉淀测序（ChIP-seq）数据，对这些位点的染色质状态进行了表征。为进一步探究DNA修饰与组蛋白修饰之间的关联，我们提出假说：结合乙酰化组蛋白的溴结构域蛋白4（BRD4）可阻止部分位点发生从头甲基化。为验证该假说，我们使用BRD4的小分子抑制剂JQ1处理了4株雌性LCLs，但结果显示JQ1处理仅对胞嘧啶修饰水平产生了微小影响，绝大多数在Xi上免于甲基化的位点仍维持未甲基化状态。这表明其他表观遗传机制或转录因子或许在保护Xi上的这些位点免于从头甲基化的过程中发挥了更核心的作用。