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.

在众多关于基因表达或修饰胞嘧啶的全基因组研究中，由于性别偏差，定位在X染色体的探针数据常被从分析中剔除。先前，我们利用全基因组阵列测量修饰胞嘧啶，观察到了高加索人与非洲淋巴母细胞株（LCLs）在常染色体上胞嘧啶修饰的种群差异。DNA甲基化在女性中X染色体的失活建立与维持中起着至关重要的作用。因此，我们推断通过研究X染色体上的胞嘧啶修饰模式，我们可以获得关于在基因组广泛分析中常被忽视的染色体的重要信息。我们研究了高加索人与非洲LCLs在X染色体上的胞嘧啶修饰模式的种群差异，并确定了在女性中X染色体失活（Xi）上逃避甲基化的新位点。通过整合ENCODE生成的广泛的组蛋白修饰ChIP-seq数据，我们表征了这些位点的染色质状态。为了进一步探索DNA与组蛋白修饰之间的关系，我们提出了BRD4蛋白，一种结合乙酰化组蛋白的蛋白，可能阻止某些位点成为去甲基化的假设。为验证这一假设，我们对4个女性LCLs进行了JQ1处理，JQ1是一种BRD4的小分子抑制剂，但发现JQ1处理仅导致了胞嘧啶修饰水平的小幅变化，而大多数在Xi上逃避甲基化的位点仍保持未甲基化状态。这表明，其他表观遗传机制或转录因子可能在保护这些位点免受Xi上的去甲基化方面发挥着更重要的作用。