Epigenetic Pathways in ATRX-Mediated CTCF Regulation

项目摘要 ATRX是一种染色质重构体，在基因调控和基因组稳定性中具有多种重要作用。 ATRX基因突变与神经发育疾病和胶质瘤有关。ATRX调节了几个 每条表观遗传途径都有可能影响关键染色质CTCF的定位 建筑蛋白。ATRX抑制共转录形成的R-环、DNA：RNA杂交结构 与CTCF共定位于基因终止区。ATRX还调节DNA甲基化，这是 能抑制CTCF结合，与DAXX共同沉积异染色组蛋白变异体H3.3， 它与CTCF共同定位于印记基因。我们已经生成了ATRX击倒鼠标 胚胎干细胞(ATRX KD MESCs)，并发现这些细胞中ATRX的缺失改变了CTCF的定位 在全基因组的数百个位置。我们建议检验ATRX损失影响CTCF的假设 通过多条表观遗传途径的失调进行定位。我们将分两个阶段检验这一假设 旨在使用一些全基因组测序方法来揭示表观遗传途径是如何改变的 在ATRX损失之后。在目标1中，我们将研究ATRX缺失对R环形成和基因的影响 表情。这个目标将使用BisMapR，这是一种检测全基因组R-环的新技术，具有高 分辨率和链特异性，以及RNA-Seq来测量转录。在目标2中，我们将阐明 DNA甲基化和组蛋白H3.3在CTCFATRX调控中的作用这一目标将使用亚硫酸氢盐 焦解测序法和增强还原亚硫酸氢盐测序法检测DNA甲基化 ATRX和H3.3基因敲除细胞，切割并运行检测H3.3基因敲除细胞中CTCF的结合。我们 期待这项研究的结果能够阐明ATRX的表观遗传功能是如何对一种新的 在CTCF调节中的作用，以及进一步了解ATRX缺失在疾病中的后果。

Project Summary ATRX is a chromatin remodeler with diverse and essential roles in gene regulation and genomic stability. Mutations in ATRX are associated with neurodevelopmental disease and glioma. ATRX regulates several epigenetic pathways that each have the potential to impact localization of CTCF, a key chromatin architecture protein. ATRX suppresses R-loops, DNA:RNA hybrid structures that form co-transcriptionally and that co-localize with CTCF at gene terminator regions. ATRX also regulates DNA methylation, which can inhibit CTCF binding, and together with DAXX deposits the heterochromatic histone variant H3.3, which co-localizes with CTCF at imprinted genes. We have generated ATRX knockdown mouse embryonic stem cells (ATRX KD mESCs) and found that ATRX loss in these cells alters CTCF localization at hundreds of sites genome-wide. We propose to test the hypothesis that ATRX loss impacts CTCF localization through the dysregulation of multiple epigenetic pathways. We will test this hypothesis in two aims, using a number of genome-wide sequencing methods to reveal how epigenetic pathways are altered upon ATRX loss. In Aim 1, we will examine the consequences of ATRX loss on R-loop formation and gene expression. This aim will use BisMapR, a novel technique that detects genome-wide R-loops with high resolution and strand specificity, and RNA-Seq to measure transcription. In Aim 2, we will elucidate the roles of DNA methylation and histone H3.3 in ATRX regulation of CTCF. This aim will use bisulfite pyrosequencing and enhanced reduced representation bisulfite sequencing to assay DNA methylation in ATRX and H3.3 knockdown cells, and CUT&RUN to detect CTCF binding in H3.3 knockdown cells. We expect the results of this study to illuminate how the epigenetic functions of ATRX contribute to a novel role in CTCF regulation, and further understanding of the consequences of ATRX loss in disease.