Epigenetic Pathways in ATRX-Mediated CTCF Regulation

ATRX 介导的 CTCF 调节中的表观遗传途径

• 批准号: 10314271
• 负责人: Phillip Wulfridge
• 金额: $ 6.64万
• 依托单位: WISTAR INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-01 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10314271
• 关键词: Epigenetic; Pathways; ATRX; Mediated; CTCF

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.

项目摘要 ATRX是一种染色质重塑剂，在基因调控和基因组稳定性中具有多种重要作用。 ATRX突变与神经发育疾病和胶质瘤相关。ATRX调节几个 表观遗传途径，每个都有可能影响CTCF，一个关键的染色质定位 结构蛋白ATRX抑制R环，DNA：RNA杂合结构， 并且与CTCF共定位于基因终止子区域。ATRX还调节DNA甲基化， 能抑制CTCF结合，并与DAXX一起沉积异染色质组蛋白变体H3.3， 与CTCF共定位于印记基因。我们已经产生了ATRX敲低小鼠 胚胎干细胞（ATRX KD mESCs），并发现这些细胞中ATRX的丢失改变了CTCF的定位 在全基因组范围内的数百个位点。我们建议测试ATRX损耗影响CTCF的假设 通过多种表观遗传途径的失调定位。我们将分两部分来检验这个假设 目的是利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丢失时。在目标1中，我们将研究ATRX丢失对R环形成和基因表达的影响。 表情这一目标将使用BisMapR，这是一种新的技术，可以检测基因组范围内的R环， 分辨率和链特异性，以及用于测量转录的RNA-Seq。在目标2中，我们将阐明 DNA甲基化和组蛋白H3.3在ATRX调节CTCF中的作用。该目标将使用亚硫酸氢盐 焦磷酸测序和增强的还原代表性亚硫酸氢盐测序来测定 ATRX和H3.3敲低细胞，以及CUT&RUN以检测H3.3敲低细胞中的CTCF结合。我们 我希望这项研究的结果能够阐明ATRX的表观遗传功能如何有助于一种新的 在CTCF调节中的作用，并进一步了解ATRX损失在疾病中的后果。