CTCF-dependent mechanisms of ATRX in neuronal differentiation

ATRX 在神经元分化中的 CTCF 依赖性机制

基本信息

批准号：
10625522
负责人：
Kavitha Sarma
金额：
$ 53.06万
依托单位：
WISTAR INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-01 至 2027-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10625522
关键词：
3-Dimensional ATRX gene Affect Alleles Architecture Behavior Binding Binding Sites Biochemical Brain CRISPR/Cas technology Cell Differentiation process Cells Chromatin Complex DNA Methylation Data Defect Deposition Development Disease Engineering Epigenetic Process Exhibits Gene Expression Genes Genital Genitalia Genome Genomic Segment Genomics Genotype Histones Impairment Lead Link Maintenance Malignant Neoplasms Masks Mediating Molecular Mus Mutate Mutation Neurodevelopmental Disorder Neurologic Neuronal Differentiation Oncogene Deregulation Outcome PHD Finger Patients Pattern Phenotype Polycomb Positioning Attribute Process Proteins Psychomotor Impairments Reagent Regulation Role Severity of illness Site Syndrome Testing Variant chromatin remodeling embryonic stem cell epigenetic silencing genome wide methylation genome-wide helicase imprint knock-down mutant nerve stem cell neural novel prevent programs severe intellectual disability

项目摘要

PROJECT SUMMARY ATRX is an epigenetic factor that is mutated in ATRX syndrome. ATRX is required for the maintenance of multipotent neuroprogenitor cells (NPCs), particularly as these cells initiate differentiation programs in the brain. Mutations that cause ATRX syndrome cluster within two domains: the ATRX PHD finger domain, and the helicase domain. These mediate interactions with histones, and remodel chromatin, respectively. Interestingly, mutations in the PHD finger are associated with severe intellectual disability and psychomotor impairment, while mutations in the helicase domain often manifest with milder neurodevelopmental delays but more severe genital abnormalities13. The basis for this genotype-phenotype correlation has never been investigated. ATRX has well- established roles in molecular processes that are crucial for normal brain development including histone variant H3.3 deposition and Polycomb repressive complex 2 targeting for epigenetic silencing. It also has a poorly understood role in regulating CTCF, a critical genome architectural protein that is essential for development. Our preliminary data indicate PHD finger and helicase mutations of ATRX differentially regulate CTCF localization and may underlie genotype-phenotype correlations. Our preliminary data has uncovered a genome-wide role for ATRX in CTCF localization. We found that ATRX knock down (KD) in mouse embryonic stem cells (mESCs) results in CTCF accumulation at many genomic sites, including both imprinted and non-imprinted loci. We also discovered that ATRX interacts with ADNP and DNMT3L, both of which can prevent CTCF binding. Using CRISPR/Cas9, we have generated isogenic ESCs where the endogenous Atrx allele has been replaced by point mutants found in ATRX syndrome patients. We show that mutations in the PHD finger cause significant impairment of NPC differentiation, while mutations in the helicase domain cause more subtle differentiation delays. Our preliminary findings and novel reagents uniquely position us to interrogate the consequence of ATRX mutations, both for genome organization through CTCF, and for the ability of ESCs to differentiate into NPCs. Based on our preliminary data, we propose to test the hypothesis that distinct ATRX domains regulate specific chromatin processes that impinge to different extents on the function of CTCF. In Aim 1, we will investigate the consequence of ATRX syndrome mutations to gene expression and genome organization during neuronal differentiation. In Aim 2, we will decipher the mechanisms by which ATRX regulates CTCF localization.

项目摘要 ATRX是ATRX综合征突变的表观遗传因子。维护需要ATRX 多能神经元基因细胞（NPC），特别是当这些细胞启动大脑中的分化程序时。引起ATRX综合征簇的突变在两个域中：ATRX PHD手指域和解旋酶结构域。这些分别介导与组蛋白的相互作用和重塑染色质。有趣的是，博士学位的突变与严重的智力残疾和精神病障碍有关，而解旋酶结构域中的突变通常表现出温和的神经发育延迟，但更严重的生殖器异常13。从未研究过这种基因型 - 表型相关性的基础。 ATRX有很好在包括组蛋白变异的正常脑发育至关重要的分子过程中已建立的作用 H3.3沉积和Polycomb抑制复合物2靶向表观遗传沉默。它也很差理解在调节CTCF方面的作用，CTCF是一种对开发至关重要的关键基因组结构蛋白。我们的初步数据表明ATRX的PhD手指和解旋酶突变差异调节CTCF定位并可能是基因型 - 表型相关性的基础。我们的初步数据已经发现了全基因组的作用 CTCF本地化中的ATRX。我们发现ATRX在小鼠胚胎干细胞（MESC）中撞倒（KD）导致CTCF在许多基因组部位的积累，包括印迹和非印刷基因座。我们也是发现ATRX与ADNP和DNMT3L相互作用，这两者都可以防止CTCF结合。使用 CRISPR/cas9，我们已经产生了等源性ESC，内源性ATRX等位基因已被点取代在ATRX综合征患者中发现的突变体。我们表明，博士手指中的突变会引起显着 NPC分化的损害，而解旋酶结构域中的突变会导致更细微的分化延迟。我们的初步发现和新颖试剂独特地定位了我们询问ATRX的后果突变，包括通过CTCF进行基因组组织，以及ESC分化为NPC的能力。根据我们的初步数据，我们建议测试以下假设，即不同的ATRX域调节特定染色质过程会影响CTCF功能的不同范围。在AIM 1中，我们将调查在神经元期间，ATRX综合征突变对基因表达和基因组组织的结果分化。在AIM 2中，我们将破译ATRX调节CTCF定位的机制。