ADNP mechanisms in R-loop regulation during differentiation

ADNP 在分化过程中 R 环调节的机制

基本信息

批准号：
10444141
负责人：
Kavitha Sarma
金额：
$ 43.16万
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：
2022-09-06 至 2026-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10444141
关键词：
Binding Biochemical Biological Assay Chromatin Structure Data Defect Development Disease ES Cell Line Ensure Epitopes Functional disorder Gene Expression Gene Expression Regulation Genome Homeodomain Proteins Impairment In Vitro Intellectual functioning disability Link Malignant Neoplasms Molecular Mouse Protein Mus Mutate Mutation Neuronal Differentiation Neurons Nucleic Acid Binding Nucleic Acids Proteins RNA Reagent Regulation Resolution Ribonuclease H Role Site Syndrome Tertiary Protein Structure Testing Transcription Repressor Zinc Fingers attenuation autism spectrum disorder base developmental disease disease-causing mutation embryonic stem cell gene repression genome-wide homeodomain human disease in vivo in vivo evaluation insight mutant nerve stem cell neurodevelopment novel nucleic acid structure prevent programs protein distribution protein function

项目摘要

Project Summary Aberrations in chromatin structure can be a causal mechanism of numerous human disease and developmental syndromes. R-loops are RNA containing chromatin structures that are deregulated in numerous developmental disorders and cancers. In most cases the underlying mechanisms and the functional significance of R-loop deregulation and whether they are causal to specific disease is not clear. Activity dependent neuroprotective protein (ADNP) is a homeodomain containing transcriptional repressor that is critical for neuronal differentiation and neurodevelopment. ADNP mutations cause ADNP syndrome, a condition characterized by intellectual disability and features of autism spectrum disorder. ADNP is upregulated during neurodevelopment. ADNP contains zinc finger motifs and a homeodomain, both of which can bind nucleic acids. ADNP syndrome mutations result in protein products that lack the homeodomain, underscoring the importance of this region to ADNP function. However, the precise contributions of the zinc fingers and homeodomain to ADNP localization and function in gene expression during neuronal differentiation is not well understood. Our preliminary data have uncovered a novel role for ADNP in the resolution of R-loops. Biochemical assays revealed that ADNP resolves R-loops – in a homeodomain dependent manner. ADNP also suppresses R-loops in vivo, as deletion of ADNP in mouse embryonic stem cells (mESCs) resulted in R-loop accumulation specifically at ADNP target sites, but not at sites not bound by ADNP. Last, ADNP-deficient mESCs and mESCs exclusively expressing an ADNP mutant lacking the homeodomain fail to differentiate into neural progenitor cells (NPCs), indicating an essential role for ADNP and its homeodomain in neuronal differentiation. These results suggest a potentially novel mechanism – R-loop resolution – for ADNP, and possibly other homeodomain-containing proteins, in gene regulation, which can link R-loop dysfunction to numerous disorders and diseases caused by mutations in homeodomain proteins. Based on our preliminary data, we hypothesize that ADNP drives neuronal differentiation by suppressing R-loops; as a corollary, we propose that resolving R-loops that accumulate upon ADNP loss may rescue gene expression programs to enable neuronal differentiation. We propose two aims to test our central hypothesis. In Aim 1, we will elucidate the molecular basis of R-loop resolution by ADNP by identifying roles of the ADNP protein domains and their interactions with nucleic acids. In Aim 2, we will elucidate how ADNP suppression of R-loops influences neuronal differentiation.

项目概要染色质结构的畸变可能是许多人类疾病和发育的致病机制 R 环是含有染色质结构的 RNA，在许多发育过程中失调。在大多数情况下，R 环的潜在机制和功能意义。放松管制以及它们是否导致特定疾病尚不清楚。蛋白（ADNP）是一种含有转录抑制因子的同源结构域，对神经元分化至关重要 ADNP 突变会导致 ADNP 综合征，这是一种以智力为特征的疾病。 ADNP 在神经发育过程中上调。包含锌指基序和同源结构域，两者都可以结合 ADNP 综合征突变。导致蛋白质产物缺乏同源结构域，强调了该区域对 ADNP 的重要性然而，锌指和同源域对 ADNP 定位的精确贡献和我们的初步数据还不清楚神经元分化过程中基因表达的功能。发现了 ADNP 在 R 环解析中的新作用生化测定表明 ADNP 可以解析。 R 环 – ADNP 也以同源结构域依赖性方式抑制体内 R 环，如 ADNP 缺失。小鼠胚胎干细胞 (mESC) 中导致 R 环专门在 ADNP 靶位点积累，但是最后，ADNP 缺陷型 mESC 和仅表达 ADNP 的 mESC 不存在。缺乏同源结构域的突变体无法分化为神经祖细胞（NPC），这表明 ADNP 及其同源结构域在神经元分化中的作用表明了一种潜在的新颖性。机制 – R 环解析 – 用于基因中的 ADNP 和可能的其他含有同源结构域的蛋白质调节，它可以将 R 环功能障碍与多种由 R 环突变引起的紊乱和疾病联系起来。根据我们的初步数据，我们发现 ADNP 驱动神经元。通过抑制 R 环来求导；作为推论，我们建议解决累积的 R 环 ADNP 丢失可能会拯救基因表达程序以实现神经元分化。在目标 1 中，我们将通过 ADNP 阐明 R 环解析的分子基础。在目标 2 中，我们将阐明 ADNP 蛋白结构域的作用及其与核酸的相互作用。 ADNP 对 R 环的抑制如何影响神经元分化。