ADNP mechanisms in R-loop regulation during differentiation

ADNP 在分化过程中 R 环调节的机制

• 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
• 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

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.

项目摘要 染色质结构中的畸变可以是许多人类疾病和发育的因果机制。 综合征R环是含有染色质结构的RNA，其在许多发育中被失调。 疾病和癌症。在大多数情况下，R-loop的潜在机制和功能意义 放松管制以及它们是否与特定疾病有关尚不清楚。活动依赖性神经保护 蛋白（ADNP）是一个含有转录抑制因子的同源结构域，对于神经元分化至关重要 和神经发育。ADNP突变导致ADNP综合征，这是一种以智力障碍为特征的疾病。 自闭症谱系障碍的残疾和特征。ADNP在神经发育过程中上调。ADNP 含有锌指基序和同源结构域，两者都可以结合核酸。ADNP综合征突变 导致缺乏同源结构域的蛋白质产物，强调了该区域对ADNP的重要性 功能然而，锌指和同源结构域对ADNP定位的精确贡献以及 在神经元分化过程中基因表达的功能还不清楚。我们的初步数据显示 发现了ADNP在R环解析中的新作用。生化分析显示，ADNP解决 R环-以同源结构域依赖的方式。ADNP还抑制体内R环，因为ADNP的缺失 在小鼠胚胎干细胞（mESC）中，导致R环特异性地在ADNP靶位点积累，但 而不是在不被ADNP结合的位点。最后，ADNP缺陷型mESC和仅表达ADNP的mESC 缺乏同源结构域的突变体不能分化为神经祖细胞（NPC），这表明一种重要的 ADNP及其同源结构域在神经元分化中的作用。这些结果表明， 机制-R环解析-为ADNP，并可能其他同源结构域蛋白，在基因 调节，这可以将R环功能障碍与许多由基因突变引起的疾病联系起来。 同源结构域蛋白。基于我们的初步数据，我们假设ADNP驱动神经元， 作为推论，我们提出，解决积累在 ADNP缺失可以挽救基因表达程序，使神经元分化。我们提出两个目标， 测试我们的核心假设在目标1中，我们将阐明ADNP解析R环的分子基础， 鉴定ADNP蛋白结构域的作用及其与核酸的相互作用。在目标2中，我们将阐明 ADNP对R环的抑制如何影响神经元分化。