Investigating the role of THAP transcription factor mediated regulation of cell proliferation in vertebrate development and neurodevelopmental disease

项目总结/摘要 尽管智力残疾（ID;智商< 70）的患病率很高（~2%）和社会经济成本，但 大多数病例的潜在遗传原因尚不清楚。这项计划的目的是研究新的遗传 许多脊椎动物THAP结构域含有转录因子（TF）调节神经发育。 THAP 11突变与发育迟缓和癫痫相关，THAP 1突变导致6型 肌张力障碍THAP 1/11通过募集它们的辅因子来调节基因转录， 宿主细胞因子1（HCFC 1），虽然确切的机制一直具有挑战性，以确定，因为完整的 任何一种基因的缺失都会导致小鼠胚胎死亡。另一个THAP基因THAP 7的突变与ID、癫痫、 和颅面缺损3个家系，提示THAP 7是一个新的ID基因。THAP 1/7/11的能力 调节细胞增殖的蛋白质可以解释它们在神经发育中的作用。神经祖细胞（NPC） 发育中的皮质平衡增殖和分化，以确保皮质层正常形成; 导致大脑大小改变和认知障碍。THAP 1/11通过自身束缚HCFC 1调节增殖 与ID有关的基因与细胞周期基因相关，导致染色质修饰剂的募集。THAP 7丢失不会阻止 细胞生长，表明Thap 7-/-小鼠可以存活。本研究的长期目标是调查 THAP TF在正常神经发育和ID中的作用，使用THAP 7作为模型。THAP 7如何调节增殖 仍然未知。虽然THAP 7可能作为TF并结合到一些细胞周期基因的启动子，但THAP 7可能是一种转录因子。 THAP 7在神经发育过程中作为TF的功能程度尚未测试。拟议的研究将测试 以下假设1）THAP 7通过调节皮质NPC增殖促进神经发育，和 THAP 7的突变或缺失导致异常的皮质发育，以及2）THAP 7完成 这是通过募集HCFC 1来调节细胞周期基因的表达。本提案的具体目标 如下：研究THAP调节神经母细胞瘤细胞增殖的能力，并在神经母细胞瘤细胞中充当TF， 使用功能基因组学（Aim 1）研究THAP 7的缺失、过表达或突变，并评估THAP 7的功能。 通过产生Thap 7 KO小鼠（CRISPR）和通过经由患者Thap 7对人ID进行建模， 敲入（KI）小鼠。将评估小鼠的神经发育缺陷，包括皮质NPC组成。 通过Thap 7 KO/KI的功能基因组学分析来确定THAP 7在NPC中充当TF的能力 来源于体外分化的小鼠胚胎干细胞的NPC。拟议的研究将揭示 THAP 7的分子和生物学功能，并提供有关THAP蛋白如何广泛贡献的见解 拟议的研究与培训计划相结合，其中包括规定， 提高PI的沟通、管理和领导技能，并利用丰富的环境 由NICHD提供，将确保PI充分准备过渡到独立的位置。

Project Summary/Abstract Despite the high prevalence rate (~2%) and socio-economic cost of intellectual disability (ID; IQ < 70), the underlying genetic cause of most cases is unknown. The objective of this proposal is to investigate novel genetic causes of ID. Many vertebrate THAP-domain containing transcription factors (TF) regulate neurodevelopment. THAP11 mutation is associated with developmental delay and epilepsy, and mutations in THAP1 cause type 6 dystonias. THAP1/11 contribute to these disorders by regulating gene transcription by recruiting their cofactor, host cell factor 1 (HCFC1), though the exact mechanism has been challenging to determine because complete loss of either is embryonic lethal in mice. Mutations in another THAP gene, THAP7, segregate with ID, epilepsy, and craniofacial defects in three families, suggesting THAP7 is a novel ID gene. The ability of THAP1/7/11 proteins to regulate cell proliferation may explain their role in neurodevelopment. Neural progenitors (NPCs) of the developing cortex balance proliferation and differentiation to ensure cortical layers form properly; defects lead to altered brain size and cognitive impairment. THAP1/11 regulate proliferation by tethering HCFC1, itself implicated in ID, to cell cycle genes, leading to recruitment of chromatin modifiers. THAP7 loss does not arrest cell growth, suggesting Thap7-/- mice may be viable. The long-term goal of this research is to investigate the role of THAP TFs in normal neurodevelopment and ID using THAP7 as a model. How THAP7 regulates proliferation remains unknown. While THAP7 likely acts as a TF and binds to the promoters of a few cell cycle genes, the extent of THAP7’s function as a TF during neurodevelopment is untested. The proposed research will test the following hypotheses 1) THAP7 promotes neurodevelopment by regulating cortical NPC proliferation, and that mutation or loss of THAP7 leads to aberrant cortical development and 2) that THAP7 accomplishes this by regulating expression of cell cycle genes via recruitment of HCFC1. Specific aims of this proposal are as follows: investigate THAP’s ability to regulate proliferation of and act as a TF in neuroblastoma cells upon loss, overexpression, or mutation of THAP7 using functional genomics (Aim 1) and to assess the function of THAP7 in vivo (Aim 2) by generating Thap7 KO mice (CRISPR) and by modeling human ID via patient Thap7 knock-in (KI) mice. Mice will be assessed for neurodevelopmental defects, including cortical NPC composition. The ability of THAP7 to act as a TF in NPCs will be determined via functional genomics analysis of Thap7 KO/KI NPCs derived from in vitro differentiated mouse embryonic stem cells. The proposed research will reveal the molecular and biological function of THAP7, and provide insight into how THAP proteins broadly contribute to neurodevelopment and ID. The proposed research combined with the training plan, which includes provisions to improve the PIs communication, grantsmanship, and leadership skills and leverages the rich environment provided by NICHD, will ensure that the PI is fully prepared to transition into an independent position.