Epigenetic mechanisms regulating neuron production during cortical development

皮质发育过程中调节神经元产生的表观遗传机制

• 批准号: 10683534
• 负责人: Louis-Jan Pilaz
• 金额: $ 32.48万
• 依托单位: SANFORD RESEARCH/USD
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2023-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10683534
• 关键词: 19p13; Affect; Architecture; Biological Assay; Brain region; Candidate Disease Gene; Cerebral cortex; ChIP-seq; Chromatin; Complex; Coupled; Development; Differentiated Gene; Disease; Embryonic Development; Epigenetic Process; Equilibrium; Gene Expression; Generations; Genes; Genetic; Human; Intellectual functioning disability; Lead; Luciferases; Macrocephaly; Mediating; Microcephaly; Mission; Molecular; Mus; Neurodevelopmental Disorder; Neurons; Nucleic Acid Regulatory Sequences; Pediatric Research; Pediatrics; Phenotype; Process; Production; Proteins; Rare Diseases; Reading; Regulator Genes; Research; Role; Syndrome; System; autism spectrum disorder; candidate validation; cognitive function; experimental study; insight; mouse model; nerve stem cell; novel; outcome prediction; recruit; transcription factor; transcriptome sequencing

The cerebral cortex is the brain region underlying human higher cognitive functions, such as complex decisionmaking, reading and reasoning. These functions rely on networks of neurons mainly generated during embryonic development. During that developmental period, the balance between proliferation and differentiation of neural precursors (NPs) is critical for the generation of appropriate numbers of neurons. Therefore, disruptions of NP proliferation are at the origin of numerous neurodevelopmental disorders, such intellectual disabilities, autism, microcephaly and macrocephaly. Numerous cellular and molecular processes regulate proliferation vs differentiation decisions in NPs. In the present study, we wiPll focus on epigenetic mechanisms modulating the expression of proliferation and differentiation genes. To do this, we will focus on the transcription factor ZBTB7A, known to mediate chromatin accessibility in the regulatory regions of genes implicated in proliferation and differentiation. ZBTB7A has been implicated in many different systems, but its role in NPs during cortical development is completely unknown. However, the ZBTB7A gene is located in the 19p13.3 microlocus containing 3 genes, and whose duplication or deletion lead to microcephaly and macrocephaly, respectively. Our preliminary studies in the mouse show that altered expression of ZBTB7A in NPs leads to proliferation deficits with predicted outcomes matching those observed in the 19p13.3 syndrome. Altogether these findings make ZBTB7A an outstanding candidate to discover novel epigenetic mechanisms regulating the development of the cerebral cortex. In this study we will use mouse models mimicking ZBTB7A alterations in humans to characterize how altered expression of ZBTB7A impacts NP proliferation and the establishment of cortical architecture. In a second step, we will characterize ZBTB7A target genes in NPs using ChIP-seq coupled with RNA-seq. After validation of candidate genes using luciferase assays, we will attempt genetic rescue experiments to re-establish the phenotypes caused by altered ZBTB7A levels. In a third step we will further dissect the molecular mechanisms by which ZBTB7A regulates gene expression in NPs, focusing on ZBTB7A co-factors. To do this, we will use BioID to identify proteins operating in the vicinity of ZBTB7A in cortical NPs and we will use luciferase, ChIP-PCR and co-IP assays to understand the mechanism by which ZBTB7A can affect the recruitment of those co-factors to gene regulatory region, and thus impact gene expression. This project will advance the mission of the Pediatrics and Rare Diseases group at Sanford Research, while providing new insights into the epigenetic mechanisms regulating the development of the cerebral cortex, and how disruption of these mechanisms can lead to neuropediatric diseases.

大脑皮层是人类更高认知功能的脑部区域，例如复杂的决策， 阅读和推理。这些功能依赖于主要在胚胎期间生成的神经元网络 发展。在那个发育时期，增殖与神经分化之间的平衡 前体（NP）对于生成适当数量的神经元至关重要。因此，NP的破坏 扩散是许多神经发育障碍的起源，此类智力残疾，自闭症， 小头畸形和脑畸形。许多细胞和分子过程调节增殖与分化 NPS的决定。在本研究中，我们专注于调制表达的表观遗传机制 增殖和分化基因。为此，我们将重点关注转录因子ZBTB7A，已知 介导与增殖和分化有关的基因调节区域中的染色质可及性。 ZBTB7A已与许多不同的系统有关，但其在皮质发育过程中的NP中的作用完全是 未知。但是，ZBTB7A基因位于包含3个基因的19p13.3微孔中，其 重复或缺失分别导致小头畸形和大脑畸形。我们在小鼠中的初步研究 表明NPS中ZBTB7A的表达改变会导致增殖缺陷，预测结果匹配 那些在19p13.3综合征中观察到的。这些发现完全使ZBTB7A成为 发现调节大脑皮层发育的新型表观遗传机制。在这项研究中，我们将使用 鼠标模仿人类中ZBTB7A改变的变化，以表征ZBTB7A的表达如何影响 NP扩散和皮质建筑的建立。在第二步中，我们将表征ZBTB7A目标 使用芯片序列与RNA-seq结合的NP中的基因。使用荧光素酶测定验证候选基因后，我们 将尝试尝试遗传救援实验，以重新建立由ZBTB7A水平改变引起的表型。三分之一 步骤我们将进一步剖析ZBTB7A调节NP中基因表达的分子机制，聚焦 在ZBTB7A的联合因素上。为此，我们将使用生物剂来识别在ZBTB7A附近运行的蛋白质 皮质NP，我们将使用荧光素酶，ChIP-PCR和Co-IP分析来了解ZBTB7A的机制 可以影响这些共同因素募集到基因调节区域，从而影响基因表达。这个项目 将推进Sanford Research的儿科和稀有疾病小组的任务，同时提供新的见解 进入调节大脑皮层发展的表观遗传机制，以及这些机制的破坏程度如何 机制可以导致神经病史疾病。