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.

大脑皮层是人类高级认知功能的大脑区域，例如复杂的决策、 阅读和推理。这些功能依赖于主要在胚胎时期产生的神经元网络 发展。在此发育时期，神经细胞增殖和分化之间的平衡 前体细胞（NP）对于产生适当数量的神经元至关重要。因此，NP的破坏 增殖是许多神经发育障碍的根源，例如智力障碍、自闭症、 小头畸形和大头畸形。许多细胞和分子过程调节增殖与分化 NP 中的决策。在本研究中，我们将重点关注调节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，众所周知 介导与增殖和分化相关的基因调控区域的染色质可及性。 ZBTB7A 与许多不同的系统有关，但它在皮质发育过程中 NP 中的作用完全是 未知。然而，ZBTB7A基因位于包含3个基因的19p13.3微位点，其 重复或缺失分别导致小头畸形和大头畸形。我们在小鼠中的初步研究 显示 NP 中 ZBTB7A 表达的改变会导致增殖缺陷，与预测的结果相匹配 那些在 19p13.3 综合征中观察到的情况。总而言之，这些发现使 ZBTB7A 成为杰出的候选者 发现调节大脑皮层发育的新表观遗传机制。在本研究中我们将使用 模拟人类 ZBTB7A 改变的小鼠模型，以表征 ZBTB7A 表达改变的影响 NP 增殖和皮质结构的建立。第二步，我们将描述 ZBTB7A 目标的特征 使用 ChIP-seq 与 RNA-seq 结合分析 NP 中的基因。使用荧光素酶测定验证候选基因后，我们 将尝试基因拯救实验来重建由 ZBTB7A 水平改变引起的表型。在第三个 步骤我们将进一步剖析 ZBTB7A 调节 NP 中基因表达的分子机制，重点关注 关于 ZBTB7A 辅因子。为此，我们将使用 BioID 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 检测来了解 ZBTB7A 的机制 可以影响这些辅助因子向基因调控区域的募集，从而影响基因表达。这个项目 将推进桑福德研究中心儿科和罕见疾病小组的使命，同时提供新的见解 研究调节大脑皮层发育的表观遗传机制，以及如何破坏这些机制 机制可导致神经儿科疾病。