Epigenetic mechanisms regulating neuron production during cortical development

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期产生的神经元网络 发展在此期间，神经细胞增殖和分化之间的平衡， 神经前体（NPs）对于产生适当数量的神经元至关重要。因此，NP的中断 增殖是许多神经发育障碍，如智力残疾，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 NPs的决定。在本研究中，我们将集中在调节表达的表观遗传机制， 增殖和分化基因。为了做到这一点，我们将重点放在转录因子ZBTB 7A上，已知ZBTB 7A是一种转录因子。 介导与增殖和分化有关的基因调节区域的染色质可及性。 ZBTB 7A与许多不同的系统有关，但其在皮质发育过程中的NP中的作用完全不清楚。 未知然而，ZBTB 7A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。我们在老鼠身上的初步研究 显示NPs中ZBTB 7A表达的改变导致增殖缺陷，预测结果匹配 在19p13.3综合征中观察到的那些。总之，这些发现使ZBTB 7A成为一个优秀的候选人， 发现调节大脑皮层发育的新的表观遗传机制。在这项研究中，我们将使用 模拟人类ZBTB 7A改变的小鼠模型，以表征ZBTB 7A表达的改变如何影响 NP增殖和皮质结构的建立。在第二步中，我们将表征ZBTB 7A靶标 使用ChIP-seq与RNA-seq偶联的NP中的基因。在使用荧光素酶检测验证候选基因后，我们 将尝试基因拯救实验，以重新建立由改变ZBTB 7A水平引起的表型。在第三 第一步，我们将进一步剖析ZBTB 7A调控纳米粒中基因表达的分子机制， ZBTB 7A辅助因子的研究为了做到这一点，我们将使用BioID来识别在ZBTB 7A附近工作的蛋白质， 我们将使用荧光素酶，ChIP-PCR和co-IP测定来了解ZBTB 7A 可以影响这些辅因子向基因调控区的募集，从而影响基因表达。这个项目 将推进桑福德研究所儿科和罕见疾病小组的使命，同时提供新的见解 进入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， 机制可能导致神经儿科疾病。

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.