Epigenetic mechanisms regulating neuron production during cortical development

大脑皮层是人类高级认知功能的基础大脑区域，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产生的神经元网络 发展。在那个发育时期，神经的增殖和分化之间的平衡 前体(NPs)对于产生适当数量的神经元是至关重要的。因此，NP的中断 增殖是许多神经发育障碍的根源，如智力残疾、自闭症、 小头畸形和巨头畸形。许多细胞和分子过程调节增殖和分化 NP中的决策。在目前的研究中，我们将集中在表观遗传学机制调控的表达 增殖和分化基因。为此，我们将重点研究转录因子ZBTB7A，已知的 在与增殖和分化有关的基因的调节区中调节染色质的可及性。 ZBTB7A与许多不同的系统有关，但它在皮质发育的NPs中的作用是完全的 未知。而ZBTB7A基因位于19p13.3微位点处，包含3个基因，其 复制或缺失分别导致小头畸形和巨头畸形。我们在小鼠身上的初步研究 显示ZBTB7A在NPs中的表达改变导致增殖缺陷，与预期结果匹配 在19p13.3综合征中观察到的那些。总而言之，这些发现使ZBTB7A成为一个杰出的候选者 发现调节大脑皮层发育的新表观遗传机制。在这项研究中，我们将使用 模拟人类ZBTB7A改变的小鼠模型以表征ZBTB7A改变的表达如何影响 NP的增殖和皮质结构的建立。在第二步中，我们将表征ZBTB7A目标 利用CHIP-SEQ和RNA-SEQ偶联的NPs中的基因。在使用荧光素酶分析验证候选基因后，我们 将尝试进行遗传拯救实验，以重建由ZBTB7A水平变化引起的表型。在三分之一 我们将进一步剖析ZBTB7A调控NPs基因表达的分子机制，重点 ZBTB7A共因子的研究。为了做到这一点，我们将使用BioID来识别在ZBTB7A附近工作的蛋白质 我们将使用荧光素酶、芯片-聚合酶和共IP分析来了解ZBTB7A 可以影响这些辅助因子的募集到基因调控区，从而影响基因表达。这个项目 将推进桑福德研究的儿科和罕见疾病小组的使命，同时提供新的见解 关于调节大脑皮层发育的表观遗传机制，以及这些机制如何被破坏 机制可能会导致神经儿科疾病。

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.