A Mass Spectrometry Approach to the Genetic and Epigenetic Mechanisms Controlling Neuronal Identity

摘要 破译大脑中控制细胞命运选择和维持的机制是 了解破坏性神经疾病，如小头畸形、精神分裂症和脑癌 由于这些过程中的缺陷造成的。一个悬而未决的问题是，哺乳动物的神经元身份是如何 在发育和成年期间维持和保护，特别是在转录水平上。这个 BTB/POZ和锌指转录因子RP58(又名ZBTB18)是大脑发育和 体内和体外的神经元分化；RP58基因突变与人类小头畸形症和体部相关 骨痂发育不全。我们分析了胚胎皮质有丝分裂后神经元的转录组，并显示 其他细胞系的基因标记物的表达，如肌源性谱系，在以下情况下增加 RP58缺失提示RP58通过抑制其他谱系的基因来保护神经元的同一性。我们的 总体假设是，RP58是建立和维持神经元同一性所必需的，而它的丢失 转录功能可能导致小头畸形和成人大脑中的神经退行性疾病。至 破译RP58如何控制大脑中的细胞分化，并确定RP58蛋白伙伴可能是 参与其功能，我们进行了RP58免疫沉淀与质谱联用(IP-MS)。 小鼠胚胎和出生后皮质的实验。我们的数据显示RP58与两者的成员相结合 多梳抑制复合体2(PRC2)和SWI/SNF复合体--两个重要的染色质重塑 参与大脑发育的复合体。此外，使用定量组蛋白蛋白质组学和蛋白质印迹 分析表明，Rp58在胚胎脑中的缺失导致了组蛋白在胚胎发育后的整体变化。 翻译修饰(PTM)，包括减少H3K27甲基化(H3K27me)和增加 H3K9/14乙酰化。这些结果提出了RP58控制发育和成体同一性的假设 神经元及其转录程序通过调节PRC2和SWI/SNF复合体的功能和 染色质景观。 以下目标将解决这些假设。目标1：破译RP58和染色质重塑 复合体相互作用以建立和维持神经元的同一性。目标2：破译是否以及如何需要RP58 神经细胞中的组蛋白PTMS。目的3：确定细胞和发育阶段特异的RP58蛋白 神经细胞中的相互作用网络。这笔赠款是Dahmane实验室(威尔·康奈尔) 医学)，拥有发育神经生物学和小鼠遗传学方面的专业知识，以及加西亚实验室(加州大学 宾夕法尼亚州)拥有定量质谱学方面的专业知识，因为它与表观遗传机制和 染色质调节。完成这些拟议的研究将有助于阐明细胞的类型和阶段 特定的RP58调控机制控制神经元的身份，并对遗传和 脑部发育的表观遗传调控及其在小头畸形等脑部疾病中的失调。

Abstract Deciphering the mechanisms controlling cell fate choice and maintenance in the brain is a critical step in understanding devastating neurological disorders such as microcephaly, schizophrenia and brain cancer that result from defects in these processes. An unresolved question is how the mammalian neuronal identity is maintained and protected, particularly at the transcriptional level, during development and adult life. The BTB/POZ and Zinc finger transcription factor RP58 (aka ZBTB18), is required for brain development and neuronal differentiation both in vivo and in vitro; Mutations in RP58 are linked to human microcephaly and corpus callosum agenesis. We have analyzed the embryonic cortical post mitotic neurons’ transcriptome and showed that the expression of gene markers of other cell lineages, such as myogenic lineage, is increased following Rp58 deletion suggesting that RP58 protects the neuronal identity by repressing genes of other lineages. Our overall hypothesis is that RP58 is required to establish and maintain the neuronal identity and that the loss of its transcriptional function may lead to microcephaly and to neurodegenerative diseases in the adult brain. To decipher how RP58 controls cell differentiation in the brain and to identify RP58 protein partners that may be involved in its function, we have performed RP58 immunoprecipitation coupled to mass spectrometry (IP-MS) experiments on mouse embryonic and postnatal cortices. Our data show that RP58 binds to members of both the Polycomb Repressive Complex 2 (PRC2) and the SWI/SNF complex, two critical chromatin remodeling complexes involved in brain development. In addition, using quantitative histone proteomics and western blot analyses, we show that deletion of Rp58 in the embryonic brain leads to global changes in histones post- translational modifications (PTMs) including to decreased H3K27 methylation (H3K27me) and increased H3K9/14 acetylation. These results raise the hypothesis that RP58 controls the identity of developing and adult neurons and thus their transcriptional program by modulating the PRC2 and SWI/SNF complexes function and the chromatin landscape. The following aims will address these hypotheses. Aim 1: Decipher how RP58 and chromatin remodeling complexes interact to establish and maintain neuronal identity. Aim 2: Decipher if and how RP58 is required for histone PTMs in neural cells. Aim 3: Determine the cell and developmental stage-specific RP58 protein-protein interacting network in neural cells. This grant is a collaboration between the Dahmane lab (Weill Cornell Medicine) with expertise in developmental neurobiology and mouse genetics and the Garcia lab (University of Pennsylvania) with expertise in quantitative mass spectrometry as it relates to epigenetic mechanisms and chromatin regulation. Completion of these proposed studies will lead to the elucidation of the cell type and stage specific RP58 regulatory mechanisms controlling neuronal identity and to key insights into the genetic and epigenetic regulation of brain growth and of their misregulation in brain disorders such as microcephaly.