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 了解毁灭性的神经系统疾病，如小头畸形，精神分裂症和脑癌， 这些过程中的缺陷。一个尚未解决的问题是哺乳动物的神经元身份是如何 维持和保护，特别是在转录水平，在发展和成年生活。的 BTB/POZ和锌指转录因子RP 58（又名ZBTB 18）是大脑发育所必需的， 体内和体外的神经元分化; RP 58中的突变与人类小头畸形和体 胼胝体发育不全我们分析了胚胎皮层有丝分裂后神经元的转录组， 其他细胞谱系，如肌源性谱系的基因标记物的表达在以下情况下增加： Rp 58缺失表明Rp 58通过抑制其他谱系的基因来保护神经元身份。我们 总的假设是，RP 58是建立和维持神经元身份所必需的，并且其缺失是神经元的一个重要因素。 转录功能可能导致成人脑中的小头畸形和神经变性疾病。到 破译RP 58如何控制大脑中的细胞分化，并鉴定可能与细胞分化有关的RP 58蛋白伴侣。 我们进行了RP 58免疫沉淀-质谱联用（IP-MS）， 小鼠胚胎和出生后皮质的实验。我们的数据表明，RP 58结合到两个成员， 多梳抑制复合物2（PRC 2）和SWI/SNF复合物，两个关键的染色质重塑 参与大脑发育的复合体。此外，利用定量组蛋白质组学和western blot 分析，我们表明，Rp 58在胚胎脑中的缺失导致组蛋白的整体变化后， 翻译修饰（PTM），包括降低H3 K27甲基化（H3 K27 me）和增加H3 K27甲基化（H3 K27 me）。 H3 K9/14乙酰化。这些结果提出了一个假设，即RP 58控制发育和成年的身份 通过调节PRC 2和SWI/SNF复合物功能， 染色质景观。 以下目标将解决这些假设。目的1：解读RP 58和染色质重塑 复合物相互作用以建立和维持神经元身份。目标2：破译是否以及如何需要RP 58， 神经细胞中的组蛋白PTM。目的3：确定细胞和发育阶段特异性RP 58蛋白-蛋白 神经细胞中的交互网络。这项资助是Dahmane实验室（Weill Cornell 在发育神经生物学和小鼠遗传学方面具有专长的Garcia实验室（哥伦比亚大学， 宾夕法尼亚州），具有定量质谱分析方面的专业知识，因为它与表观遗传机制有关， 染色质调节完成这些拟议的研究将导致阐明细胞类型和阶段 控制神经元身份的特异性RP 58调节机制，以及对遗传和 大脑发育的表观遗传调节及其在大脑疾病如小头畸形中的失调。

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.