Impact of histone serotonylation domain organization on neurodevelopment

项目摘要 新的证据表明，染色质机制有助于大脑发育，包括组织 H3赖氨酸4三甲基化(H3K4me3)结构域。广泛的H3K4me3结构域与细胞特异性相连 转录激活，并与神经元中的突触信号有关，H3K4me3在神经元中传播 自闭症谱系障碍(ASD)患者死后前额叶皮质(PFC)神经元受损。 因此，H3K4me3的广度可能会影响重要的神经发育过程，但这是如何发生的 不清楚。H3K4me3宽峰的扩展部分受赖氨酸甲基转移酶2E酶的调节 (Kmt2e)，其中有30多个遗传变异-包括第140位(V140I)的valine到异亮氨酸的替代 它的染色质--读博士的手指--在有智力障碍相关症状的个体中观察到 和发育迟缓。有趣的是，H3K4me3位于谷氨酰胺5旁边，谷氨酰胺5可以被5-羟色胺基化，产生 组合组蛋白翻译后修饰H3K4me3Q5序列进一步增强了容许性 与单独使用H3K4me3进行比较。我们的初步数据显示H3Q5ser增强了Kmt2e的结合 对H3K4me3和胚胎前脑中H3K4me3Q5ser的芯片测序鉴定出广泛的H3K4me3Q5ser 与神经发育相关的过程相关的领域。因此，我假设Kmt2e 通过组织H3K4me3广泛的结构域来调节大脑发育，而邻近的 H3Q5ser会影响这种相互作用。将Kmt2e和H3K4me3Q5ser绑定作为 广谱峰组织的调节因子突变Kmt2eV140I，该突变位于H3Q5ser 会在H3K4me3和Kmt2e PHD手指结合期间延伸，被选为翻译相关基因 可用于评估此交互作用的机械性影响的变量。在目标1中，我将定量评估 H3K4me3Q5ser与Kmt2eWT与Kmt2eV140I PhD指间的结合亲和力 在某些病理上可能被破坏的发育中的大脑，使用多肽免疫沉淀随后 蛋白质印迹和等温滴定量热法。在目标2中，我将在二倍体RPE1中使用CRISPR/Cas9技术 评估标记Kmt2eWT与Kmt2eV140I敲入与Kmt2e敲除对宽峰的影响 使用H3K4me3Q5ser芯片-seq的分销，使用Kmt2e芯片-seq的Kmt2e招聘，以及下游 使用RNA-seq转录。在目标3中，我将通过在子宫内使用Kmt2e来评估Kmt2e对大脑发育的影响 电穿孔转染靶向PFC中Kmt2e基因表达的人工miRNA 祖细胞，同时通过添加反向标记的Kmt2eWT和Kmt2eV140I转基因而进行拯救，使用 H3K4me3Q5ser和Kmt2e芯片序列作为表观遗传归一化读数，RNA-seq进行评价 发育基因表达计划和神经元形态分析，以评估Kmt2e对 突触发育。总之，这些研究将为一种新的表观遗传机制提供有价值的见解。 调节可能在ASD等综合症中受到干扰的神经发育。

Project Summary Emerging evidence suggests chromatin mechanisms contribute to brain development, including organization of H3 lysine 4 tri-methylation (H3K4me3) domains. Broad H3K4me3 domains are linked with cell-specific transcriptional activation and are associated with synaptic signaling in neurons, where H3K4me3 spreading was disrupted in postmortem prefrontal cortex (PFC) neurons from patients with autism spectrum disorders (ASD). Thus, H3K4me3 breadth likely influences important neurodevelopmental processes, but how this occurs is unclear. Spreading of H3K4me3 broad peaks is regulated, in part, by the enzyme Lysine methyltransferase 2e (Kmt2e), where over 30 genetic variants - including a valine-to-isoleucine substitution at position 140 (V140I) in its chromatin-reading PHD finger - were observed in individuals with symptoms related to intellectual disability and developmental delay. Interestingly, H3K4me3 sits next to glutamine 5 that can be serotonylated, producing the combinatorial histone post-translational modification H3K4me3Q5ser that further enhances permissive transcription compared to H3K4me3 alone. Our preliminary data show that H3Q5ser enhances binding of Kmt2e to H3K4me3 and ChIP-sequencing of H3K4me3Q5ser in embryonic forebrain identified broad H3K4me3Q5ser domains that associate with neurodevelopment-associated processes. Thus, I hypothesize that Kmt2e regulates brain development via organization of H3K4me3 broad domains, and that the neighboring H3Q5ser influences such interactions. To specifically interrogate Kmt2e and H3K4me3Q5ser binding as a regulator of broad peak organization, the mutant Kmt2eV140I, that contains a mutation at the site where H3Q5ser would extend during H3K4me3 and Kmt2e PHD finger binding, was selected as a translationally relevant genetic variant that can be used to assess the mechanistic impact of this interaction. In Aim 1, I will quantitatively assess the binding affinity between H3K4me3Q5ser and Kmt2eWT vs. Kmt2eV140I PHD fingers as a crucial interaction in the developing brain that may be disrupted in some pathologies, using peptide immunoprecipitation followed by western blotting and isothermal titration calorimetry. In Aim 2, I will use CRISPR/Cas9 technology in diploid RPE1 cells to assess the impact of tagged Kmt2eWT vs. Kmt2eV140I knock-in vs. Kmt2e knockout on broad peak distribution using H3K4me3Q5ser ChIP-seq, on Kmt2e recruitment using Kmt2e ChIP-seq, and on downstream transcription using RNA-seq. In Aim 3, I will assess the impact of Kmt2e in developing brain by using in utero electroporation to transfect artificial miRNA designed to specifically knockdown Kmt2e expression in PFC progenitor cells, with simultaneous ‘rescue’ by adding back tagged Kmt2eWT vs. Kmt2eV140I transgenes, using H3K4me3Q5ser and Kmt2e ChIP-seq as readouts of epigenetic normalization, RNA-seq to evaluate developmental gene expression programs, and neuronal morphology analyses to assess Kmt2e impact on synapse development. Together, these studies will provide valuable insight into a novel epigenetic mechanism regulating neurodevelopment that may be perturbed in syndromes such as ASD.