Impact of histone serotonylation domain organization on neurodevelopment

项目摘要 新出现的证据表明，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（H3 K4 me 3）结构域。广泛的H3 K4 me 3结构域与细胞特异性 转录激活，并与神经元中的突触信号传导相关，其中H3 K4 me 3扩散是神经元中的一个重要因素。 自闭症谱系障碍（ASD）患者死后前额叶皮层（PFC）神经元的破坏。 因此，H3 K4 me 3的宽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H3 K4 me 3宽峰的扩展部分受赖氨酸甲基转移酶2 e的调节 （Kmt 2 e），其中超过30种遗传变体-包括在Kmt 2 e中的位置140处的缬氨酸至异亮氨酸取代（V140 I）， 它的染色质阅读PHD手指-在与智力残疾有关的症状的个体中观察到 和发育迟缓。有趣的是，H3 K4 me 3位于可以被乙酰化的谷氨酰胺5旁边， 进一步增强允许的组蛋白翻译后修饰H3 K4 me 3Q 5ser 与单独的H3 K4 me 3相比。我们的初步数据表明，H3 Q5 ser增强Kmt 2 e的结合 H3 K4 me 3Q 5ser的ChIP测序鉴定了宽H3 K4 me 3Q 5ser 与神经发育相关过程相关的领域。因此，我假设Kmt 2 e 通过组织H3 K4 me 3广泛的结构域来调节大脑发育，并且邻近的 H3 Q5 ser影响这种相互作用。为了特异性地询问Kmt 2 e和H3 K4 me 3Q 5ser结合， 宽峰组织的调节子，突变体Kmt 2 eV 140 I，其在H3 Q5 ser 将在H3 K4 me 3和Kmt 2 e PHD指结合过程中延伸，被选为与H3 K4 me 3和Kmt 2 e PHD指结合相关的基因。 可以用来评估这种相互作用的机制影响的变量。在目标1中，我将定量评估 H3 K4 me 3Q 5ser和Kmt 2 eWT相对于Kmt 2 eV 140 I PHD指之间的结合亲和力作为H3 K4 me 3Q 5ser中的关键相互作用。 发育中的大脑可能会在某些病理中被破坏，使用肽免疫沉淀，然后 Western印迹和等温滴定量热法。在目标2中，我将在二倍体RPE 1中使用CRISPR/Cas9技术， 细胞，以评估标记的Kmt 2 eWT与Kmt 2 eV 140 I敲入与Kmt 2 e敲除对宽峰的影响 在使用H3 K4 me 3Q 5ser ChIP-seq的Kmt 2 e募集上，以及在使用H3 K4 me 3Q 5ser ChIP-seq的下游上， 使用RNA-seq.在目标3中，我将通过在子宫内使用Kmt 2 e来评估Kmt 2 e在发育中的大脑中的影响。 电穿孔以检测设计用于特异性敲低PFC中Kmt 2 e表达的人工miRNA 祖细胞，同时通过添加回标记的Kmt 2 eWT与Kmt 2 eV 140 I转基因进行“拯救”，使用 H3 K4 me 3Q 5ser和Kmt 2 e ChIP-seq作为表观遗传标准化的读数，RNA-seq用于评估 发育基因表达程序和神经元形态学分析，以评估Kmt 2 e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 调节神经发育，可能在ASD等综合征中受到干扰。

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.