Impact of histone serotonylation domain organization on neurodevelopment

组蛋白血清酰化结构域组织对神经发育的影响

基本信息

批准号：
10387512
负责人：
Jennifer C Chan
金额：
$ 6.86万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-09 至 2024-09-08
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10387512
关键词：
ASD patient Affinity Autopsy Back Binding Biochemical Biological Biological Assay Brain Brain region CRISPR/Cas technology Calorimetry Cell Culture Techniques Cell Line Cell Proliferation Cells ChIP-seq Child Chromatin Complex Confocal Microscopy Coupled DNA delivery Data Deposition Development Developmental Delay Disorders Developmental Gene Diploidy Disease Doxycycline Electroporation Embryo Embryonic Development Enzymes Epigenetic Process Epilepsy Etiology Exhibits Family member Female Gene Expression Genes Genetic Transcription Genome Glutamine Hela Cells Histone H3 Histones Human Image Immunoprecipitation In Vitro Individual Infant Intellectual functioning disability Isoleucine Knock-in Knock-out Life Link Lysine MLL gene Macrocephaly Methylation Methyltransferase MicroRNAs Modification Molecular Morphology Mus Mutation Neurodevelopmental Disorder Neuronal Differentiation Neurons Neurotransmitters Nuclear Extract Outcome PHD Finger Pathology Pathway interactions Patients Pattern Peptides Positioning Attribute Post-Translational Protein Processing Prefrontal Cortex Process Prosencephalon Reader Reading Regulation Research Resistance Risk Role Serotonin Signal Transduction Site Symptoms Synapses Syndrome System Tail Testing Tissues Titrations Training Transcriptional Activation Transgenes Valine Western Blotting age related autism spectrum disorder chromatin immunoprecipitation combinatorial design epigenetic regulation genetic variant genome editing in utero in vivo insight knock-down male molecular phenotype monoamine mutant nervous system development neurodevelopment neuron development neuropathology novel nuclease plasmid DNA prenatal programs receptor recruit stem cells tool transcriptome sequencing

项目摘要

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.

项目摘要新兴证据表明染色质机制有助于大脑的发育，包括组织 H3赖氨酸4三甲基化（H3K4me3）域。广泛的H3K4ME3域与细胞特异转录激活，与神经元中的合成信号传导有关，其中H3K4ME3扩散为自闭症谱系障碍患者（ASD）患者的后额叶前皮层（PFC）神经元中断。这是H3K4ME3广度可能会影响重要的神经发育过程，但是这种情况的发生是不清楚。 H3K4me3宽峰的传播部分由赖氨酸甲基转移酶2E调节（KMT2E），其中30多种遗传变异 - 包括在140（V140I）中的阀到异亮氨酸替代它的染色质阅读博士手指 - 在与智障症状有关的个体中观察到并发展延迟。有趣的是，H3K4Me3位于谷氨酰胺5旁边，可以用谷氨酰胺5，产生组合Hisstone翻译后修饰H3K4ME3Q5SER，进一步增强了允许的与仅H3K4me3相比，转录。我们的初步数据表明，H3Q5SER增强了KMT2E的结合在胚胎前脑中H3K4me3Q5SER的H3K4ME3和芯片序列确定了宽H3K4Me3Q5Ser 与神经发育相关的过程相关的域。那我假设KMT2E 通过组织H3K4Me3广泛领域来调节大脑的发育，而邻近 H3Q5SER会影响这种相互作用。专门询问KMT2E和H3K4ME3Q5SER绑定为一个 Broad Peak组织的调节器，突变体KMT2EV140I，该突变在H3Q5SER的位置中包含突变将在H3K4ME3和KMT2E PHD手指结合期间延伸，以翻译相关的通用。可用于评估这种相互作用的机械影响的变体。在AIM 1中，我将定量评估 H3K4Me3Q5SER和KMT2EWT与KMT2EV140I博士手指之间的结合亲和力是至关重要的相互作用使用胡椒免疫沉淀后，然后蛋白质印迹和等温滴定量热法。在AIM 2中，我将在二倍体RPE1中使用CRISPR/CAS9技术细胞评估标记的KMT2EWT与KMT2EV140I的影响使用H3K4ME3Q5SER芯片seq，在KMT2E上使用KMT2E募集的分布，并在下游使用RNA-Seq转录。在AIM 3中，我将通过使用子宫来评估KMT2E在发育大脑中的影响电穿孔以翻译人工miRNA，设计用于特异性敲低kmt2e在PFC中的表达祖细胞使用简单的“救援”，通过使用后添加标记的KMT2EWT与KMT2EV140I TRESS基因 H3K4ME3Q5SER和KMT2E芯片seq作为表观遗传归一化的读数，RNA-Seq评估发展基因表达程序和神经元形态分析，以评估KMT2E对突触发展。总之，这些研究将为一种新颖的表观遗传机制提供宝贵的见解调节在ASD等综合征中可能会干扰的神经发育。