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：
10490842
负责人：
Jennifer C Chan
金额：
$ 7.17万
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/10490842
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.

项目概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传导相关，其中 H3K4me3 传播是自闭症谱系障碍 (ASD) 患者死后前额皮质 (PFC) 神经元受到破坏。因此，H3K4me3 的宽度可能会影响重要的神经发育过程，但这种情况是如何发生的尚不清楚目前尚不清楚 H3K4me3 宽峰的扩散部分受赖氨酸甲基转移酶 2e 的调节。 (Kmt2e)，其中超过 30 个遗传变异 - 包括 140 位 (V140I) 处的缬氨酸至异亮氨酸取代它的染色质读取 PHD 手指 - 在具有智力障碍相关症状的个体中观察到隐含地，H3K4me3 位于可被血清素化的谷氨酰胺 5 旁边，产生组合组蛋白翻译后修饰 H3K4me3Q5ser 进一步增强了许可性与单独的 H3K4me3 转录相比，我们的初步数据表明 H3Q5ser 增强了 Kmt2e 的结合。胚胎前脑中 H3K4me3 和 H3K4me3Q5ser 的 ChIP 测序鉴定出广泛的 H3K4me3Q5ser 因此，我领导了 Kmt2e。通过组织 H3K4me3 广泛的结构域来调节大脑发育，并且邻近的 H3Q5ser 影响此类相互作用。宽峰组织的调节因子，突变体 Kmt2eV140I，在 H3Q5ser 所在位点包含突变将在 H3K4me3 和 Kmt2e PHD 指结合期间延伸，被选为翻译相关遗传可用于评估这种相互作用的机械影响的变体在目标 1 中，我将定量评估。 H3K4me3Q5ser 和 Kmt2eWT 与 Kmt2eV140I PHD 手指之间的结合亲和力作为关键相互作用使用肽免疫沉淀法，在某些病理情况下可能会破坏发育中的大脑在目标 2 中，我将在二倍体 RPE1 中使用 CRISPR/Cas9 技术。细胞以评估标记的 Kmt2eWT 与 Kmt2eV140I 敲入与 Kmt2e 敲除对宽峰的影响使用 H3K4me3Q5ser ChIP-seq 进行分布，使用 Kmt2e ChIP-seq 进行 Kmt2e 招募，以及下游在目标 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。电穿孔转染人工 miRNA，旨在特异性敲低 PFC 中 Kmt2e 的表达祖细胞，通过添加反向标记的 Kmt2eWT 与 Kmt2eV140I 转基因同时进行“救援”，使用 H3K4me3Q5ser 和 Kmt2e ChIP-seq 作为表观遗传标准化的读数，RNA-seq 进行评估发育基因表达程序和神经元形态分析以评估 Kmt2e 对这些研究将为新的表观遗传机制提供有价值的见解。调节自闭症谱系障碍等综合症中可能受到干扰的神经发育。