Exploring the Roles of Neuron-Specific Histone Methylation Dynamics

探索神经元特异性组蛋白甲基化动力学的作用

• 批准号: 9394215
• 负责人: Robert Scott Porter
• 金额: $ 3.56万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9394215
• 关键词: Address; Affect; Alternative Splicing; Autistic Disorder; Basic Science; Binding; Biochemical; Biological Assay; Brain; Cells; Childhood; Chromatin; Cognition Disorders; Cognitive deficits; Complementary DNA; Complex; DNA; Data; Development; Disease; Down-Regulation; Embryo; Enzymes; Epigenetic Process; Functional disorder; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Genetic study; Goals; Histone H3; Histones; Human; Individual; Intellectual functioning disability; KDM1A gene; Kabuki Make-Up Syndrome; Knockout Mice; Knowledge; Lead; Learning; Light; Literature; Luciferases; Lysine; Mediating; Medical; Memory; Methylation; Milk; Morphology; Neonatal; Nervous system structure; Neurodevelopmental Disorder; Neurons; Nucleosomes; Nucleotides; PHD Finger; Pathogenesis; Pathway interactions; Patients; Phenotype; Physicians; Population; Property; Protein Isoforms; Protein Methylation; Proteins; Reaction; Reader; Regulation; Reporter; Role; Scientist; Signal Pathway; Substrate Specificity; Synapses; Synaptic Potentials; Syndrome; System; Testing; Time; Training; Transcriptional Regulation; Transfection; Translational Research; Work; base; brain abnormalities; brain morphology; career; cell type; cognitive development; demethylation; epigenetic regulation; exhaustion; genome-wide analysis; histone demethylase; histone methylation; histone modification; loss of function; mouse model; nervous system disorder; postnatal; programs; synaptic function; synaptogenesis; targeted treatment; transcriptome; transcriptome sequencing; ward

PROJECT SUMMARY Neurodevelopmental disorders (NDDs), including syndromes of intellectual disability (ID) are early-presenting cognitive disorders that affect 1-8% of the population. Recent genome-wide studies that have sought the genetic basis of ID have implicated genes responsible for synaptic function, transcriptional regulation, and enzymes that modulate the post-translational methylation of histones, proteins around which DNA is wrapped. This proposal seeks to address the gap in knowledge around why disruption of histone methylation dynamics frequently leads to cognitive deficits by investigating brain-specific alternative splicing of histone-regulating genes. Recent work has shown that the histone demethylase, LSD1, has a neuronal isoform leading to altered substrate specificity. PHF21A is a histone reader that acts in complex with LSD1 such that it canonically recognizes the product of the LSD1 reaction. Loss of function of LSD1 and PHF21A lead to ID syndromes, Kabuki Syndrome and Potocki Shaffer Syndrome (PSS), respectively. My preliminary data shows that PHF21A also has a neuronal-specific isoform (PHF21A-n). Through an RNA-Seq study of PSS patients, I also found that loss of PHF21A function leads to transcriptional downregulation of signaling pathways important for learning and memory. Our lab previously generated a Phf21a homozygous null mouse that died as a result of an inability to suckle milk. No structural brain abnormalities or neuron morphological abnormalities, but synaptic formation, a phenotype relevant to NDDs, was not assessed. Given the literature and my preliminary data, my central hypothesis is that PHF21A-n has unique histone binding properties that allow for proper synaptic formation in maturing neurons. In this proposal, I will test this hypothesis by (1) biochemical analysis of PHF21A-n function by performing binding, transcriptional reporter, and demethylation assays, in combination with canonical and neuronal isoforms of LSD1. I will next (2) assess the contributions of each PHF21A isoform in synaptic development using a neuron culture system with Phf21a null cultured neurons with each isoform individually replaced by transfection. I will evaluate changes in synaptic development immunohistochemically and identify programs of PHF21A isoform-specific transcriptional regulation using RNA-Seq. Completion of this work will benefit our understanding of the underlying mechanisms of NDDs given that this proposal aims to study several pathways known to be affected in NDDs: synaptic formation, epigenetic regulation, and alternative splicing. Additionally, completion of this work will provide me with the scientific, technical, and medical training, as detailed in my training plan, which will propel me into a successful career as a physician scientist studying the epigenetic basis of pediatric neurological disease.

项目总结 神经发育障碍(NDDS)，包括智力残疾综合征(ID)，是早期表现 影响1-8%人口的认知障碍。最近的全基因组研究寻求 ID的遗传基础涉及负责突触功能、转录调控和 调节组蛋白翻译后甲基化的酶，组蛋白是包裹DNA的蛋白质。 这项建议试图解决关于为什么组蛋白甲基化动力学被破坏的知识差距 通过研究组蛋白调节的大脑特定的选择性剪接经常导致认知障碍 基因。最近的研究表明，组蛋白去甲基酶LSD1具有神经元异构体，导致改变 底物专一性。PHF21A是一种组蛋白读取器，它与LSD1形成复合体，从而规范地 识别LSD1反应的产物。LSD1和PHF21A功能丧失导致ID综合征， 歌舞伎综合征和波托奇雪弗综合征(PSS)。我的初步数据显示PHF21A 也有神经元特异性的亚型(PHF21A-n)。通过对PSS患者的RNA-Seq研究，我还发现 PHF21A功能的丧失会导致重要的信号通路转录下调 学习和记忆。我们的实验室之前培育了一只Phf21a纯合子空小鼠，该小鼠因 不能吸奶。没有脑部结构异常或神经元形态异常，但 突触形成，一种与NDDS相关的表型，没有被评估。 根据文献和我的初步数据，我的中心假设是PHF21a-n具有独特的组蛋白 结合特性，允许成熟神经元形成适当的突触。在这个提案中，我将测试这一点 假设(1)通过执行结合、转录报告、 以及去甲基化分析，结合LSD1的规范和神经元异构体。我将评估下一(2)个 利用Phf21a神经元培养系统研究PHF21A各亚型在突触发育中的作用 空白培养的神经元，每一种亚型都被单独的转染法取代。我将评估突触的变化 PHF21A异构体特异性转录程序的免疫组织化学建立及鉴定 使用RNA-Seq进行调控。这项工作的完成将有助于我们对潜在的 鉴于本提案的目的是研究已知的几条在非药物依赖性药物中受到影响的途径，非药物依赖性药物的作用机制： 突触形成、表观遗传调控和选择性剪接。此外，这项工作的完成将 为我提供科学、技术和医学培训，如我的培训计划中详细说明的那样，这将推动 我进入了一个成功的职业生涯，成为一名研究儿科神经病学表观遗传学基础的内科科学家 疾病。