Exploring the Roles of Neuron-Specific Histone Methylation Dynamics

项目摘要 神经发育障碍（NDD），包括智力残疾（ID）综合征是早期表现的 认知障碍影响1-8%的人口。最近的全基因组研究， ID的遗传基础涉及负责突触功能、转录调节和 调节组蛋白（DNA包裹的蛋白质）翻译后甲基化的酶。 该提案旨在解决关于为什么组蛋白甲基化动力学的差距 通过研究脑特异性组蛋白调节的选择性剪接， 基因.最近的研究表明，组蛋白去甲基化酶，LSD 1，有一个神经元亚型，导致改变， 底物特异性PHF 21 A是一种组蛋白阅读器，与LSD 1复合作用，因此它符合规范 识别LSD 1反应的产物。LSD 1和PHF 21 A的功能丧失导致ID综合征， Kabuki综合征和Potocki Shaffer综合征（PSS）。我的初步数据显示PHF 21 A 还具有神经元特异性同种型（PHF 21 A-n）。通过对PSS患者的RNA-Seq研究，我还发现 PHF 21 A功能的丧失导致信号通路的转录下调， 学习和记忆。我们的实验室以前产生了一个Phf 21 a纯合子无效小鼠，死亡的结果， 不能挤奶没有脑结构异常或神经元形态异常，但 突触形成，一种与NDD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PHF 21 A-n具有独特的组蛋白， 结合特性允许成熟神经元中适当的突触形成。在这份提案中，我将测试这一点 通过（1）通过进行结合、转录报告子 和去甲基化测定，与LSD 1的典型和神经元同种型组合。接下来（2）评估 使用具有Phf 21 a的神经元培养系统，每个PHF 21 a亚型在突触发育中的贡献 用转染单独替换每种同种型的空培养神经元。我会评估突触的变化 PHF 21 A亚型特异性转录调控基因的构建和鉴定 使用RNA-Seq.这项工作的完成将有助于我们了解潜在的 NDD的机制，因为该提案旨在研究已知在NDD中受影响的几种途径： 突触形成、表观遗传调节和可变剪接。此外，这项工作的完成将 为我提供科学，技术和医疗培训，如我的培训计划所述，这将推动 我成为一名成功的医生科学家，研究儿科神经系统的表观遗传基础， 疾病

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.