Identification and characterization of mCpH binding proteins in neurons

神经元中 mCpH 结合蛋白的鉴定和表征

基本信息

批准号：
10676980
负责人：
Yijing Su
金额：
$ 66.2万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-05 至 2027-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10676980
关键词：
ATAC-seq Adult Affinity Aliquot Amyotrophic Lateral Sclerosis Bar Codes Behavioral Binding Binding Proteins Biological Assay Biological Process Brain Brain Diseases Cells ChIP-seq Chromatin Cytoplasmic Granules Cytosine DNA DNA Library DNA Methylation DNA Sequence DNA Transposable Elements DNMT3a Data Development Dideoxy Chain Termination DNA Sequencing Dinucleoside Phosphates EMSA Effectiveness Elements Epigenetic Process Foundations Fragile X Syndrome Funding Gel Gene Expression Gene Expression Regulation Genes Genetic Transcription Genome Genomic Imprinting Germ Lines Goals Guanine High-Throughput Nucleotide Sequencing Human In Vitro Incubated Kinetics Knockout Mice Lead Libraries Ligation Literature Longevity Luciferases Maintenance Mediating Methyl-CpG-Binding Protein 2 Methylation Methyltransferase Modification Molecular Mus Mutation Nervous System Neurodevelopmental Disorder Neurons Neurophysiology - biologic function Outcome Phase Phenotype Play Pluripotent Stem Cells Protein Array Proteins Reader Regulation Reporting Resolution Resources Rett Syndrome Role Series System Technology Testing Tissues Transcription Alteration Transcription Repressor Transcriptional Regulation Validation Viral X Inactivation base bisulfite candidate selection candidate validation carcinogenesis conditional knockout deep sequencing dentate gyrus digital embryonic stem cell gain of function gene repression high throughput screening histone modification in vitro Assay in vivo induced pluripotent stem cell innovation insight loss of function mammalian genome motor impairment mouse model nervous system disorder new therapeutic target postmitotic postnatal recruit stem cells success synaptogenesis transcription factor transcriptome sequencing

项目摘要

PROJECT SUMMARY DNA methylation is a major epigenetic modification that plays an important role in key biological processes, including genomic imprinting, X-chromosome inactivation, suppression of transposable elements, and carcinogenesis. Although it has been traditionally considered to be restricted to CpG dinucleotides in metazoan genomes, emerging evidence over the past decade has shown that CpH (H=A/C/T) methylation is present in mammalian genomes, including cultured pluripotent stem cells, embryonic stem cells, induced pluripotent stem cells, the mouse germ line, and especially at a relatively high level in human and mouse brains. Given that CpGs only represent 4% of the metazoan genomes, CpH methylation greatly expands the proportion of the genome that is subject to regulation by cytosine methylation and represents a new mechanism of transcriptional regulation. In our previous studies, we generated neuronal DNA methylation profiles at a single base-resolution of the adult mouse dentate gyrus in which 80-90% of the cells are NeuN positive granule neurons, and our team was one of the first to show that ~25% of cytosine methylations are located in the CpH context. Notably, we identified the first mCpH reader, MeCP2, both in vitro and in postmitotic neurons in vivo. In addition, we found that CpH methylation was established postnatally and required DNMT3A for its active maintenance in neurons in vivo. Mutations on both the reader and writer lead to neurodevelopmental disorders, such as fragile X syndrome (FXS), amyotrophic lateral sclerosis (ALS), and Rett syndrome. Loss of either Dnmt3A or MeCP2 in the mouse models causes overlapping and distinct phenotypes in behavioral and molecular tests, suggesting the existence of additional mCpH-binding proteins. We believe that a critical step towards understanding the biological functions of mCpH is to identify its binding proteins. In this proposal, our goal is to identify additional mCpH binding proteins. We hypothesize that mCpH regulates transcription directly or indirectly via recruiting sequence-independent and/or -dependent mCpH-binding proteins in neurons. We will use protein array (Aim 1) and Digital Affinity Profiling via Proximity Ligation (DAPPL; Aim 2) to identify sequence-independent and -dependent mCpH-binding proteins and validate candidates using gel-shift (EMSA), OCTET and luciferase assays in vitro. We will employ a viral in vivo delivery system and high- throughput sequencing technologies to characterize their roles in transcriptional and chromatin regulation in the adult mouse brain (Aim 3). The effectiveness of our strategy will be rigorously evaluated via a series of in vitro and in vivo assays. If funded, the success of this project is expected to provide a rich resource of sequence-dependent and independent mCpH-binding proteins that will lay the foundation to elucidate the roles of CpH methylation in neurons, stem cells and other tissues. The insights into the mechanism of CpH methylation is expected to provide novel drug targets for treating neurodevelopmental disorders.

项目概要 DNA甲基化是一种主要的表观遗传修饰，在关键生物过程中发挥着重要作用，包括基因组印记、X染色体失活、转座元件抑制，以及致癌作用。尽管传统上被认为仅限于后生动物中的 CpG 二核苷酸在基因组中，过去十年的新证据表明 CpH (H=A/C/T) 甲基化存在于哺乳动物基因组，包括培养的多能干细胞、胚胎干细胞、诱导多能干细胞细胞、小鼠生殖系，尤其是在人类和小鼠大脑中水平相对较高。鉴于 CpG仅占后生动物基因组的4%，CpH甲基化大大扩大了后生动物基因组的比例基因组受到胞嘧啶甲基化的调节，代表了一种新的机制转录调控。在我们之前的研究中，我们在单个成年小鼠齿状回的碱基分辨率，其中 80-90% 的细胞为 NeuN 阳性颗粒神经元，我们的团队是最早证明约 25% 的胞嘧啶甲基化位于 CpH 中的团队之一语境。值得注意的是，我们在体外和体内有丝分裂后神经元中鉴定出了第一个 mCpH 读取器 MeCP2。此外，我们发现 CpH 甲基化是在出生后建立的，并且需要 DNMT3A 才能发挥其活性体内神经元的维持。读者和作者的突变都会导致神经发育障碍，例如脆性 X 综合征 (FXS)、肌萎缩侧索硬化症 (ALS) 和雷特综合征。失去其中任何一个小鼠模型中的 Dnmt3A 或 MeCP2 导致行为和行为方面重叠和不同的表型分子测试表明存在额外的 mCpH 结合蛋白。我们相信这是关键的一步为了了解 mCpH 的生物学功能，需要鉴定其结合蛋白。在这个提案中，我们的目标是鉴定其他 mCpH 结合蛋白。我们假设 mCpH 直接调节转录或间接通过在神经元中募集序列无关和/或依赖的 mCpH 结合蛋白。我们将使用蛋白质阵列（目标 1）和通过邻近连接的数字亲和力分析（DAPPL；目标 2）来识别序列独立和依赖的 mCpH 结合蛋白，并使用凝胶迁移验证候选物 (EMSA)、OCTET 和体外荧光素酶测定。我们将采用病毒体内传递系统和高通量测序技术来表征其在转录和染色质调控中的作用成年小鼠大脑（目标 3）。我们战略的有效性将通过一系列的严格评估体外和体内测定。如果获得资助，该项目的成功预计将提供丰富的资源序列依赖性和独立性 mCpH 结合蛋白将为阐明其作用奠定基础神经元、干细胞和其他组织中 CpH 甲基化的研究。对CpH作用机制的深入探讨甲基化有望为治疗神经发育障碍提供新的药物靶点。