Identification and characterization of mCpH binding proteins in neurons

神经元中 mCpH 结合蛋白的鉴定和表征

• 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
• 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

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.

项目摘要 DNA甲基化是一种主要的表观遗传修饰，在关键的生物学过程中发挥重要作用， 包括基因组印记、X染色体失活、转座因子的抑制，以及 致癌作用尽管传统上认为它仅限于后生动物中的CpG二核苷酸， 基因组，在过去的十年中出现的证据表明，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、诱导的多能干细胞 细胞，小鼠生殖系，特别是在人类和小鼠大脑中相对较高的水平。鉴于 CpGs仅代表后生动物基因组的4%，CpH甲基化极大地扩大了 基因组受到胞嘧啶甲基化的调控，代表了一种新的基因表达机制。 转录调控在我们以前的研究中，我们在一个单一的细胞中产生了神经元DNA甲基化谱， 成年小鼠齿状回的基本分辨，其中80-90%的细胞为NeuN阳性颗粒 我们的研究小组是最早发现约25%的胞嘧啶甲基化位于CpH的研究小组之一。 上下文值得注意的是，我们确定了第一个mCpH读者，MeCP 2，在体外和体内有丝分裂后神经元。 此外，我们发现CpH甲基化是在出生后建立的，并且需要DNMT 3A才能激活。 在体内神经元中的维持。阅读器和书写器的突变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（ALS）和Rett综合征。失去任何一个 小鼠模型中的Dnmt 3A或MeCP 2引起行为和免疫学上的重叠和不同表型， 分子测试，表明存在额外的mCpH结合蛋白。我们认为关键的一步 了解mCpH的生物学功能的关键是鉴定其结合蛋白。在本建议中，我们的 目的是鉴定额外的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非依赖性和/或依赖性mCpH结合蛋白。我们 将使用蛋白质阵列（Aim 1）和通过邻近连接的数字亲和分析（DAPPL; Aim 2）来鉴定 序列非依赖性和依赖性mCpH结合蛋白，并使用凝胶位移验证候选物 （EMSA）、OCTET和荧光素酶测定。我们将采用病毒体内输送系统和高- 通量测序技术，以表征其在转录和染色质调控中的作用， 成年小鼠大脑（Aim 3）。我们的战略的有效性将通过一系列 体外和体内测定。如果得到资助，该项目的成功预计将为非洲提供丰富的资源， 序列依赖性和非依赖性的mCpH结合蛋白，这将为阐明mCpH的作用奠定基础 在神经元、干细胞和其他组织中的CpH甲基化。对CpH机制的认识 甲基化有望为治疗神经发育障碍提供新的药物靶点。