Identification and characterization of mCpH binding proteins in neurons

项目总结 DNA甲基化是一种主要的表观遗传修饰，在关键的生物过程中发挥着重要作用， 包括基因组印迹，X染色体失活，抑制转座元件，以及 致癌。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，过去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，小鼠的生殖系，尤其是在人类和小鼠的大脑中处于相对较高的水平。考虑到 CPGS只占后生动物基因组的4%，CPH甲基化大大扩大了 受胞嘧啶甲基化调节的基因组，代表了一种新的 转录调控。在我们之前的研究中，我们在一个单独的 成年小鼠齿状回80%~90%细胞为NeuN阳性颗粒的碱基分辨 神经元，我们的团队是首批证明~25%的胞嘧啶甲基化位于CPH中的人之一 背景。值得注意的是，我们在体外和体内的有丝分裂后神经元中都发现了第一个mCpH阅读器MeCP2。 此外，我们还发现，CPH甲基化是在出生后建立的，需要DNMT3A才能发挥其活性 活体内神经元的维持。读者和作者的基因突变都会导致神经发育障碍， 如脆性X综合征(FXS)、肌萎缩侧索硬化症(ALS)和Rett综合征。失去其中任何一个 DNMT3A或MeCP2在小鼠模型中引起重叠和不同的表型，在行为和 分子测试，表明存在额外的mCpH结合蛋白。我们认为，关键的一步是 要了解mCpH的生物学功能，就必须鉴定其结合蛋白。在这项提案中，我们的 目的是鉴定更多的mCpH结合蛋白。我们假设mCpH直接调节转录。 或间接地通过在神经元中招募序列无关和/或依赖的mCpH结合蛋白来实现。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)来识别 序列非依赖和依赖的mCpH结合蛋白及凝胶移位验证候选蛋白 (EMSA)、八倍体和荧光素酶体外检测。我们将采用病毒体内递送系统和高- 吞吐量测序技术表征它们在转录和染色质调节中的作用 成年小鼠脑(目标3)。我们战略的有效性将通过一系列的 体外和体内检测。如果获得资金，该项目的成功有望提供丰富的资源 序列依赖和独立的mCpH结合蛋白，将为阐明这些作用奠定基础 神经元、干细胞和其他组织中CPH甲基化的研究。对CPH机制的几点认识 甲基化有望为治疗神经发育障碍提供新的药物靶点。

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.