Dissecting mechanistic links between MAPK signaling, genomic hypomethylation and naive pluripotency

剖析 MAPK 信号传导、基因组低甲基化和初始多能性之间的机制联系

基本信息

批准号：
10094448
负责人：
Konrad Hochedlinger
金额：
$ 51.16万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-01 至 2026-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10094448
关键词：
Address Affect Alleles Attenuated BRAF gene Cells Chemicals Chromosomal Instability Chromosomal Stability Cues Cultured Cells DNA DNA Methylation Data Defect Dependence Development Embryo Environment Epiblast Epigenetic Process Exhibits Female Genes Genetic Transcription Genome Stability Genomic Imprinting Genomic Instability Genomics Goals Human Impairment In Vitro Karyotype Knock-out LIF gene Lead Link MAP Kinase Gene MAP2K1 gene MAPK phosphatase MEKs Maintenance Measures Methylation Mitogen-Activated Protein Kinase Inhibitor Mitogen-Activated Protein Kinase Kinases Molecular Mus Mutation Analysis Pathway interactions Pharmacology Phenotype Play Pluripotent Stem Cells Process Proteomics Protocols documentation Research Personnel Role Serum Sex Chromosomes Sex Differences Signal Transduction Somatotype System Testing Titrations Up-Regulation Work X Chromosome base blastocyst cell type embryonic stem cell epigenome genome wide methylation human embryonic stem cell human stem cells implantation imprint improved in vivo induced pluripotent stem cell inhibitor/antagonist male methylation pattern natural Blastocyst Implantation phosphoproteomics pluripotency preimplantation preservation self renewing cell self-renewal sex stem cell model stem-like cell tool upstream kinase

项目摘要

SUMMARY Embryonic stem cells (ESCs) self-renew indefinitely in culture while retaining the capacity to produce all cell types of the body. Mouse ESCs are typically maintained in serum and LIF, which capture a state resembling the normally methylated, post-implantation epiblast, whereas culture of ESC in the presence of inhibitors of MEK1/2 and GSK3, termed “2i”, captures a hypomethylated, naïve state that resembles the pre-implantation epiblast. As Wnt activation (via GSK3 inhibitor) and MAPK suppression (via MEK1/2 inhibitor) recapitulates the signaling environment of early embryos, 2i-induced hypomethylation offers a tractable and powerful ex vivo system to study the reprogramming of genomic methylation patterns within the pre-implantation embryo. Notably, methylation patterns are not only influenced by external signals but also by sex chromosomes, with female ESCs being hypomethylated compared to male ESCs. The process of female-specific hypomethylation and its connection to the naïve state remain incompletely understood. We recently discovered that suppression of the MAPK pathway through pharmacological inhibition of MEK1/2 or upregulation of the X-linked MAPK phosphatase DUSP9 underlies 2i-induced and female-specific hypomethylation, respectively. Unexpectedly, we found that suppression of the MAPK pathway also compromises genomic stability and the developmental potential of ESCs. Here, we outline 3 complementary aims to dissect the mechanisms by which the MAPK pathway influences DNA methylation in pluripotent cells through either sex chromosomes or external signals. In SPECIFIC AIM 1, we will narrow down the upstream and downstream components of the MAPK pathway responsible for hypomethylation and test candidate targets identified by proteomics approaches. We will further explore the molecular consequences of loss of genomic hypomethylation within the naïve epiblast. In SPECIFIC AIM 2, we will test candidate targets of DUSP9 in female ESCs and integrate results with Aim 1 to define similarities and differences between sex-dependent and environment (2i)-induced hypomethylation. We will further characterize the self- renewal defect we uncovered in ESCs lacking both Dusp9 alleles and assess its dependence on DNA methylation. Lastly, we will determine whether sex-specific methylation differences in ESCs originate from pre- or post-implantation embryos. In SPECIFIC AIM 3, we will investigate whether the mechanistic connection we observed between MAPK signaling and DNA methylation is conserved in naïve human ESCs and whether this information can be exploited to grow more stable human cells. Specifically, we will assess whether the titration of inhibitors that target MAPK signaling or the use of alternative MEK inhibitors increases DNA methylation and decreases genomic instability. Collectively, our work will explore molecular links between MAPK signaling and DNA methylation, genomic stability and developmental potential in pluripotent cells with the goal to dissect basic mechanisms and define improved conditions for human stem cells.

概括胚胎干细胞 (ESC) 在培养物中无限期地自我更新，同时保留产生所有细胞的能力小鼠 ESC 通常保存在血清和 LIF 中，捕获类似于人体的状态。正常甲基化，植入后外胚层，而在 MEK1/2 抑制剂存在下培养 ESC GSK3，称为“2i”，捕获类似于植入前外胚层 As 的低甲基化、幼稚状态。 Wnt 激活（通过 GSK3 抑制剂）和 MAPK 抑制（通过 MEK1/2 抑制剂）概括了信号传导在早期胚胎的环境中，2i诱导的低甲基化提供了一个易于处理且强大的离体系统研究植入前胚胎内基因组甲基化模式的重编程。甲基化模式不仅受到外部信号的影响，还受到性染色体的影响，女性ESCs 与男性 ESC 相比，女性特有的低甲基化过程及其。我们最近发现了对天真的状态的抑制。通过药理学抑制 MEK1/2 或上调 X 连锁 MAPK 磷酸酶的 MAPK 通路 DUSP9 分别是 2i 诱导的低甲基化和女性特异性低甲基化的基础，我们意外地发现了这一点。 MAPK 通路的抑制还会损害基因组稳定性和 ESC 的发育潜力。在这里，我们概述了 3 个互补的目标来剖析 MAPK 通路影响 DNA 的机制在具体目标 1 中，我们将通过性染色体或外部信号在多能细胞中进行甲基化。缩小负责低甲基化的 MAPK 通路的上游和下游组件的范围并测试通过蛋白质组学方法确定的候选靶点，我们将进一步探索分子生物学。在 SPECIFIC AIM 2 中，我们将测试幼稚外胚层内基因组低甲基化缺失的后果。女性 ESC 中 DUSP9 的候选目标，并将结果与目标 1 相结合，以确定相似点和差异我们将进一步描述性别依赖性和环境（2i）诱导的低甲基化之间的关系。我们在缺乏 Dusp9 等位基因的 ESC 中发现了更新缺陷，并评估了其对 DNA 的依赖性最后，我们将确定 ESC 中的性别特异性甲基化差异是否源自前体。在特定目标 3 中，我们将研究是否存在机械连接。在原始人类 ESC 中观察到的 MAPK 信号传导和 DNA 甲基化之间是否保守具体来说，我们将评估滴定是否可以利用信息来培养更稳定的人体细胞。针对 MAPK 信号传导的抑制剂或使用替代 MEK 抑制剂会增加 DNA 甲基化，总的来说，我们的工作将探索 MAPK 信号传导之间的分子联系。以及多能细胞中的 DNA 甲基化、基因组稳定性和发育潜力，目标是剖析基本机制并定义人类干细胞的改善条件。