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

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

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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）在培养中无限期地自我更新，同时保留产生所有细胞的能力身体类型。鼠标ESC通常保持在血清和LIF中，捕获类似于该状态的状态通常是甲基化的，植入后的培养细胞，而在MEK1/2的抑制剂存在下ESC的培养物 GSK3称为“ 2i”，捕获了类似于植入前植入植物的非甲基化，幼稚的状态。作为 Wnt激活（通过GSK3抑制剂）和MAPK抑制（通过MEK1/2抑制剂）概括了信号传导早期胚胎的环境，2-i诱导的低甲基化提供了一个可拖动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。尤其，甲基化模式不仅受外部信号的影响，而且还受性染色体的影响，女性ESC 与男性ESC相比，低甲基化。女性特异性低甲基化及其过程与幼稚状态的联系仍然不完全理解。我们最近发现了对 MAPK途径通过MEK1/2的药物抑制或X连锁MAPK磷酸酶上调 DUSP9分别是2-i诱导的和女性特异性低甲基化的基础。出乎意料的是，我们发现 MAPK途径的抑制也损害了基因组稳定性和ESC的发育潜力。在这里，我们概述了3个完整的目的是剖析MAPK途径影响DNA的机制通过性染色体或外部信号中的多能细胞中的甲基化。在特定目标1中，我们将缩小负责低甲基化的MAPK途径的上游和下游组件并通过蛋白质组学方法确定的测试候选目标。我们将进一步探索分子幼稚的层细胞内基因组低甲基化丧失的后果。在特定目标2中，我们将测试女性ESC中DUSP9的候选目标和AIM 1的整合结果，以定义相似性和差异在性别依赖性和环境之间（2i）诱导的低甲基化。我们将进一步描述自我我们在缺乏dusp9等位基因的ESC中发现的更新缺陷和评估其对DNA的依赖性甲基化。最后，我们将确定ESC的性别特异性甲基化差异是否源于前或植入后胚胎。在特定的目标3中，我们将调查我们是否机械连接在MAPK信号传导和DNA甲基化之间观察到的人类ESC保守了可以探索信息以增加更稳定的人类细胞。具体来说，我们将评估是否滴定靶向MAPK信号传导或使用替代MEK抑制剂的抑制剂会增加DNA甲基化和减少基因组不稳定性。总的来说，我们的工作将探索MAPK信号之间的分子联系和DNA甲基化，基因组稳定性和多能细胞中的发育潜力，其目标是解剖基本机制并定义改善人类干细胞的条件。