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Role of S-nitrosylation in Transdifferentiation

S-亚硝基化在转分化中的作用

基本信息

批准号：
9906255
负责人：
JOHN P COOKE
金额：
$ 40.38万
依托单位：
METHODIST HOSPITAL RESEARCH INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-04-01 至 2022-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9906255
关键词：
Binding Bioinformatics Cell Lineage Cell Nucleus Cells ChIP-seq Chromatin Consensus Consensus Sequence DNA DNase I hypersensitive sites sequencing Data Data Set Endothelial Cells Enzymes Epigenetic Process Equilibrium Fibroblasts Genes Genetic Global Change Histone Deacetylase Histones Immune signaling Immunoblot Analysis Inflammatory Innate Immune Response Messenger RNA Methods NOS2A gene Nitric Oxide Synthase Nobel Prize Nuclear Nuclear Translocation Paper Pathologic Pattern recognition receptor Peptides Pharmacology Phenotype Play Process Proteins Publishing Reporting Retroviral Vector Role Signal Transduction Somatic Cell TLR3 gene Testing Therapeutic Transcriptional Regulation Transfection Viral Viral Vector Work c-myc Genes cell transformation data integration histone acetyltransferase immune activation induced pluripotent stem cell insight matrigel nuclear reprogramming pluripotency transcriptome sequencing transdifferentiation

项目摘要

PROJECT SUMMARY We discovered that innate immune activation facilitates nuclear reprogramming to pluripotency or to a different cell lineage. Our characterization of this process (Lee et al, Cell 2012) 1 has produced new insights into the mechanisms of nuclear reprogramming. We discovered that innate immune activation causes global changes in the expression of epigenetic modifiers, associated with histone markings that favor nuclear reprogramming. We intend to further elucidate the mechanisms by which innate immune activation facilitates direct reprogramming (transdifferentiation). A major factor involved in innate immune response is the inducible enzyme nitric oxide synthase (iNOS). Our data indicates that iNOS translocates to the nucleus during transdifferentiation and may directly bind and S-nitrosylate epigenetic modifiers. Accordingly, we intend to: 1. Assess the direct effects of S-nitrosylation on epigenetic regulators during transdifferentiation. We will assess the effect of S-nitrosylation on the activity of selected epigenetic factors, and/or their binding to co- factors or chromatin, using immunoblot analyses, IP and ChIP, and the effect of genetic or pharmacologic manipulation of iNOS. We will also elucidate the importance of iNOS translocation and its physical association with epigenetic modifiers in transdifferentiation using genetic and pharmacological methods to disrupt iNOS translocation and binding to selected epigenetic modifiers. 2. Characterize the effects of S-nitrosylation on DNA accessibility and fidelity of transdifferentiation. We will characterize open chromatin regions using DNAse-Seq and Mnase-Seq; bivalent chromatin markings by ChIP-seq, integrated with a global assessment of the transcriptional regulation (RNA-Seq) and determine the importance of iNOS in the fidelity of epigenetic changes required for activation of cell identity genes.

项目摘要我们发现，先天免疫激活促进核重编程为多能性或不同的功能。细胞谱系我们对这一过程的表征（Lee等人，Cell 2012）1产生了对这一过程的新见解。核重组机制。我们发现先天性免疫激活导致了在表观遗传修饰剂的表达中，与有利于核重编程的组蛋白标记相关。我们打算进一步阐明先天性免疫激活促进直接免疫的机制。转分化（transdifferentiation）参与先天免疫反应的一个主要因素是可诱导的一氧化氮合酶（iNOS）。我们的数据表明，iNOS易位到细胞核中，转分化，并可能直接结合和S-亚硝基化表观遗传修饰。因此，我们打算： 1.评估转分化过程中S-亚硝基化对表观遗传调节因子的直接影响。我们将评估S-亚硝基化对所选表观遗传因子的活性和/或其与共因子或染色质，使用免疫印迹分析，IP和ChIP，以及遗传或药理学的影响，操纵iNOS。我们还将阐明iNOS易位的重要性及其物理关联在转分化中使用表观遗传修饰剂，使用遗传和药理学方法破坏iNOS 易位和结合到选定的表观遗传修饰剂。 2.表征S-亚硝基化对DNA可及性和转分化保真度的影响。我们将使用DNAse-Seq和Mnase-Seq表征开放的染色质区域;二价染色质标记通过ChIP-seq，与转录调控的全球评估（RNA-Seq）相结合并确定 iNOS在细胞身份基因激活所需的表观遗传变化的保真度中的重要性。