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Dissecting the Mechanisms of Pioneer Factor Facilitated Chromatin Opening

剖析先锋因子促进染色质开放的机制

基本信息

批准号：
10501478
负责人：
Evgenia Nikolaevna Nikolova
金额：
$ 34.39万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-01 至 2027-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10501478
关键词：
Address Affect B-DNA Base Pairing Binding Binding Sites Biochemical Biological Assay Cell physiology Cells Chemicals Chromatin Chromatin Fiber Chromatin Structure Chromosomes Complex DNA DNA Packaging DNA Polymerase II DNA Sequence DNA-Binding Proteins Data Dependence Deposition Development Digestion Disease Embryonic Development Enzymes Epigenetic Process Event Future Gel Gene Activation Gene Expression Gene Expression Regulation Genetic Transcription Genome Genomic DNA Goals Histone H3 Histones Human In Vitro Investigation Kinetics Lead Link MLL gene Malignant Neoplasms Mediating Methods Methylation Modification Molecular Molecular Conformation Monitor Mutate Mutation N-terminal NMR Spectroscopy Nuclear Magnetic Resonance Nucleic Acid Regulatory Sequences Nucleosomes Organism Physiology Play Positioning Attribute Post-Translational Protein Processing Protein Dynamics Proteins RNA Regulation Regulator Genes Resolution Role Signal Transduction Site Solvents Somatic Cell Stem Cell Research Structure Tail Testing Transcription Initiation Transcriptional Activation Variant Work Writing adult neurogenesis chromatin remodeling embryonic stem cell flexibility genetic regulatory protein histone methyltransferase improved in vivo insight link protein nervous system disorder novel therapeutic intervention nuclease pluripotency preference programs self-renewal therapeutic target three dimensional structure transcription factor

项目摘要

Project Summary Nucleosomes cover most of the eukaryotic genome and present a barrier for the competent binding of many regulatory proteins that control gene expression. A special set of transcription factors called pioneer factors can bind their target sites on nucleosomal DNA, loosen chromatin, and promote the activity of other chromatin factors and enzymes, leading to nucleosome repositioning and gene activation. Sox2 and Oct4 are pioneer factors that orchestrate changes in cell fate during embryogenesis and adult neurogenesis and are major players in the reprogramming of somatic cells into a pluripotent state. Mutation and deficiency in these proteins are linked to neurological disorders, while their enrichment is tied to cancer. Despite ample studies, little is known about how to Sox2, Oct4 and other pioneer factors exert their pioneering activity at the molecular level. Our goal is to address key unresolved questions about how Sox2 and Oct4 engage with nucleosomes in distinct binding modes and alter the structure and dynamics of nucleosomes, and how these changes affect the downstream action of chromatin modifying and remodeling enzymes. Towards this goal, we propose to integrate chemical probing assays with high-resolution nuclear magnetic resonance (NMR) spectroscopy to obtain site-specific structural and dynamic information about pioneer factor interaction with nucleosomes and the perturbations they elicit in DNA and histones to potentially regulate other factors. In Aim 1, we will utilize in vitro binding assays to characterize distinct binding modes of Sox2 and Oct4 to artificial and natural nucleosomes and their dependence on DNA sequence, co-factor cooperativity, and histone composition. We will also adapt methyl TROSY NMR, suitable for studies of super-large biomolecules, to interrogate the site-specific binding conformation and dynamics of these proteins to select nucleosomes with atomic detail. In Aim 2, we will examine the effect of Sox2 and Oct4 binding in various nucleosome positions on the conformation and accessibility of nucleosomal DNA and the disordered histone tails by using NMR, chemical and enzymatic probing methods sensitive to structural and dynamic perturbations. We will further assess whether Sox2 and Oct4, through direct contact and nucleosome perturbations, stimulate the activity of histone methyltransferase enzymes that are found to collaborate in vivo with these pioneer factors. Finally, in Aim 3, we will adapt our approach to characterize how a methyl-DNA binding protein and proposed pioneer factor Kaiso binds to and alters nucleosomes, providing insight into the recognition and function of epigenetic DNA marks in chromatin regulation. The proposed work will lay the basis for future investigations of other pioneer factors and the role of DNA context, synergistic action, histone variants and epigenetic modifications into pioneer factor-mediated chromatin opening. This will improve our ability to modify pioneer factor function and lead to novel approaches for therapeutic targeting.

项目总结