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Assessing the basis of chromatin opening by hematopoietic pioneer factors

通过造血先锋因子评估染色质开放的基础

基本信息

批准号：
9907544
负责人：
Megan A. Frederick
金额：
$ 4.5万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-03-01 至 2023-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9907544
关键词：
Address Affinity Binding Biochemical Biology CCAAT-Enhancer-Binding Protein-alpha Cell Fate Control Cells Chromatin Chromatin Structure Chromosomes DNA DNA Binding Domain DNA Sequence Data Deoxyribonuclease I Dependence Development Ectopic Expression Eukaryotic Cell Expression Profiling Fibroblasts Gene Expression Gene Expression Regulation Generations Genomic approach Goals Hematopoietic Hematopoietic stem cells Histones Hypersensitivity In Vitro Label Length Liver Maps Mass Spectrum Analysis Molecular Myelogenous Nucleosomes Pattern Positioning Attribute Process Proteins Regulation Research Role Structural Protein Tertiary Protein Structure Testing Therapeutic activating transcription factor cell type cellular development clinically relevant crosslink deletion analysis genome-wide in vivo insight macrophage mutant nanomolar nanopore programs protein structure reconstitution screening transcription factor

项目摘要

Project Summary/Abstract The goal of this proposal is to investigate the intrinsic ability of hematopoietic transcription factors (TFs) to engage closed chromatin and initiate chromatin opening during cell fate changes. Cell fate control is a fundamental process in biology and de novo generation of clinically relevant cell types has enormous implications for research and therapeutics; however, the general principles by which closed and unmarked chromatin is initially accessed by these factors are not fully understood. In eukaryotic cells, chromatin compaction serves as a mechanism for gene regulation by modulating the accessibility of TFs to target DNA sequences. Development of macrophages from hematopoietic stem cells requires coordinated expression of PU.1 with myeloid TFs C/EBPα and C/EBPβ, and ectopic expression of PU.1 and C/EBPα/β converts fibroblasts to the macrophage lineage. I hypothesized that PU.1, C/EBPα, and C/EBPβ act as “pioneer” factors and that initiating development requires these factors to directly bind compacted chromatin, initiate chromatin opening, and allow subsequent binding of additional TFs that activate gene expression. Our lab identified nucleosomes targeted by PU.1, C/EBPα, and C/EBPβ in vivo, and reconstituted these nucleosomes in vitro. To assess the interactions of these factors with higher-order chromatin substrates, I reconstituted fluorescently end-labeled synthetic nucleosome arrays and inserted a central nucleosome capable of binding by PU.1, C/EBPα, and C/EBPβ. I have found that PU.1, C/EBPα, and C/EBPβ bind and initiate accessibility of H1- compacted nucleosome arrays with different efficiencies. Furthermore, I have found that the DNA binding domains of PU.1 and C/EBPα open chromatin with less efficiency compared to the full-length proteins. I am now investigating the ability of these pioneer factors to open chromatin and how their mechanisms of action compare to another established pioneer factor, FoxA. I hypothesize that PU.1, C/EBPα, and C/EBPβ possess protein domains that enable chromatin interactions, and that initiating chromatin accessibility is fundamental to the ability of these factors to direct cell fate in vivo. To address this hypothesis I will 1) use nanopore sequencing to define TF-chromatin interactions and determine if PU.1, C/EBPα, and C/EBPβ interact with histones using crosslinking mass spectrometry 2) perform a deletion analysis of PU.1, C/EBPα, and C/EBPβ to map domains required for chromatin opening in vitro and determine if chromatin opening by pioneer factors is required for TF directed macrophage transdifferentiaiton. My project will provide insights into the mechanisms of chromatin structure regulation by hematopoietic TFs during cellular development and reprogramming. Furthermore, biochemical analysis will provide details of the molecular features that endow TFs with pioneering activity.

项目总结/文摘