Assessing the basis of chromatin opening by hematopoietic pioneer factors

项目总结/摘要 本研究的目的是研究造血转录因子（TF）的内在能力， 在细胞命运改变期间接合封闭的染色质并启动染色质开放。细胞命运控制是 生物学中的基本过程和临床相关细胞类型的从头产生具有巨大的 对研究和治疗的影响;然而，封闭和未标记的一般原则 染色质最初是由这些因子进入的，目前还不完全清楚。在真核细胞中， 致密化通过调节TF对靶DNA的可接近性作为基因调控的机制 序列的从造血干细胞发育成巨噬细胞需要协调表达 PU.1与髓系TF C/EBPα和C/EBPβ的关系，以及PU.1和C/EBPα/β的异位表达转化 成纤维细胞到巨噬细胞谱系。我假设PU.1、C/EBPα和C/EBPβ作为“先锋”因子 启动发育需要这些因子直接结合紧密的染色质，启动染色质 开放，并允许随后结合额外的TF，激活基因表达。我们的实验室发现 1、C/EBPα和C/EBPβ靶向的核小体，并在体外重建这些核小体。到 为了评估这些因子与高阶染色质底物的相互作用，我用荧光重建了 末端标记的合成核小体阵列，并插入能够被PU.1结合的中心核小体， C/EBPα和C/EBPβ。我发现PU.1、C/EBPα和C/EBPβ结合并启动H1- 不同效率的紧凑核小体阵列。此外，我发现DNA结合 PU.1和C/EBPα结构域打开染色质的效率低于全长蛋白质。我是 现在正在研究这些先驱因子打开染色质的能力以及它们的作用机制 与另一个已建立的先驱因子FoxA相比。我假设PU.1、C/EBPα和C/EBPβ具有 蛋白质结构域，使染色质相互作用，并启动染色质的可及性是根本的， 这些因子在体内指导细胞命运的能力。为了解决这个假设，我将1）使用纳米孔 测序以确定TF-染色质相互作用，并确定PU.1、C/EBPα和C/EBPβ是否与 使用交联质谱法的组蛋白2）进行PU.1、C/EBPα和C/EBPβ的缺失分析 绘制体外染色质开放所需的结构域，并确定先驱因子是否导致染色质开放 是TF指导的巨噬细胞转分化所必需的。我的项目将提供深入了解 在细胞发育过程中造血TF调节染色质结构的机制， 重新编程此外，生物化学分析将提供分子特征的细节， 具有开拓性活动的TF。

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.