Molecular impediments to fate-specifying pioneer factor activity during development

项目摘要/摘要 转录因子通过与DNA结合，在发育过程中作为细胞分化的特定因子 增强子序列并激活它们以控制发育基因的表达。增强子激活是 通常与核小体的去除有关，核小体修饰真核基因组并通常包裹 大约150个碱基对的DNA在高度稳定的配置中。一个长期存在的发育基因之谜 调控是指当转录因子最初包裹在核小体中时，它们如何结合和激活其靶向增强子， 它们通常会抑制转铁蛋白结合。一种假说认为，一类特殊的“先锋因子”能够将 它们的靶标在核小体包裹的背景下，并取代它们结合以激活和 暴露增强子以进行下游转铁蛋白结合。然而，要证实这一点非常困难 体内存在核小体结合的“先锋活性”，使先锋因子在体内的发育作用 问题。我们最近使用高分辨率表观基因组图谱来鉴定核小体结合的实例，通过 先锋因子在增强子上富含次优基序结合序列，呈现 耐人寻味的可能性是，先锋活动是一种机制，以确保增强子激活在 容易受到当地染色质环境中自然波动的影响。先锋因素往往在早期发挥作用 发展，它保持高保真，尽管染色质结构的自然变化，是敏感的 细胞的代谢状态。因此，先锋因素可能在绝缘发育中起到直接作用。 针对新陈代谢差异的过渡。然而，先锋因素在发展保真度中的潜在作用 到目前为止，针对代谢异质性的缓冲作用尚未被发现。在本提案中，我将使用 受控先锋因子表达系统研究先锋因子如何驱动发育变化 缓冲了故意的染色质和新陈代谢干扰。在目标1中，我将检验先驱的假设 活动通过观察在遗传上强制实施染色质障碍后的发育来促进发育保真度 先锋因子结合和失活特定先锋因子的核小体结合先锋活性。在……里面 目标2，我将使用代谢控制发育的模型系统来理解先锋因子是如何结合的 对代谢变化的反应，以及特定的先锋因子-增强子激活事件如何导致不同 作为回应的发展成果。这些目标将揭示这种差距的机械性解释。 分子水平上基因调控过程的差异与细胞命运结果的精确度之间的关系 在发育水平上，我的发现将对癌症等疾病产生直接影响 极端的异质性压倒了对细胞命运的制衡。K99/R00大奖将具有重要意义 在回答这些问题并为我提供高水平的新方法和生物学理论培训方面 这将使我做好准备，继续从事与先锋因子和染色质控制相关的主要研究途径 在我未来的独立职业生涯中有很大的发展空间。

Project Summary/Abstract Transcription factors act as specifying agents of cell differentiation during development by binding to DNA enhancer sequences and activating them to control developmental gene expression. Enhancer activation is typically associated with the removal of nucleosomes, which decorate eukaryotic genomes and normally wrap roughly 150 base pairs of DNA in a highly stable configuration. A persistent puzzle of developmental gene regulation is how TFs bind and activate their target enhancers when they are initially wrapped in nucleosomes, which typically inhibit TF binding. One hypothesis posits that a special class of “pioneer factors” are able to bind their targets in the context of nucleosomal wrapping and displace the nucleosomes they bind to activate and expose the enhancer for downstream TF binding. However, it has been exceedingly difficult to confirm the presence of nucleosome binding “pioneer activity” in vivo, leaving the developmental roles of pioneer factors in question. We recently used high-resolution epigenome profiling to identify instances of nucleosome binding by pioneer factors that were enriched at enhancers with suboptimal motif binding sequences, presenting the intriguing possibility that pioneer activity is a mechanism to ensure the fidelity of enhancer activation at sites that are vulnerable to natural fluctuations in the local chromatin environment. Pioneer factors often function in early development, which maintains high fidelity despite natural variation in chromatin structure that is sensitive to the metabolic state of the cell. Therefore, pioneer factors may play a direct role in insulating developmental transitions against metabolic variance. However, the potential roles of pioneer factors in developmental fidelity and buffering against metabolic heterogeneity have not been uncovered to date. In this proposal, I will use a controlled pioneer factor expression system to study how pioneer factor-driven developmental changes are buffered against deliberate chromatin and metabolic perturbations. In Aim 1, I will test the hypothesis that pioneer activity facilitates developmental fidelity by observing development after genetically enforcing chromatin barriers to pioneer factor binding and inactivating the nucleosome binding pioneer activity of a specific pioneer factor. In Aim 2, I will use a model system of metabolic control of development to understand how pioneer factor binding responds to metabolic changes, and how specific pioneer factor-enhancer activation events underlie different developmental outcomes in response. These Aims will uncover mechanistic explanations for the disparity between variance in gene regulatory processes on the molecular level and the precision of cell fate outcomes on the developmental level, and my findings will be of direct consequence to diseases such as cancer where extreme heterogeneity overwhelms the checks and balances on cell fate. A K99/R00 Award will be instrumental in addressing these questions and furnishing me with high level training in new methods and biological theory that will prepare me to continue to pursue major research avenues related to pioneer factor and chromatin control of development in my future independent career.