Molecular impediments to fate-specifying pioneer factor activity during development

项目总结/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指定因子 增强子序列并激活它们以控制发育基因表达。增强子激活是 通常与核小体的去除有关，核小体装饰真核基因组，通常包裹 大约有150个碱基对的DNA，高度稳定。一个持续的发育基因之谜 调节是当TF最初被包裹在核小体中时，TF如何结合并激活其靶增强子， 其通常抑制TF结合。一种假设认为，一类特殊的“先驱因子”能够结合 在核小体包裹的背景下，它们的靶点并取代它们结合以激活的核小体， 使增强子暴露于下游TF结合。然而，很难证实 体内核小体结合“先锋活性”的存在，使先锋因子的发育作用， 问题我们最近使用高分辨率表观基因组分析来识别核小体结合的实例， 先锋因子在增强子处富集了次优基序结合序列，呈现出 一种有趣的可能性是，先锋活性是一种机制，以确保增强子激活的保真度在网站， 易受局部染色质环境的自然波动影响。先锋因素通常在早期 发育，尽管染色质结构的自然变化，但它仍保持高保真度，这是敏感的。 细胞的代谢状态。因此，先锋因子可能在隔离发育中起直接作用。 代谢变异的转换。然而，先驱因素在发展保真度中的潜在作用 以及对代谢异质性的缓冲作用至今尚未发现。在这个建议中，我将使用一个 受控的先锋因子表达系统，以研究先锋因子驱动的发育变化是如何 缓冲防止有意的染色质和代谢扰动。在目标1中，我将检验一个假设， 活性通过在遗传上加强染色质屏障后观察发育来促进发育保真度 涉及先锋因子结合和灭活特定先锋因子的核小体结合先锋活性。在 目的2，我将使用一个模型系统的代谢控制的发展，以了解先锋因子如何绑定 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的 发展成果的回应。这些目标将揭示这种差异的机械解释 在分子水平上基因调控过程的差异和细胞命运结果的精确性之间 在发展水平上，我的发现将直接影响到癌症等疾病， 极端的异质性破坏了对细胞命运的制衡。K99/R 00奖将发挥重要作用 在解决这些问题，并提供我在新方法和生物学理论的高水平培训 这将使我准备继续从事与先锋因子和染色质控制有关的主要研究途径 在我未来的独立职业生涯中。

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.