Mechanisms and function of Tet1 regulated gene networks in intestinal stem cell biology

摘要 肠干细胞（ISCs）位于小肠中称为隐窝的腺体结构的基部， 上皮ISCs是由自我更新和多能性的功能特征定义的，被认为存在于 作为多个不同的群体，并表现出显着的可塑性，在稳态以及在损伤后 再生这种可塑性的遗传调节，特别是关于无数的染色质调节因子， 元素，仍然不完全理解。在这个职业生涯阶段，我的主要目标是补充我的 在ISC生物学的专业知识与基因组学的新培训，以建立一个独立的职业生涯，重点是 染色质调控。我的初步数据表明Tet 1，这是一个关键的调节DNA 在与活性和储备ISC一致的细胞群体中，脱甲基化特异性上调。 此外，PRC 2复合物的亚基，其被TET 1募集到靶位点， 在这两个ISC群体之间表达。本研究的中心假设是Tet 1建立了 不同的基因调控网络在活跃和储备ISCs。为了验证这个假设， 目标，我将采取双管齐下的方法：（1）通过RNA表征Tet 1/PRC 2基因调控网络- 和ChIP-seq（目标1和2）和（2）应用这些基因组规模的发现来理解功能结果 在Tet 1-null ISC中。这一策略将使我获得专业知识，在实验，概念和实践 成功实现我的独立职业目标所必需的方法，以及提供充足的数据来建立一个 为后续的独立融资奠定基础。

ABSTRACT Intestinal stem cells (ISCs) reside at the base of glandular structures, termed crypts, in the small intestinal epithelium. ISCs are defined by functional characteristics of self-renewal and multipotency, are thought to exist as multiple distinct populations, and exhibit remarkable plasticity during homeostasis as well in post-damage regeneration. The genetic regulation of this plasticity, especially in regards to myriad chromatin regulator elements, remains incompletely understood. At this career stage, my central goal is to supplement my expertise in ISC biology with new training in genomics in order to establish an independent career focused on chromatin regulation in ISCs. My preliminary data demonstrate that Tet1, which is a critical regulator DNA demethylation, is specifically upregulated in cell populations consistent with active and reserve ISCs. Additionally, subunits of the PRC2 complex, which is recruited to target sites by TET1, are differentially expressed between these two ISC populations. The central hypothesis of this study is that Tet1 establishes distinct gene regulatory networks in active and reserve ISCs. To test this hypothesis and meet my training goals, I will take a two-pronged approach to: (1) characterize the Tet1/PRC2 gene regulatory network by RNA- and ChIP-seq (Aims 1 and 2) and (2) apply these genome-scale findings to understanding functional outcomes in Tet1-null ISCs. This strategy will allow me to gain expertise in the experimental, conceptual, and practical approaches necessary to succeed in my independent career goals, as well as provide ample data to build a foundation for subsequent independent funding.