Determining the role of Tet1 in intestinal stem cell differentiation and self-renewal

摘要 肠道干细胞(ISCs)驻留在肠道隐窝中，经历持续的增殖、自我更新和 分化以维持肠上皮的功能，肠上皮大约每隔一年更换一次。 在成年后的每一周。对自我更新和分化之间的细胞命运决定的精确调控 对ISC动态平衡和损伤反应至关重要，但染色质调节在平衡这些方面的作用 决策的特点仍然很差。我们目前资助的K01专注于解剖基因组 Tet1的调控网络，催化DNA去甲基化，在ISC人群中特异富含。 这些研究的初步数据表明，Tet1的缺失会导致显著快速的隐窝增生 Tet1消融后5d内，ISC功能增强。的核心假说 建议的研究是，Tet1通过促进分化来负向调节ISC的活性。为了测试这一点 假设和扩展我们的K01，我们将：(1)表征Tet1在ISC增殖和表型中的作用 体内分化，消融后5天和4周(目标1)和(2)绘制依赖于Tet1的基因组图 在ISCs中，TET1催化活性的主要产物5-羟甲基胞嘧啶(5HmC)的景观， 祖细胞和有丝分裂后细胞(目标2)。总体而言，这些目标将扩展并与我们的 正在进行的K01研究为未来R01资助的Tet1介导的研究提供足够的初步数据 参与ISC动态平衡和再生的机制。

ABSTRACT Intestinal stem cells (ISCs) reside in intestinal crypts and undergo continual proliferation, self-renewal, and differentiation in order to maintain function of the intestinal epithelium, which is replaced approximately once a week throughout adult life. Precise regulation of cell fate decisions between self-renewal and differentiation is critical for ISC homeostasis and damage response, but the role of chromatin regulation in balancing these decisions remains poorly characterized. Our currently funded K01 focuses on dissecting the genomic regulatory network of Tet1, which catalyzes DNA demethylation and is specifically enriched in ISC populations. Preliminary data from these studies demonstrate that loss of Tet1 results in remarkably rapid crypt hyperplasia and increased ISC function, which occurs within 5 days of Tet1 ablation. The central hypothesis of the proposed study is that Tet1 negatively regulates ISC activity by promoting differentiation. To test this hypothesis and expand on our K01, we will: (1) characterize the phenotypic role of Tet1 in ISC proliferation and differentiation in vivo, at 5 days and 4 weeks post-ablation (Aim 1) and (2) map the Tet1-dependent genomic landscape of 5-hydroxymethylcytosine (5hmC), the primary product of TET1 catalytic activity, in ISCs, progenitors, and post-mitotic cells (Aim 2). Collectively, these Aims will expand on and integrate with our ongoing K01 studies to provide sufficient preliminary data for future, R01-funded investigation of Tet1-mediated mechanisms involved in ISC homeostasis and regeneration.