Repression of the hTERT gene during cell differentiation

描述(由申请人提供)：我们的长期目标是了解hTERT基因在发育过程中的关键调控步骤。HTERT基因编码人类端粒酶限速亚基，在胚胎组织和干细胞中高水平表达，但在大多数成体细胞分化时被抑制。虽然已经对hTERT启动子进行了广泛的研究，但染色质环境和顺式元件对内源性hTERT转录的抑制作用尚未确定。 以前的结果，包括我们自己的结果，已经表明天然染色质在hTERT转录调控中起着关键作用。HTERT转录可以通过抑制组蛋白脱乙酰酶可逆地诱导，这种诱导伴随着hTERT启动子的染色质重塑。此外，我们和其他人已经证明，瞬时转移的质粒报告程序不是在体细胞中抑制内源性hTERT基因的理想模型。基于这些发现，我们假设染色质环境是抑制天然hTERT启动子的调控机制的关键组成部分。在这里，我们计划以TPA诱导U937细胞分化为模型，在染色质背景下研究hTERT抑制的分子细节。我们建议追求三个相互关联的目标：(1)确定全球染色质环境在分化过程中hTERT抑制中的作用；(2)确定在分化过程中发生在hTERT核心启动子区域的核因子招募和组蛋白修饰的顺序事件。(3)构建一种新的基于染色体的报告系统，分析顺式调控元件在hTERT抑制中的作用。

DESCRIPTION (provided by applicant): Our long-term goal is to understand the critical steps of hTERT gene regulation during development. The hTERT gene, which encodes the rate-limiting subunit of human telomerase, is expressed at a high level in embryonic tissues and stem cells, but is repressed upon differentiation in the majority of adult somatic cells. Although the hTERT promoter has been studied extensively, the contribution of chromatin environment and the requirement of cis-elements have not been determined for the repression of the endogenous hTERT transcription. Previous results, including our own, have indicated that native chromatin plays a critical role in the regulation of hTERT transcription. hTERT transcription can be reversibly induced by inhibition of histone deacetylases and this induction is accompanied by chromatin remodeling at the hTERT promoter. Furthermore, we and others have shown that transiently transfected plasmid reporters are not ideal models for the repression of endogenous hTERT gene in somatic cells. Based on these findings, we hypothesize that chromatin environment is a critical component of the regulatory mechanisms for the repression of the native hTERT promoter. Here, we plan to study the molecular details of hTERT repression in a chromatin context using TPA-induced U937 cell differentiation as a model. We propose to pursue three interconnected aims: (1) To determine the role of global chromatin environment in hTERT repression during differentiation; (2) To determine sequential events of nuclear factor recruitment and histone modifications that occur at the hTERT core promoter region during differentiation. (3) To create a novel chromosome-based reporter system and dissect the roles of cis-regulatory elements in hTERT repression.