Repression of the hTERT gene during cell differentiation

描述（由申请人提供）：我们的长期目标是确定发育过程中端粒酶调控的机制。hTERT基因编码人端粒酶的限制性亚基，主要在转录水平上受到调控。它在多能干细胞中高度表达，但在大多数成体体细胞中受到严格抑制。尽管过去十年进行了深入的研究，但其抑制机制（包括顺式调节元件和反式作用因子）仍有待阐明。我们以前报道，内源性hTERT基因座是嵌入在一个浓缩的染色质结构域在许多体细胞，而这样的结构域不存在于较少抑制的小鼠TERT基因。与染色质在其紧密调控中的重要作用一致，我们还发现，人成纤维细胞中的附加型hTERT基因座不受抑制，而染色体整合的hTERT基因座重现了其天然调控。因此，我们假设1）远端元件和核心启动子在其天然染色质背景下的相互作用对于hTERT抑制是重要的;和2）这种抑制的部分丧失导致细胞永生化期间的hTERT转录。为了研究hTERT抑制的机制，我们开发了一种新的技术平台，重组酶介导的BAC靶向或RMBT方法，用于将单拷贝BAC报告基因靶向整合到指定的染色体位点。使用这种技术，我们证明，染色体整合的BAC构建体含有hTERT基因座导致建立一个替代染色质设置中的hTERT启动子被紧紧抑制，并重演其内源性基因在人成纤维细胞。在本申请中，我们计划追求以下具体目标：1）描绘人成纤维细胞中参与hTERT抑制的顺式元件。2)鉴定和表征人成纤维细胞中参与hTERT抑制的蛋白质因子。3)确定mESC中赋予mTERT基因人源化调节的顺式元件。 端粒是线性染色体的末端，对细胞的长期增殖和存活至关重要。端粒酶是一种延长端粒的酶，在癌症和衰老相关疾病中起着重要作用。本基金申请旨在研究人类体细胞端粒酶调控的机制。

DESCRIPTION (provided by applicant): Our long-term goal is to determine the mechanisms of telomerase regulation during development. The hTERT gene, which encodes the limiting subunit of human telomerase, is primarily regulated at the level of transcription. It is highly expressed in pluripotent stem cells, but stringently repressed in most adult somatic cells. Despite intensive investigation in the past decade, mechanisms of its repression, including cis-regulatory elements and trans-acting factors remain to be elucidated. We previously reported that the endogenous hTERT locus was embedded in a condensed chromatin domain in many somatic cells, while such a domain did not exist in the less repressed mouse TERT gene. Consistent with the vital role of chromatin in its tight regulation, we also found that an episomal hTERT locus in human fibroblasts was not subjected to repression, whereas a chromosomally integrated hTERT locus recapitulated its native regulation. Thus, we hypothesize that 1) the interplay between distal elements and core promoter in their native chromatin context is important for hTERT repression; and 2) partial loss of this repression leads to hTERT transcription during cellular immortalization. To study the mechanisms of hTERT repression, we developed a novel technical platform, the recombinase-mediated BAC targeting or RMBT method, for targeted integration of single-copy BAC reporters into specified chromosomal sites. Using this technique, we demonstrated that chromosomal integration of a BAC construct containing the hTERT locus resulted in the establishment of a surrogate chromatin setting in which the hTERT promoter was tightly repressed and recapitulated its endogenous gene in human fibroblasts. In this application, we plan to pursue the following specific aims: 1) Delineate cis elements involved in hTERT repression in human fibroblasts. 2) Identify and characterize protein factors involved in hTERT repression in human fibroblasts. 3) Determine cis elements that confer humanized regulation of the mTERT gene in mESCs. PUBLIC HEALTH RELEVANCE: Telomeres are ends of linear chromosomes and essential for long-term cell proliferation and survival. Telomerase, the enzyme that elongates telomeres, plays an important role in cancers and aging-related diseases. This grant application is proposed to determine the mechanisms of telomerase regulation in human somatic cells.