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端粒酶在发育过程中的调节机制。编码人类端粒酶限制性亚基的hTERT基因主要在转录水平上受到调节。它在多能干细胞中高度表达，但在大多数成年体细胞中受到严格抑制。尽管在过去十年中进行了深入的研究，但其抑制机制，包括顺式调控因素和反式作用因素仍有待阐明。我们之前报道过，在许多体细胞中，内源性的hTERT位点嵌入在一个浓缩的染色质结构域中，而在较少抑制的小鼠TERT基因中不存在这样的结构域。与染色质在其严格调控中的重要作用相一致，我们还发现人类成纤维细胞中的外体hTERT位点不受抑制，而染色体整合的hTERT位点则重现其天然调控。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hTERT抑制很重要；2)在细胞永生化过程中，这种抑制的部分缺失导致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，即重组酶介导的BAC靶向或RMBT方法，用于将单拷贝BAC报告基因靶向整合到特定的染色体位点。使用这种技术，我们证明了含有hTERT位点的BAC构建体的染色体整合导致了替代染色质设置的建立，其中hTERT启动子被严格抑制，并在人类成纤维细胞中再现了其内源性基因。在这项应用中，我们计划追求以下具体目标：1)描述参与人类成纤维细胞中hTERT抑制的顺式元件。2)鉴定和表征人成纤维细胞中参与hTERT抑制的蛋白因子。3)确定在mESCs中赋予mTERT基因人源化调控的顺式元件。

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.