Repression of the hTERT gene during cell differentiation

摘要 我们的长期目标是确定端粒酶在发育过程中的调控机制。的hTERT 编码人端粒酶限制亚基的基因主要在 转录。它在多能干细胞中高度表达，但在大多数成人体细胞中被严格抑制。 细胞尽管在过去的十年中进行了深入的研究，但其抑制机制，包括顺式调节， 元素和反式作用因子仍有待阐明。我们以前报道过， 在许多体细胞中，hTERT位点嵌入在一个浓缩的染色质结构域中，而这样的结构域 不存在于较少抑制的小鼠TERT基因中。与染色质在其紧密结合中的重要作用相一致 调控，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环境中的相互作用是重要的 对于hTERT抑制;和2）这种抑制的部分丧失导致细胞内hTERT转录， 永生为了研究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，用于单拷贝BAC报告基因的靶向整合 插入特定的染色体位点使用这种技术，我们证明了染色体整合的一个 含有hTERT基因座的BAC构建体导致在细胞中建立替代染色质设置。 hTERT启动子在人成纤维细胞中被紧密抑制并重现其内源基因。 在本申请中，我们计划实现以下具体目标：1）描述hTERT中的顺式元件 人成纤维细胞中的抑制。2)鉴定和表征参与hTERT抑制的蛋白质因子， 人成纤维细胞。3)确定mESC中赋予mTERT基因人源化调节的顺式元件。

Abstract 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.