Repression of the hTERT gene during cell differentiation

摘要 我们的长期目标是确定发育过程中端粒酶的调节机制。HTERT 编码人类端粒酶限制性亚基的基因主要在 抄写。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。 细胞。尽管在过去十年中进行了深入的调查，但其抑制机制，包括顺式调控 其作用元件和反式作用因子仍有待阐明。我们之前报道过内源性的 在许多体细胞中，hTERT基因座嵌入了一个浓缩的染色质结构域，而这个结构域 在抑制较少的小鼠TERT基因中不存在。与染色质在其致密的 我们还发现，人成纤维细胞中的一个上体hTERT基因座不受抑制， 然而，染色体上整合的hTERT基因座重现了它的天然调控。因此，我们假设 1)远端元件和核心启动子之间在其天然染色质环境中的相互作用是重要的 用于hTERT抑制；以及2)这种抑制的部分丧失导致hTERT在细胞内转录 永垂不朽。为了研究端粒酶逆转录酶的抑制机制，我们开发了一个新的技术平台， 重组酶介导的BAC靶向或RMBT方法，用于靶向整合单拷贝BAC记者 进入特定的染色体位置。使用这项技术，我们证明了一种染色体整合 含有hTERT基因座的BAC构建导致在 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。 在这项申请中，我们计划追求以下具体目标：1)描述hTERT中涉及的顺式元件 人成纤维细胞的抑制作用。2)鉴定和鉴定参与hTERT抑制的蛋白质因子 人类成纤维细胞。3)确定mESCs中mTERT基因人源化调控的顺式元件。

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.