Repression of the hTERT gene during cell differentiation

细胞分化过程中 hTERT 基因的抑制

• 批准号: 8534147
• 负责人: JIYUE ZHU
• 金额: $ 10.72万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8534147
• 关键词: Address; Adult; Aging; Applications Grants; Beryllium; Cancer Model; Cell Differentiation process; Cell Proliferation; Cell Survival; Characteristics; Chromatin; Chromosomes; Consensus; Data; Dependency; Development; Disease; Distal; Elements; Embryonic Development; Environment; Enzymes; Epigenetic Process; Fibroblasts; Funding; Gene Expression Regulation; Genes; Genetic Engineering; Genetic Screening; Genetic Transcription; Genomics; Goals; Human; Indium; Investigation; Malignant Neoplasms; Mediating; Methods; Mouse Strains; Mus; Play; Pluripotent Stem Cells; Proteins; RNA Interference; Regulation; Regulatory Element; Reporter; Reporting; Repression; Repressor Proteins; Role; Sequence-Specific DNA Binding Protein; Site; Somatic Cell; Specific qualifier value; T cell differentiation; TERT gene; Techniques; Telomerase; Testing; Tissues; Trans-Activators; Transcriptional Regulation; Transgenic Mice; Work; Yin-Yang; abstracting; age related; base; cancer cell; embryonic stem cell; expectation; genetic regulatory protein; improved; innovation; mouse model; novel; osteogenic; promoter; recombinase; stem cell differentiation; telomere; tumorigenesis

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.

抽象的 我们的长期目标是确定发育过程中端粒酶的调节机制。端粒酶 编码人类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，但其压制机制，包括顺式监管 元素和反式作用因子仍有待阐明。我们之前报道过，内源性 hTERT 位点嵌入在许多体细胞中的浓缩染色质结构域中，而该结构域 不存在于抑制较少的小鼠 TERT 基因中。与染色质在其紧密结合中的重要作用一致 调节，我们还发现人成纤维细胞中的附加型 hTERT 位点没有受到抑制， 而染色体整合的 hTERT 位点则重现了其天然调控。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很重要 用于 hTERT 抑制； 2) 这种抑制的部分丧失导致细胞内 hTERT 转录 永生化。为了研究 hTERT 抑制机制，我们开发了一个新颖的技术平台， 重组酶介导的 BAC 靶向或 MBT 方法，用于单拷贝 BAC 报告基因的靶向整合 进入指定的染色体位点。使用这种技术，我们证明了染色体整合 含有 hTERT 位点的 BAC 构建体导致在 其中 hTERT 启动子被严格抑制并在人成纤维细胞中重演其内源基因。 在此应用中，我们计划实现以下具体目标：1）描绘hTERT中涉及的顺式元件 人成纤维细胞的抑制。 2) 鉴定和表征参与 hTERT 抑制的蛋白质因子 人类成纤维细胞。 3) 确定赋予 mESC 中 mTERT 基因人源化调控的顺式元件。