Molecular studies on mammalian telomerase

端粒酶是一种核糖核蛋白复合体，可合成端粒富含G的链DNA。端粒酶是一种特殊的逆转录酶，由催化成分TERT和模板RNA Tr组成。为了更好地了解端粒酶的调控机制，本研究首先建立了TERT^&lt；-1-&gt；基因敲除小鼠。第一代TERT^-lt；-1-&gt；基因敲除小鼠没有表现出任何表型，这表明端粒酶催化蛋白的缺失本身并不影响小鼠的发育。然而，当TERT^-lt；-1-gt；小鼠交配以保持该品系没有端粒酶时，第五代小鼠表现出显著的生殖系细胞营养不良。这些结果表明生殖细胞对端粒长度缩短特别敏感。我们和其他人已经证明，TERT基因表达水平在调节细胞中的端粒酶活性方面起着主要作用。尽管已有报道包括Myc、Sp1、ER等几种转录因子参与了…在TERT的表达方面，调控TERT的机制，特别是在正常的体细胞中的细节，仍然很大程度上是未知的。我们以正常人外周血淋巴细胞为模型系统进行了研究。我们在体外发现转录因子USF1和USF2正向调控TERT启动子。已知截短形式的USF1和2，mini-USF1和mini-USF2是由相同的基因通过选择性剪接产生的。有趣的是，我们在体外证明了mini-USF1和2对启动子的负调控。人的外周淋巴细胞大多处于静息状态，不具有端粒酶活性。当外界刺激静止的淋巴细胞时，它们开始增殖，同时开始显示端粒酶活性。我们发现，负性调节因子迷你USFs存在于静止细胞中，而不存在于激活的淋巴细胞中。相反，正向调节的全长USFs存在于静止和生长的淋巴细胞中。这些结果表明，在控制TERT表达方面，微型USFs可能对完整的USFs有明显的负面影响。较少

Telomerase is a ribonucleoprotein complex that synthesizes telomeric G-rich strand DNA de novo. It is a specialized reverse transcriptase, containing the catalytic component, TERT and the template RNA, TR.To better understand regulation mechanisms of telomerase, in this study, we first established TERT^<-1-> knockout mice. The first generation of TERT^<-1-> knockout mice did not show any phenotype, indicating that the absence of telomerase catalytic protein by itself does not affect mouse development. However, when TERT^<-1-> mice were mated to keep the line absent for telomerase, the fifth generation mice showed significantly hypotrophic germ line cells. These results indicate that germ cells are particularly sensitive to telomere length reduction. We and others have already demonstrated that TERT gene expression levels have a primary role in regulating telomerase activity in cells. Although it has been reported that several transcription factors, including Myc, Sp1, ER, are involved … More in TERT expression, details of mechanisms regulating TERT, especially in normal somatic cells, remain largely unknown. We have studied normal human peripheral lymphocytes as a model system. We found that the transcription factors USF1 and 2 positively regulate the TERT promoter in vitro. Truncated forms of USF1 and 2, mini-USF1 and mini-USF2, are known to be produced from the same genes by alternative splicing. Interestingly, we demonstrated that mini-USF1 and 2 negatively regulate the promoter in vitro. Human peripheral lymphocytes are mostly resting and do not possess telomerase activity. Upon external stimuli applied to resting lymphocytes, they start proliferation and simultaneously begin to show telomerase activity. We found that the negative regulator, mini-USFs are present in resting cells but not in activated lymphocytes. In contrast, the positive regulator, full length USFs are present both in resting and growing lymphocytes. These results suggest that mini-USFs might have dominant negative effects on full USFs in controlling TERT expression. Less