RNAi screen for telomerase reverse transcriptase transcriptional regulators identifies HIF1α as critical for telomerase function in murine embryonic stem cells
RNAi screen for telomerase reverse transcriptase transcriptional regulators identifies HIF1α as critical for telomerase function in murine embryonic stem cells
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DOI：
10.1073/pnas.0913834107
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发表时间：
2010-08-03
影响因子：
11.1
通讯作者：
Allsopp, Richard
中科院分区：
文献类型：
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作者：
Coussens, Matthew;Davy, Philip;Allsopp, Richard
在各种类型的干细胞中，包括胚胎干（ES）细胞和造血干细胞，端粒酶的功能是通过维持端粒储备来确保长期自我更新能力。端粒酶的催化组分端粒酶逆转录酶（Tert）的表达是端粒酶活性所必需的，其在许多类型的细胞中是有限的，因此在建立端粒酶活性水平中起重要作用。然而，调节Tert在细胞（包括干细胞）中表达的机制目前知之甚少。在本研究中，我们试图通过使用靶向鼠转录调节因子的shRNA表达文库在鼠ES（mES）细胞中进行筛选来鉴定参与干细胞中Tert表达调节的基因。在该筛选中鉴定的18种候选Tert表达转录调节因子中，只有一种候选物，缺氧诱导因子1 α（Hif1 α），对mES细胞形态、存活率或生长速率没有显著影响。shRNA介导的Hif1 α表达的直接敲低证实了Hif1 α水平的抑制伴随着Tert mRNA和端粒酶活性水平的降低。此外，在Hif1 α靶向mES细胞的广泛增殖期间观察到逐渐的端粒磨损。将Hif1 α靶向mES细胞切换到低氧环境中，在很大程度上恢复了Hif1 α水平，以及Tert表达，端粒酶活性水平和端粒长度。总之，这些发现表明Hif1 α对mES细胞中端粒酶调节的直接作用，并暗示Hif1 α可能在维持干细胞中端粒酶的功能水平方面具有生理相关作用。
In various types of stem cells, including embryonic stem (ES) cells and hematopoietic stem cells, telomerase functions to ensure long-term self-renewal capacity via maintenance of telomere reserve. Expression of the catalytic component of telomerase, telomerase reverse transcriptase (Tert), which is essential for telomerase activity, is limiting in many types of cells and therefore plays an important role in establishing telomerase activity levels. However, the mechanisms regulating expression of Tert in cells, including stem cells, are presently poorly understood. In the present study, we sought to identify genes involved in the regulation of Tert expression in stem cells by performing a screen in murine ES (mES) cells using a shRNA expression library targeting murine transcriptional regulators. Of 18 candidate transcriptional regulators of Tert expression identified in this screen, only one candidate, hypoxia inducible factor 1 alpha (Hif1 alpha), did not have a significant effect on mES cell morphology, survival, or growth rate. Direct shRNA-mediated knockdown of Hif1 alpha expression confirmed that suppression of Hif1 alpha levels was accompanied by a reduction in both Tert mRNA and telomerase activity levels. Furthermore, gradual telomere attrition was observed during extensive proliferation of Hif1 alpha-targeted mES cells. Switching Hif1 alpha-targeted mES cells to a hypoxic environment largely restored Hif1 alpha levels, as well as Tert expression, telomerase activity levels, and telomere length. Together, these findings suggest a direct effect of Hif1 alpha on telomerase regulation in mES cells, and imply that Hif1 alpha may have a physiologically relevant role in maintenance of functional levels of telomerase in stem cells.