Molecular Mechanism for cellular senescence and immortalization

I.染色体末端、端粒与TTAGGG序列的串联重复序列和各种端粒蛋白通过形成大的复合物而紧密结合，在保护染色体末端免受DNA损伤反应中发挥重要作用。TRF 1和TRF 2作为同源二聚体直接与双链端粒DNA结合。另一种端粒蛋白TIN 2与TRF 1和TRF 2相互作用并使它们稳定在端粒上。此外，TIN 2与PTOP相互作用，PTOP将单链端粒DNA结合蛋白POT 1拴在端粒上。我们已经建立了小鼠TRF 1条件性敲除ES细胞，并证明TRF 1起着关键作用的“功能性”端粒结构的维护和组织这些端粒蛋白的端粒上的本地化。在新的研究中，我们进一步描述了TRF 1和其他端粒蛋白的功能相互作用，特别是对H2 AX磷酸化（H2 AX）和细胞生长的调节。首先，我们产生了鸡和小鼠融合TRF 1（cmTRF 1）， ...更多信息 N端结构域来源于鸡TRF 1，C端来源于小鼠，因为发现鸡TRF 1与小鼠Tin 2不相关。cmTRF 1蛋白与mTin 2无关，也不能挽救任何TRF 1缺陷型。接着，将全长或截短的TIN 2和PTOP与cmTRF 1融合并表达到TRF 1条件性KO ES细胞中。除了不能挽救POT 1的端粒定位的融合分子外，所有的TIN 2-或PTOP-cmTRF 1融合分子都抑制DH 2AX在端粒上的积累。总的来说，我们认为POT 1和/或PTOP是调节DH 2AX端粒的关键。然而，TIN 2-或PTOP-cmTRFl均不抑制TRFl缺陷型ES细胞的异常端粒信号。这些数据表明TRF 1对“功能性端粒”具有至少两种不同的作用，一种是依赖于TRF 1的，另一种是依赖于TRF 1相关的端粒分子。一些永生细胞使用替代端粒延长（ALT）途径来维持其端粒而不是端粒酶。以往的研究表明，同源重组（HR）有助于ALT途径。为了进一步阐明分子机制，我们在小鼠ALT胚胎干（ES）细胞中灭活了参与HR的Rad 54。尽管Rad 54缺陷型ALT ES细胞表现出与预期一致的放射敏感性，但细胞生长和端粒维持超过200次细胞分裂。此外，虽然MMC刺激的姐妹染色单体交换（SCE）被抑制在Rad 54缺陷ALT ES细胞，ALT相关的端粒SCE不受影响。这是第一个遗传学证据表明小鼠Rad 54是ALT途径的启动子。少

I. The ends of chromosomes, telomeres cohsist of the tandem repeat arrays of TTAGGG sequences and various telomeric proteins by forming a large complex which play an important role to protect the chromosomal ends from DNA damage response. TRF1 and TRF2 directly bind to double-stranded telomeric DNA as a homodimer. Another telomeric protein, TIN2 interacts with both TRF1 and TRF2 and stabilizes them on telomere. In addition, TIN2 interacts with PTOP which tethers single-stranded telomeric DNA binding protein, POT1 to telomere. We have established mouse TRF1 conditional knockout ES cells and demonstrated that TRF1 plays critical roles for the maintenance of the "functional" telomere structure and organizes the localization of these telomeric proteins on telomere. In the new study, we further describe functional interaction of TRF1 and other telomere proteins, especially on regulation of H2AX phosphorylation (H2AX) and cell growth.First, we generated chicken and mouse fusion TRF1(cmTRF1), … More N-terminal domain is derived from chicken TRF1 and C-terminal is mouse, because it was found that chicken TRF1 is not associated with mouse Tin2. The cmTRF1 protein was not associated with mTin2 and didn't rescue any TRF 1-deficient phenotypes. Next, full-length or truncated TIN2 and PTOP were fused with cmTRF1 and expressed into the TRF1 conditional KO ES cells. All the TIN2-or PTOP-cmTRF1 fusion molecules excepting the one which did not rescue POT1 of telomere localization, suppressed DH2AX accumulation on telomeres. Taken collectively, we suggest that POT1 arid/or PTOP are critical for regulation of DH2AX on telomeres. However, none of TIN2-or PTOP-cmTRFl suppressed abnormal telomere signals of TRF1-deficient ES cells. This data suggests that TRF1 possesses at least two distinct roles for "functional telomere", one is TRF 1-dependent and the other is dependent on TRF 1-associating telomere molecules.II. Some immortal cells use the alternative lengthening of telomeres (ALT) pathway to maintain their telomeres instead of telomerase. Previous studies revealed that homologous recombination (HR) contributes to the ALT pathway. To further elucidate molecular mechanisms, we inactivated Rad54 involved in HR, in mouse ALT embryonic stem (ES) cells. Although Rad54-deficient ALT ES cells showed radiosensitivity in line with expectation, cell growth and telomeres were maintained for more than 200 cell divisions. Furthermore, although MMC-stimulated sister chromatid exchange (SCE) was suppressed in the Rad54-deficient ALT ES cells, ALT-associated telomere SCE was not affected. This is the first genetic evidence that mouse Rad54 is dispensable for the ALT pathway. Less