Molecular Mechanism for cellular senescence and immortalization

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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