Although mechanisms of telomere protection are well-defined in differentiated cells,how stem cells sense and respond to telomere dysfunction,in particular telomeric double-strand breaks(DSBs),is poorly characterized.H...Although mechanisms of telomere protection are well-defined in differentiated cells,how stem cells sense and respond to telomere dysfunction,in particular telomeric double-strand breaks(DSBs),is poorly characterized.Here,we report the DNA damage signaling,cell cycle,and transcriptome changes in human induced pluripotent stem cells(iPSCs)in response to telomere-internal DSBs.We engineer human iPSCs with an inducible TRF1-FokI fusion protein to acutely induce DSBs at telomeres.Using this model,we demonstrate that TRF1-FokI DSBs activate an ATR-dependent DNA damage response,which leads to p53-independent cell cycle arrest in G2.Using CRISPR–Cas9 to cripple the catalytic domain of telomerase reverse transcriptase,we show that telomerase is largely dispensable for survival and lengthening of TRF1-FokI-cleaved telomeres,which instead are effectively repaired by robust homologous recombination(HR).In contrast to HR-based telomere maintenance in mouse embryonic stem cells,where HR causes ZSCAN4-dependent extension of telomeres beyond their initial lengths,HR-based repair of telomeric breaks is sufficient to maintain iPSC telomeres at a normal length,which is compatible with sustained survival of the cells over several days of TRF1-FokI induction.Our findings suggest a previously unappreciated role for HR in telomere maintenance in telomerase-positive iPSCs and reveal distinct iPSC-specific responses to targeted telomeric DNA damage.展开更多
基金supported by the NIH(R01HL148821 and F31CA260918)University of Pennsylvania Abramson Cancer Center(Patel Scholar Award)Pennsylvania Department of Health(Health Research Formula Fund).
文摘Although mechanisms of telomere protection are well-defined in differentiated cells,how stem cells sense and respond to telomere dysfunction,in particular telomeric double-strand breaks(DSBs),is poorly characterized.Here,we report the DNA damage signaling,cell cycle,and transcriptome changes in human induced pluripotent stem cells(iPSCs)in response to telomere-internal DSBs.We engineer human iPSCs with an inducible TRF1-FokI fusion protein to acutely induce DSBs at telomeres.Using this model,we demonstrate that TRF1-FokI DSBs activate an ATR-dependent DNA damage response,which leads to p53-independent cell cycle arrest in G2.Using CRISPR–Cas9 to cripple the catalytic domain of telomerase reverse transcriptase,we show that telomerase is largely dispensable for survival and lengthening of TRF1-FokI-cleaved telomeres,which instead are effectively repaired by robust homologous recombination(HR).In contrast to HR-based telomere maintenance in mouse embryonic stem cells,where HR causes ZSCAN4-dependent extension of telomeres beyond their initial lengths,HR-based repair of telomeric breaks is sufficient to maintain iPSC telomeres at a normal length,which is compatible with sustained survival of the cells over several days of TRF1-FokI induction.Our findings suggest a previously unappreciated role for HR in telomere maintenance in telomerase-positive iPSCs and reveal distinct iPSC-specific responses to targeted telomeric DNA damage.
