Lentivirale shRNA-Bibliothek mit Pipettier-Roboteranlage
Final Report Abstract
Checkpoints that limit stem cell self-renewal in response to DNA damage could influence cancer and aging but remain yet to be delineated at molecular level. Using in vivo RNAi (shRNA) screens, we show that DNA damage limits self-renewal of hematopoietic stem cells (HSCs) by upregulation of the “Basic leucine zipper transcription factor, ATF-like” (BATF). The study shows that BATF controles lymphoid differentiation of normal, non-damaged HSCs and DNA damage enhances differentiation of HSCs by inducing BATF expression in a G-CSF/STAT3 dependent manner. Inhibition of this pathway ameliorates HSC depletion in response to gamma-irradiation or telomere dysfunction. An analysis of bone marrow biopsies of patients with myelodysplastic syndromes indicates that DNA damage dependent induction of BATF is conserved in human HSCs. Together, these results provide the first experimental evidence that BATF controls lymphoid differentiation of HSCs and this molecular circuit serves as a checkpoint limiting the self-renewal of HSCs in response to DNA damage. The tumour suppressor p53 activates Puma-dependent apoptosis and p21-dependent cell-cycle arrest in response to DNA damage. Deletion of p21 improved stem-cell function and organ maintenance in progeroid mice with dysfunctional telomeres, but the function of Puma has not been investigated in this context. Here we show that deletion of Puma improves stem- and progenitor-cell function, organ maintenance and lifespan of telomere-dysfunctional mice. Puma deletion impairs the clearance of stem and progenitor cells that have accumulated DNA damage as a consequence of critically short telomeres. However, further accumulation of DNA damage in these rescued progenitor cells leads to increasing activation of p21. RNA interference experiments show that upregulation of p21 limits proliferation and evolution of chromosomal imbalances of Puma-deficient stem and progenitor cells with dysfunctional telomeres. These results provide experimental evidence that p53-dependent apoptosis and cell-cycle arrest act in cooperating checkpoints limiting tissue maintenance and evolution of chromosomal instability at stem- and progenitor-cell levels in response to telomere dysfunction. Selective inhibition of Puma-dependent apoptosis can result in temporary improvements in maintenance of telomere-dysfunctional organs.
Publications
- Puma and p21 represent cooperating checkpoints limiting self-renewal and chromosomal instability of somatic stem cells in response to telomere dysfunction. Nat Cell Biol. 2011 Dec 4;14(1):73-9
Sperka et al.
(See online at https://doi.org/10.1038/ncb2388) - A differentiation checkpoint limits hematopoietic stem cell self-renewal in response to DNA damage. Cell. 2012 Mar 2;148(5):1001-14
Wang et al.
(See online at https://doi.org/10.1016/j.cell.2012.01.040)
