The role of p16INK4a in cellular senescence caused by mitotic stress after loss of Lin9
Final Report Abstract
We have recently identified the DREAM/B-MYB multiprotein complex in mammalian cells that regulates transcription during the cell cycle. DREAM consists of a core module of five proteins that dynamically associates with E2Fs, pocket proteins and the B-MYB transcription factor during the cell cycle. In quiescent cells DREAM binds to the repressors p130 and E2F4. In S-phase this binding is lost and B-MYB is incorporated into the complex. DREAM with E2F4 and p130 contributes to the repression of E2F-target genes in G0/G1 whereas DREAM/ B-MYB activates genes in G2/M. To analyze the in vivo function of DREAM, we have generated a conditional knockout model for LIN9, a conserved core unit of DREAM. We found that loss of LIN9 leads to embryonic lethality at the peri-implantation stage and that LIN9 is also essential for survival of adult mice. Deletion of Lin9 in adult mice results in rapid atrophy of the epithelium in the small intestine because of reduced proliferation. In mouse embryonic fibroblasts, loss of LIN9 resulted in severe defects in progression through mitosis and cytokinesis leading to activation of the p16INK4a-pRB and p19ARF-p53-p21CIP pathways. This ultimately results in cellular senescence. To address the contribution of both pathways to the phenotype upon deletion of LIN9, we performed experiments with the SV40 large T oncogene to inactivate the p53 and pRB pathway as well as with p16INK4A and p19ARF knockout mice. These experiments showed that both pathways are necessary to mediate the senescence phenotype upon inactivation of LIN9. In the second part of the project we focused on the mechanisms that result in activation of the pRB/p53 pathways and in the induction of senescence. In order to create defects that resemble those induced by loss of LIN9, we inhibited the mitotic kinase Aurora B and monitored activation of p21CIP1. By performing an unbiased siRNA screen of human kinases, we found that activation of p21CIP1 after Aurora B inhibition requires the MAP kinase p38. When p38 activation is prevented, p21CIP1 is no longer induced and cells fail to arrest in G1. We found that p38 MAPK is not required for stabilization of p53 or for recruitment of p53 to the p21CIP1 promoter but has a specific role in promoting transcriptional elongation of p21CIP1 in response to Aurora B inhibition. Further experiments indicate that p38 associates with the p21CIP1 gene locus and that activation of p38 MAPK activation involves reactive oxygen species. Furthermore p21CIP1 is activated in mononucleated cells after inhibition of Aurora B and binucleation is not required. This suggests that mitotic stress as opposed to tetraploidy is responsible for p21CIP1 activation. Finally, we demonstrated that the p38 MAPK pathway is also activated after inactivation of Lin9, indicating that the pathway is not specific to inhibition of Aurora B. These data suggest that senescence upon the loss of LIN9 is caused by errors in mitosis and cytokinesis because of reduced expression of proteins involved in mitotic progression. In the final part of the project we were therefore interested in identifying novel target genes of DREAM that are involved in mitotis. Transcriptional profiling identified the uncharacterized GAS2L3 protein, a tubulin and actin-binding protein, as novel target of DREAM. We found that GAS2L3 is required for cytokinesis and abscission, suggesting that its downregulation contributes to the phenotype of LIN9 knockout cells. Overall, our studies provide novel insights into the pathways that lead to senescence after mitotic stress. The pathways we identified are relevant for cancer research and for the aging process.
Publications
- (2012): GAS2L3, a target gene of the DREAM complex, is required for proper cytokinesis and genomic stability. Journal of Cell Science, 125: 2393-2406
Wolter P, Schmitt K, Fackler M, Kremling H, Probst L, Hauser S, Gruss O, and Gaubatz S
(See online at https://doi.org/10.1242/jcs.097253) - (2012): Loss of LIN9, a member of the DREAM complex, cooperates with SV40 large T antigen to induce genomic instability and anchorage-independent growth. Oncogene, 31: 1859-1868
Hauser S, Ulrich T, Wurster S, Schmitt K, Reichert N and Gaubatz S
(See online at https://doi.org/10.1038/onc.2011.364) - A role for p38 in transcriptional elongation of p21 (CIP1) in response to Aurora B inhibition (2013): Cell Cycle; 12:2051–60
Kumari G, Ulrich T, and Gaubatz S
(See online at https://doi.org/10.4161/cc.25100) - (2014): Induction of p21CIP1 Protein and Cell Cycle Arrest after Inhibition of Aurora B Kinase Is Attributed to Aneuploidy and Reactive Oxygen Species. J Biol Chem; 289:16072-84
Kumari G, Ulrich T, Krause M, Finkernagel F, Gaubatz S
(See online at https://doi.org/10.1074/jbc.M114.555060) - The GAR domain of GAS2L3 mediates binding to the chromosomal passenger complex and is required for localization of GAS2L3 to the constriction zone during abscission (2014): FEBS J; 281:2123-35
Fackler M, Wolter P, Gaubatz S
(See online at https://doi.org/10.1111/febs.12766)
