Abstract
Although the p53 tumor suppressor can elicit cell-cycle arrest or apoptosis in hematopoietic cells upon DNA damage, its function during steady-state hematopoiesis is largely unknown. We demonstrated that the Ets transcription factor MEF/ELF4 regulates both HSC proliferation/self-renewal and quiescence, as Mef null mice exhibit greater numbers of hematopoietic stem cells and the Mef null HSCs are more quiescent than normal. As such, the hematopoietic compartment of Mef null mice shows significant resistance to chemotherapy and radiation (Lacorazza et al., Cancer Cell, 2006). In this study, we have investigated the mechanisms utilized by MEF/ELF4 to regulate the quiescence and self-renewal of hematopoietic stem cells, identifying p53 as a key regulator. We have recently found that Mef null mouse embryonic fibroblasts (mefs) accumulate p53 and undergo premature senescence; MEF appears to surpress the expression of p53 by directly upregulating Mdm2 (G. Sashida et al., submitted). We hypothesized that p53 may play a role in the enhanced stem cell quiescence or the increased HSC frequency seen in Mef null mice. To examine this, we generated p53−/− Mef −/− mice and compared HSC biology in the double knock out mice (p53−/− Mef −/−) vs p53 null mice, Mef null mice and wt mice. Loss of p53 decreased the fraction of Pyronin Ylow Lin-Sca-1+ cells, suggesting fewer quiescent HSCs, and staining of CD34-LSK cells for the proliferation marker Ki67 also showed enhanced HSC proliferation in the absence of p53 (with fewer quiescent cells present). These data suggest that p53 promotes quiescence in HSCs, and in the absence of p53, HSCs more readily enter the cell cycle. When we analyzed the DKO (p53−/− Mef −/−) mice, we observed that the percentage of G0 HSCs returned to normal, indicating that p53 is essential for maintaining the enhanced quiescence of MEF null HSCs. p21 is a major target gene of p53 in cells, and has been shown to play an important role in maintaining HSC quiescence. As expeceted, we found elevated levels of p21 mRNA in MEF null LSK cells and reasoned that p21 may account for their enhanced quiescence. We generated p21 −/− Mef −/− mice, which are viable, born at normal mendelian frequency and appear grossly normal. However, cell cycle analysis of HSCs obtained from p21 −/− Mef −/− mice showed that the enhanced quiescence in MEF null HSCs did not depend on p21, indicating that other p53 target genes play an important role in maintaining stem cell quiescence. We therefore utilized transcript profiling (Microarray studies and quantitative PCR analysis) to identify potential p53-regulated genes that may be differentialy expressed in LSK cells isolated from wild type, p53−/−, Mef −/−, and p53−/− Mef −/− mice. By ChiP and luciferase reporter assays, we show for the first time that Gfi-1 and Necdin are direct transcriptional targets of p53 in HSCs and both Gfi-1 and Necdin regulate the enhanced quiescence exhibited in MEF null HSCs. Taken together, our work defines a novel role for p53 in the maintenance of HSC quiescence. Furthermore, HSC quiescence and self-renewal appear to be mediated by different p53 target genes during steady state hematopoiesis.
Stem cell quiescence: the challenging path to activation