Here we probed the mechanism of HFSC quiescence in normal tissue and in stress conditions of tumors and cell culture. We found several CDKis (p21, p57, p27, and p15), known to play overlapping roles in halting the cell cycle (3, 4) transcriptionally up-regulated either in the bulge at all stages or at the onset of HF quiescence. Using pulse-chase H2B-GFP mice (10) we find that p21 represses HFSC rates of proliferation in vitro and limits the HFSC extent of proliferation at catagen onset in vivo, but does not affect HFSC self-renewal rates at anagen. Overall p21 KO bulge cells undergo approximately one to two more divisions at catagen, but increased apoptosis maintains the bulge cell pool at a constant size. Mathematical modeling suggests a temporary escape from quiescence in a fraction of the KO bulge cells (SI Materials and Methods). Interestingly, loss of Dacapo, the Drosophila p21 homolog, also leads to an additional round of cell division before developmental arrest (34). The p21 KO bulge cells eventually enter quiescence likely via concerted action of other bulge up-regulated CDKis (p27, p57, and p15).
One of the upstream players in the mechanism of CDKi control in vivo appears to be the transcription factor Runx1, which may repress p21, p27, p15, and p57 mRNA production and is directly bound to the p21 promoter where it promotes H3K27me3 accumulation. We propose that p21 (and possibly p57) derepression due to down-regulation of the Runx1 protein at catagen promotes the timely onset of WT HFSC quiescence and limits the expansion of their pool (this work). Conversely, Runx1 protein expression in the bulge at anagen represses p21 expression and promotes self-renewal (16).
The Runx1/p21 dKO mice display a surprising extension of HFSC quiescence in vivo, whereas keratinocytes in culture grow normally (16). Tumors showed an intermediate effect, suggesting that additional Runx1 targets, of which Stat3 is an important player (31), are at play to promote tumor growth. These data exposed a robust SC control in vivo that seemingly limits the normal SC pool size and enforces quiescence. A potential factor in this mechanism is another CDKi, p15, which is up-regulated specifically in the bulge only in the Runx1/p21 dKO mice. Our data suggest that when cultured cells are crowded, transcriptional up-regulation of this CDKi (p15) is driven by direct synergistic derepression of its promoter via loss of p21 and Runx1. Previously, p21 and Runx1 were shown capable of acting as transcriptional repressors (14, 33, 35, 36), and we find that they bind to the predicted DNA sites on the p15 promoter. Additionally, p15 expression is able to hamper cell cycle progression of cultured keratinocytes. These data reveal the case of one CDKi as a direct transcriptional repressor of another, to finely tune the extent of quiescence (Fig. 4I).
Because SCs in tissues need not only to be quiescent to protect their genome, but also to activate rapidly when needed, it may be that high steady-state levels of CDKis are undesirable. An intriguing mechanism to keep CDKi levels in check may involve the dual role of some CDKis as CDK inhibitor and transcriptional repressors. Recently p27 was also shown to repress transcription, although direct targeting by p27 of another CDKi did not surface from this study (37). It will be interesting to know how general is the ability of one CDKi to directly repress another, such that when one is inadvertently lost, another is up-regulated to maintain proliferation control. For example, if p15 could repress p27 and p57, this would explain why the latter two were not expressed in the context of the Runx1/p21 dKO when the level of p15 was high. Clearly there is more to learn about CDKis beyond their traditional function in CDK inhibition. Testing triple-KO p21/Runx1/p15 mice will begin to address this model in vivo, although additional control layers may further enforce quiescence and possibly complicate the analysis.
Collectively, our study uncovers a complex and robust mechanism in vivo that enforces SC quiescence and a constant SC pool size and is synergistically tempered by Runx1 and, unexpectedly, by its downstream CDKi target (p21). Moreover, we unveil a role of a CDKis (p21) to antagonize proliferation by direct transcriptional repression of another CDKi (p15) in vitro, thereby modulating the strength of cell cycle arrest.
