br Acknowledgments The authors thank

Acknowledgments The authors thank Kathryn D. Rodgers for critically reading the manuscript. The authors also thank Anika Voss, Department of Molecular and Cellular Sport Medicine, German Sport University Cologne, Germany, for technical assistance. The study was funded by a grant from STORZ Medical AG, Tägerwilen, Switzerland. This manuscript is dedicated to memories of Helmut Neuland (†) for his tireless effort to realize this project.
Introduction Fanconi anemia (FA) is a bone marrow failure disorder caused by the disruption of FA-BRCA network, which consists of at least 15 FA genes (including FANCD2 and FANCC) and FA-associated genes (Bagby and Alter, 2006; Kim and D\'Andrea, 2012). Bone marrow failure is the primary cause of early mortality for FA patients (Kutler et al., 2003). It has been reported that a subset of FA patients with lower levels of CHK1 and p53 expression and abrogation of the G2 Deferoxamine cost checkpoint display much milder bone marrow deficiency (Ceccaldi et al., 2011). Further mechanistic studies established that an overactive p53 response to cellular stress and DNA damage drives the progressive elimination of hematopoietic stem and progenitor cells (HSPC) in FA patients, suggesting p53 down-regulation as a potential target for FA drug design (Ceccaldi et al., 2012). It is also known that loss of p53 promotes carcinogenesis in both FA mouse models and human FA patients (Ceccaldi et al., 2011; Houghtaling et al., 2005; Freie et al., 2003). Given the detrimental tumorigenic effects of p53 loss, it is desirable to shut down only those p53-target genes specifically involved in HSPC elimination in FA. Further understanding the molecular targets of p53-mediated HSPC elimination could guide the future design of therapeutic regimens exploiting this approach. p21, encoded by the Cdkn1a gene, is a key p53-target gene and the main factor responsible for p53-mediated cell cycle arrest and apoptosis (Abbas and Dutta, 2009). p21 could even be the sole mediator of the overactive p53 response to DNA damage in FA HSPC, pointing to p21 inhibition as a way to prevent the progressive HSPC loss in FA (Murray et al., 2010; Sax et al., 2002). Interestingly, p21 deletion is already known to rescue stem cell self-renewal from mice suffering from DNA damage provoked by dysfunctional telomerase (Choudhury et al., 2007). On the other hand, it has also been reported that p21 has an important role in the regulation of FA-BRCA pathway activation (Rego et al., 2012). Therefore, the precise outcome of p21 deletion in FA patients warrants further investigation. mice on the 129S4 genetic background recapitulate major FA patient phenotypes, displaying tumor susceptibility and hematopoietic defects (Houghtaling et al., 2003; Zhang et al., 2010). Here we characterized p21 and Fancd2 double-knockout mice to understand whether p21 is involved in the FA pathway and whether the rescue effects of p53 deletion on HSC maintenance are p21-related.
Methods
Results and Discussion We reported previously that mice have readily detectable hematopoietic defects, including fewer cKit+Sca1+Lin (KSL) hematopoietic stem and progenitor cells, compromised HSC repopulating capacity, and abnormal cell cycle status of KSL cells (loss of quiescence and increased cycling) (Zhang et al., 2010). To determine whether lack of p21 has an impact on any of these parameters in mice, we characterized hematopoietic phenotypes of mice first by measuring the size of their KSL pool. As compared to wild-type controls, both mice and mice suffered a significant reduction in the size of the KSL HSPC pool (Fig. 1A). mice had the smallest HSPC population among the different genotypes. This observation indicates that p21 and Fancd2 deletion independently cause loss of hematopoietic stem and progenitor cells, suggesting that both Fancd2 and p21 are important for maintaining the normal size of HSPC pool.