Micro RNA Regulate Developmental Deficits In The Hematopoietic Stem Cell Compartment In Fanconi Anemia

Hematopoietic failure is the predominant manifestation of Fanconi anemia (FA), a recessively inherited disorder in DNA double strand break (DSB) repair. The etiology underlying the progressive loss of hematopoietic stem and progenitor cells (HSPC) in FA remains to be fully clarified, but is widely considered to be mediated by the “stress activation” of p53. In a murine model of FA (Fancc), we recently showed that the onset of hematopoietic failure precedes the developmentally timed HSPC migration to the bone marrow (BM), corroborating prior reports of reduced cord blood progenitor frequency and work in human ES cell lines. Comparison of genotype frequencies at E14.5 and postnatally (25% vs 19%; p<0.004) further indicated that hematopoietic failure coincides with fetal loss during late gestation. This data has now been replicated in Fancd2 animals. The quantitative and qualitative severity of fetal HSPC pool defects in Fancc and Fancd2 mice suggested to us the possibility of a unique developmental phenotype. Fetal liver HSPC are characterized by specific molecular and functional features to support the high self-renewal capacity during developmental pool expansion. To determine the molecular basis of fetal HSPC losses in FA, we systematically studied progenitor cell populations from Fancc E14.5 fetal liver and postnatal bone marrow for the expression of candidate genes involved in HSPC function. Results confirmed in utero genotype-specific p53 pathway activation and differences in key cell cycle regulators (p21, p27 and p57). Among a group of developmentally dysregulated genes, we found the non-histone chromatin remodeling factor Hmga2 substantially upregulated in FA versus WT E14.5 cells. As an important developmental regulator of self-renewal activity, Hmga2 is not expressed postnatally, which we confirmed in FA bone marrow cells. To dissect the FA phenotype further, we investigated the expression of other components of the developmental signaling cascade, in particular micro RNA (miR) let7b, the major let7 family member accounting for translational suppression of Hmga2 during late gestation. Results indicate that let7b is downregulated in E14.5 Fancc and Fancd2 progenitor cells. To reconcile the in utero activation of a major pathway promoting self-renewal and HSPC expansion with the functional and numeric defects we found in fetal FA HSPC, we investigated other upstream components. Systematic dissection of the signaling cascade revealed an 80% decline by q RT-PCR of miR-125b, a stem cell regulatory miRNA and nodal point that targets let7b (indirectly via Lin28 suppression), but also prevents inappropriate p53 activation. A relatively more profound decrease in miR-125b expression in Sca-1+ cells substantiate this as a stem/progenitor cell phenotype. The data are further consistent with our results showing the upregulation of p21 in E14.5 FL cells (as an indicator of p53 pathway activation), suggesting that the loss of miR-125b expression is a proximal mechanism for p53 upregulation in FA HSPC. Finally, when we tested for evidence of DNA damage, we observed gamma-H2AX foci in sorted HSPC as well as increased transcriptional activation of DNA repair via non-homologous end-joining (Ku70, Ku80, Prkdc transcripts) in Sca-1 enriched Fancc and Fancd2 FL cells. Based on the results to-date, we propose a model whereby relatively decreased expression of miR-125b serves as a key regulatory step for the activation of a critical self-renewal pathway (let7b-Hmga2), but also dysregulates p53 signaling during FA hematopoietic development. Accruing and unresolved DNA damage in repair-deficient fetal FA HSPC curtails their net expansion. The functional compromise in the murine HSPC pool reveals a regulatory role for miRNA in FA HSPC failure and provides a unique model for the mechanistic study of HSPC loss under physiologic conditions and without the experimental induction via exogenous cytokines, aldehydes or alkylating agents.