REGULATION OF THE HEMATOPOIETIC SELF-RENEWAL AND LINEAGE CHOICE: ROLE OF PBX1 AND MICRO-RNAS

Hematopoiesis is a highly regulated process (Orkin and Zon, 2008). For a constant supply of short-lived terminally differentiated blood cells, and for rapid response to hematopoietic stresses, HSCs are endowed with the ability to continuously provide more mature progenitors while properly maintaining their pool size, without exhaustion, throughout life. This equilibrium is finely maintained on one hand by precisely balancing self-renewal and differentiation in HSCs, and on the other hand by regulating proliferation and differentiation also of their downstream progenitors, including lineage choice. Thus, understanding mechanisms of regulation of self-renewal versus differentiation of HSCs and downstream progenitors is a central issue in stem cell field, since tiny alteration of these mechanisms may lead to hematopoietic failure and disease. Pbx1 is a transcription factor that positively regulates post-natal HSC quiescence. Its absence in HSCs causes an excessive proliferation that leads to their exhaustion, indicating a profound self-renewal defect, and premature myeloid differentiation at the expenses of lymphoid differentiation (Ficara et al., 2008). However, the precise molecular mechanisms through which Pbx1 exerts its function in HSCs and its role in progenitor biology are two still unexplored issues. In particular, it is not known whether Pbx1 function is also mediated by micro-RNAs, crucial new players in the regulation of proliferation and differentiation in several tissues, including the hematopoietic system. Taking advantage of Pbx1 conditional KO mice, in this study, we demonstrate that Pbx1 functions as a brake on cell differentiation not only in HSCs but also in multi-potent and myeloid-restricted progenitors, to maintain progenitor reservoirs and lymphoid potential. In absence of Pbx1, both myeloid and lymphoid progenitors are able to differentiate into mature progeny but with higher efficiency and premature kinetic for the myeloid lineage and a decreased efficiency in the lymphoid (and erythroid) lineage. Pbx1 acts also upstream to lineage restricted progenitors, affecting lineage choice of multipotent progenitors (MPPs) by restraining myeloid differentiation and allowing lymphoid differentiation. Moreover, we show that in the absence of Pbx1 HSCs display an altered micro-RNA profile, which resembles the normal MPP profile, suggesting a role for miRNAs in maintaining HSC identity. Pbx1-null HSCs and MPPs show specific lists of DE miRNAs, with substantial overlaps with the normal HSC-to-MPP transition. Combining miRNA data with transcriptional profile data of the same populations (Ficara et al., 2008) allowed searching for miRNA predicted targets whose change in expression inversely correlates with those of mRNAs. This analysis, coupled with extensive bioinformatics studies (promoter analysis, co-targeting and GSEA) allowed selection of very few candidate miRNAs to be further studied for their specific role in maintaining HSC self-renewal, and their possible regulation by Pbx1. In addition to a list of Pbx1-dependent miRNAs in HSCs and MPPs, we also analyzes for the first time miRNAs characteristic of the normal transition from HSCs to the first MPP stage, which represent an important finding for understanding miRNAs physiologically involved at the apex of hematopoietic system. Overall, this analysis set the basis for the discovery of miRNAs involved in the regulation of self-renewal versus differentiation of HSCs.