Genetic Dissection of Self-Renewal Pathways during Hematopoietic Differentiation.

Abstract

Hematopoietic stem cells (HSCs) differentiate into mature blood cells; while maintaining themselves for the lifetime of the organism because of their ability to self-renew. Bmi1 is required for the maintenance of HSCs and lymphocytes. While examining the deletion of Bmi1 targets in Bmi1-/- mice, we found that p16Ink4a deletion did not alleviate defects in HSCs and lymphocytes, whereas p19Arf deletion partially restored lymphocytes but not HSCs. Deletion of both p16Ink4a and p19Arf partially restored HSC functions. Trp53 deletion also partially rescued the lymphocytes and HSCs. However, none of these deletions completely restored maintenance of HSCs and lymphocytes. We conclude that the targets of Bmi1 differentially maintain subsets of hematopoietic cells. In HSCs these results suggest that Bmi1 might not require the direct action of p19Arf for regulation of Trp53, and could possibly directly regulate Trp53 turnover or mark Trp53 transcriptional targets for epigenetic silencing. We went on to examine the impact of deleting p19Arf, p16Ink4a/p19Arf, Trp53, and p16Ink4a/p19Arf/Trp53 on hematopoietic cells. There were no defects in the output of mature cells, suggesting that these deletions do not compromise differentiation. We show that bone marrow cells from mice deficient in three genes downstream of Bmi1 - p16Ink4a, p19Arf, and Trp53 (triple mutant mice)- have an approximately 10 fold increase in cells able to reconstitute hematopoiesis. This increase is associated with the acquisition of long-term reconstitution capacity by cells whose phenotype is c-kit+Sca-1+Flt3+CD150-CD48-Lineage-, which defines multipotent progenitors (MPPs) in wild-type mice. The pattern of triple mutant MPP response to growth factors resembles that of wild-type MPPs but not wild-type HSCs. MPPs lack the ability to self-renew and are destined to stop proliferating after a finite number of cell divisions. The molecular mechanisms that limit the proliferation capacity of MPPs and more mature progenitors are not understood. These results demonstrate that Ink4a/Arf and Trp53 play a central role in limiting the expansion potential of MPPs. This has implications for oncogenesis due to the fact that these pathways are commonly repressed in cancer, suggesting a mechanism by which early progenitor cells could gain the ability to self-renew and become malignant with further oncogenic mutations.