Functional analysis of selected hox homeobox genes in hematopoiesis

Hematopoiesis is an ordered process of differentiation and proliferation, leading to the generation of mature blood cells of multiple distinctive lineages from totipotent hematopoietic stem cells. Regulation of this impressive machinery occurs at multiple levels, involving both extrinsic and intrinsic regulators. The members of the Hox homeobox gene family of transcription factors, known for their roles in determination of cell identity and pattern formation in a variety of structures during embryogenesis, have recently come under scrutiny as important regulators of hematopoiesis. In normal hematopoietic cells Hox genes have been shown to be expressed at levels that vary both as a function of the stage of differentiation of the cells, and of the specific Hox gene examined. Thus, some genes like HOXB3 and HOXB4 are preferentially expressed in the most primitive fraction of hematopoietic cells, and others like HOXA10 and HOXB9 show a broader range of expression with downregulation at later stages of hematopoietic differentiation. Furthermore, our group has recently shown that retroviral overexpression of HOXB4 in murine hematopoietic cells causes selective expansion of primitive hematopoietic cells, most profoundly the totipotent hematopoietic stem cell (HSC), without altering either myeloid or lymphoid differentiation or predisposing to leukemia. Taken together, these results strongly implicate Hox genes in the regulation of early hematopoietic cells. The major objective of this thesis was to investigate further the possible roles of Hox genes in the regulation of hematopoietic cell growth and differentiation, and to determine whether these roles are Hox gene-specific. The strategy taken was to independently engineer the overexpression of selected Hox genes in murine bone marrow cells. Two Hox genes were chosen, HOXB3 and HOXA10, based on their divergent expression patterns in hematopoietic cells. The subsequent effects of these manipulations on the proliferation and differentiation of various populations of myeloid and lymphoid cells were then analyzed in a transplantation model and various in vitro cultures. Overexpression of either of these two Hox genes was found to impact on both the processes of proliferation and differentiation of hematopoietic cells, involving multiple lineages. Specifically, overexpression of HOXA10 led to enhanced formation of megakaryocytic progenitors in vivo and in vitro, diminished numbers of macrophage and B lymphoid progenitors, and the generation of myeloid leukemias in a significant proportion of mice 5 to 8 months after transplantation. In contrast, overexpression of HOXB3 led to an almost complete block in the thymic production of CD4+CD8+ T lymphocytes accompanied by an expansion of yS-TCR+ thymocytes, impaired B lymphoid development, and enhanced myelopoiesis leading to a myeloproliferative disorder. The hematopoietic perturbations generated by overexpression of HOXB3and HOXAWwere thus strikingly different from each other and also from those previously reported for the overexpression of HOXB4, which did not detectably perturb myeloid, B or T cell differentiation but did induce expansion of myeloid and lymphoid progenitor cells, and enhanced up to 47-fold the regeneration of the HSC. This thesis work was also aimed at delineating further the effects of HOXB4 overexpression on the regenerative potential of HSC. For that purpose, the size and the clonal composition of the regenerated pool of HSCs in mice transplanted with bone marrow cells overexpressing HOXB4 was analyzed at various time points (16 to 52 weeks) after transplantation. These studies, in addition to confirming our initial observation that HOXB4 overexpression enhances the regeneration of HSC following transplantation, show that this effect is long-lasting, and that the expansion of HSCs appears both to be controlled and to involve multiple HSCs. Taken together, these results presented in this thesis add to the recognition of Hox genes as important regulators of hematopoiesis. These results suggest a role for Hox genes in the regulation of proliferation of the most primitive hematopoietic cells, and in lineage-commitments and proliferation of myeloid and lymphoid progenitor cells. Furthermore, these results also point to Hox gene-specific roles in the regulation of hematopoiesis.Medicine, Faculty ofMedical Genetics, Department ofGraduat