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Expression and function of Hox homeobox transcription factors in early hematopoiesis

University of British Columbia

The Hox genes, first identified in Drosophila, are now recognized as key determinants of mammalian development. Recent evidence of Hox gene expression in leukemic cell lines have suggested that these transcription factors also play important roles in regulation of hematopoiesis. Using an improved RT/PCR technique which enables the generation of extended-length and representative cDNA from fewer than 1,000 cells, the expression pattern of Hox genes in highly purified primitive hematopoietic subpopulations was examined. Two different approaches were used. The first exploited the presence of a highly conserved region in the DNA-binding domain of Hox genes that could be targeted with degenerate primers for amplification from cDNA obtained from each purified population and the amplified produced were subsequently subcloned and sequenced. Over 150 Hox sequence containing clones were characterized. HOXA9, A6, A5, A4, A2, B3, B7 B9 and C9 were detected in a subpopulation which was highly enriched for very primitive cells detected as long-term culture-initiating cells (LTC-IC) and depleted for clonogenic progenitors. HOXA1O, A9, A7 A5, A4 and B7 were detected in a subpopulation highly enriched for clonogenic myeloid progenitors, and HOXA1O, A9, A7, A6, A5, B7and C8 were found in an erythroid progenitor enriched subpopulation. These data suggested that HOX A cluster genes are widely expressed amongst early hematopoietic subpopulations; in contrast, some genes of the B cluster appear to be restricted to the more primitive LTC-lC containing subpopulation. To better assess the potential differential expression of Hox genes, the initial amplified cDNA from five purified CD34 subpopulations of bone marrow cells was analyzed by Southern blot using probes for specific Hox genes. With this approach, it was possible to show that expression of HOXB3 and B4 was markedly higher (up to 40 fold) in the most primitive subpopulation than in the more mature subpopulations whereas that of other Hox genes such as HOXA1O, A9 and B9 was constant in all populations. Taken together, these data suggest that many Hox genes are active in early hematopoiesis and that some of them (i.e. HOXB3 and B4) are possible candidates for regulating stem cell function. To gain further insight into the role Hox genes may play in early hematopoiesis, the effect of overexpression of HOXB4 was studied in a murine model using a myeloproliferative sarcoma-based retroviral vector carrying the human HOXB4 cDNA under the control of the 5’ viral long terminal repeat. Overexpression of HOXB4 had proliferative effects on clonogenic progenitors, day 12 CFU-S and cells with marrow repopulating ability (MRA) as assessed by colony replating or recovery from seven day liquid cultures (up to 200 fold over neo-transduced control). To study the possible effects of HOXB4 overexpression on hematopoietic cells maintained for prolonged periods in vivo, HOXB4 or nec-transduced marrow cells were transplanted into lethally irradiated syngeneic recipients and reconstitution of various hematopoietic populations analyzed. At 20 weeks post-transplantation, recipients of HOXB4- transduced marrow had -5 fold more clonogenic progenitors per femur than neo controls. To determine if HOXB4 overexpression affected the expansion of the earliest hematopoietic stem cells (HSC), their numbers were determined by using the competitive repopulation unit (CRU) assay. CRU numbers in recipients of HOXB4-transduced bone marrow cells had recovered to 140% of normal levels found in untransplanted mice or some 50 fold higher than in recipients of nec-transduced marrow; all recipients of HOXB4-transduced marrow however had normal peripheral blood counts. Southern blot analysis of unique proviral integration patterns in DNA isolated from bone marrow and thymus of secondary recipients confirmed that HQXB4-transduced hematopoietic repopulating cells were totipotent and that they extensively self-renewed. This dramatic effect of HOXB4 on HSC self-renewal was maintained in serial transplantation experiments. Together, these results indicate HOXB4 to be an important regulator of very early but not late hematopoietic cell proliferation. The ability of HOXB4 to reverse the severe decline in HSC numbers suggest an exciting new approach to the controlled amplification of genetically modified hematopoietic stem cell populations.

Thesis/Dissertation

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2009-04-24

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http://hdl.handle.net/2429/7552

Experimental Medicine

University of British Columbia

UBCV

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