Hox genes were first recognized for their role in embryonic development and may also play important lineage-specific functions in a variety of somatic tissues including the hematopoietic system. We have recently shown that certain members of the Hox A and B clusters, such as HOXB3 and HOXB4, are preferentially expressed in subpopulations of human bone marrow that are highly enriched for the most primitive hematopoietic cell types. To assess the role these genes may play in regulating the proliferation and/or differentiation of such cells, we engineered the overexpression of HOXB4 in murine bone marrow cells by retroviral gene transfer and analyzed subsequent effects on the behavior of various hematopoietic stem and progenitor cell populations both in vitro and in vivo. Serial transplantation studies revealed a greatly enhanced ability of HOXB4.transduced bone marrow cells to regenerate the most primitive hematopoietic stem cell compartment resulting in 50-fold higher numbers of transplantable totipotent hematopoietic stem cells in primary and secondary recipients, compared with serially passaged neo-infected control cells. This heightened expansion in vivo of HOXB4.transduced hematopoietic stem cells was not accompanied by identifiable anomalies in the peripheral blood of these mice. Enhanced proliferation in vitro of day-12 CFU-S and clonogenic progenitors was also documented. These results indicate HOXB4 to be an important regulator of very early but not late hematopoietic cell proliferation and suggest a new approach to the controlled amplification of genetically modified hematopoietic stem cell populations.[Key Words: Homeo box genes~ bone marrow transplantation~ retroviral gene transfer]

Class I homeobox (Hox) genes encode a major group of transcription factors controlling embryonic development and have been implicated in the continuing process of hematopoietic cell differentiation. They are clustered on four chromosomes and, in early development, exhibit spatially restricted expression with respect to their 3'-> 5' chromosomal position. By using an improved PCR-based method for amplifying total cDNA derived from limited cell numbers, we now describe the expression of class I Hox genes in highly purified CD34+ cell subpopulations isolated from normal human bone marrow that represent functionally distinct stem and progenitor cell compartments. Our data indicate that at least 16 different Hox genes, mainly from the A and the B clisters, are expressed in one or more of these subpopulations of human hematopoietic cells. Moreover, markedly elevated expression of some of the Hox genes found at the 3' end of the A and B clusters (e.g., HoxB3) was a unique feature of the subpopulations that contained the most primitive functionally defined cells, whereas genes located in the S' region of each cluster (e.g., HoxA10) were found to be expressed at nearly equal levels in the CD34+ subpopulations analyzed. In contrast to the findings for CD34+ cells, expression of two selected Hox genes, HoxB3 and HoxA10, was virtually extinguished in the CD34-fraction of bone marrow cells. These results demonstrate the expression of a broad range of Hox genes in primitive hematopoietic cells and point to the existence of a regulated program of Hox gene expression during their normal development.

with HoxA10 or Drosophila even-skipped. Site-directed mutagenesis showed that the hexapeptide motif (IYPWMK) upstream of the Hox homeo domain was essential for HoxB6 and B7 to cooperatively bind DNA with Pbx proteins. Engraftment of the HoxB7 hexapeptide onto HoxA10 endowed it with robust cooperative properties, demonstrating a functional role for the highly conserved hexapeptide element as one of the molecular determinants delimiting Hox-Pbx cooperativity. The Pbx homeo domain was necessary but not sufficient for cooperativity, which required conserved amino acids carboxy-terminal of the homeo domain. These findings demonstrate that interactions between Hox and Pbx proteins modulate their DNA-binding properties, suggesting that Pbx and Hox proteins act in parallel as heterotypic complexes to regulate expression of specific subordinate genes.

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