The Expression of Genes 5′ in the Hox-A Cluster Is Co-Ordinated Both in Normal and Leukemic Hematopoiesis.

Hox genes are known to play critical roles in hematopoiesis and are probably important in the pathogenesis of leukemias. Disruption of the Hoxa9 gene in mice leads to defects in erythroid, lymphoid and myeloid hematopoiesis while over-expression of Hoxa9 leads to leukemia in mice. Transcription of Hoxa9 is partly regulated by the Mixed lineage leukemia gene (Mll) product. Bone marrow cells of mice carrying the leukemic Mll-AF9 fusion gene as a knock-in mutation (henceforth called Mll), display an increase in expression of several Hox genes - Hoxa5, Hoxa6, Hoxa7, Hoxa9 and Hoxa10, when compared to age matched wild type mice (Kumar et. al, 2004, Blood). Since the over-expressed Hox genes all belong to the same Hox-a cluster, we hypothesized that these genes might be co-regulated during normal hematopoiesis. Co-regulation of neighboring genes within the same cluster has been reported by others for the Hox-b and c clusters. To test this hypothesis for the 5′ Hox-a cluster genes, we compared expression levels of Hoxa5, Hox7 and Hoxa10 in Hoxa9 homozygous knockout (Hoxa9^−/−) and wild type bone marrow by real-time quantitative RT-PCR using Taqman primers/probe sets (Applied Biosystems, CA). Expression levels of Hoxa5, Hoxa7, and Hoxa10 were all reduced by 65% ± 2% in Hoxa9^−/− mice compared to wild type mice. In contrast, levels of Hoxb4 and Meis1 - homeobox genes that are not part of the Hox-a cluster - were identical in Hoxa9^−/− and wild type mice. These results show no compensatory increases in expression of other 5′ Hox-a genes in the absence of Hoxa9, but instead demonstrate that disruption of Hoxa9 decreases the expression of neighboring genes in the Hox-a cluster. The hematopoietic defects seen in Hoxa9^−/− mice (leucopenia, lymphopenia and blunted granulocytic response to G-CSF) might thus be attributable to the deficiency of multiple Hox-a gene products, rather than of Hoxa9 alone. To further evaluate the extent of this co-regulation, we studied the expression levels of these genes in Mll mice that lacked Hoxa9 (Hoxa9^−/−/Mll-AF9^+/−, henceforth called Mll/Hox). The Mll/Hox mice develop leukemia at the same rate and time course as the Mll mice, but with a phenotype that is relatively more immature. In Mll/Hox mice, the expression levels of Hoxa5 and Hoxa7 were increased 13 fold and 4 fold respectively, while those of Hoxa10 remained decreased at 35% of wild type. These results indicate that the decreased expression of neighboring Hox-a genes in Hoxa9^−/− mice was reversed by Mll-AF9 for Hoxa5 and Hoxa7, but not for Hoxa10, suggesting a second level of co-regulation for Hoxa9 and Hoxa10. Overall, our findings demonstrate a co-regulated relationship between the 5′ Hox-a cluster genes during normal hematopoiesis, and provide evidence that deregulation of a single Hoxa9 gene significantly alters the expression of neighboring Hox-a cluster genes with implications for understanding the pathogenetic mechanisms of leukemia.