Activation Of Stem-Cell Specific Genes By Hoxa9 And Hoxa10 Homeodomain Proteins In Cd34+Human Cord Blood Cells

Abstract There is growing evidence for a role of HOX homeodomain (HD) proteins in normal hematopoiesis. Several HOX genes, including HOXA9 and HOXA10, are expressed in primitive hematopoietic cells, and that expression is down-regulated as cells mature, implying a role in early hematopoietic differentiation. In addition, retrovirally enforced over-expression of either of these two HD proteins results in dramatic expansion of hematopoietic stem cells (HSC). Our laboratory recently published a description of the HOXA9 transcriptome in human leukemic cell lines, using a transient over-expression strategy (Dorsam et al. Blood 2004). In that study we observed changes in the mRNA levels of a large number of genes within 24 hours of introduction of a HOXA9 expression vector in these cells. The modulated targets represented a wide variety of functional groups, including oncogenes, cell-cycle proteins, enzymes, membrane proteins and other transcription factors. Interestingly, a number of these putative targets are known to be part of the transcriptome of normal HSC. This previous study raises questions as to whether the HOXA9 targets identified in aneuploid immortalized myeloid cell lines would match the gene targets in normal hematopoietic cells. To answer this question and to compare target genes of two closely related HD proteins, human CD34+ umbilical cord blood cells were transduced with MSCV-based vectors expressing either HOXA9 or HOXA10, and RNA isolated from these cells was amplified and analyzed with cDNA microarrays. Statistical analysis using the Significance Analysis of Microarrays (SAM) algorithm revealed a common signature of several hundred modulated genes, demonstrating that the transcriptomes of HOXA9 and HOXA10 largely overlap in this cellular context. In a second step, CLUSTER analysis was performed, introducing threshold values to increase the likelihood of the identified genes being truly regulated and of biological significance. Using these more stringent criteria, 115 genes were modulated by one or both of the over-expressed genes. Several genes that were up-regulated by both HOX proteins were validated by quantitative real-time RT-PCR. HOXA9 and HOXA10 showed positive regulation of genes in the Wnt pathway, including Wnt 10b and two Wnt receptors Frizzled 1 and Frizzled 5, a important pathway for hematopoietic stem cell (HSC) self renewal. Other validated targets included Ets-related gene (ERG),, alcohol dehydrogenase 1 (ALDH1) and very long chain acyl-CoA synthetase (VLCS-H1), all of which are known to be expressed in normal CD34+ cells. One down-regulated gene in this survey is CYBB, a respiratory burst oxidase component pg91(phox), which is expressed in myeloid cells, and has previously been shown to be repressed by HOXA10. GenMAPP pathway analysis indicated that HOXA9 and HOXA10 repressed expression of several genes involved in heme biosynthesis and three globin genes, indicating a general suppression of the erythroid maturation program. HOXA9 and HOXA10 both appear to activate many HSC-specific genes while repressing genes involved in hematopoietic differentiation.