Activation of stem-cell specific genes by HOXA9 and HOXA10 homeodomain proteins in CD34+ human cord blood cells.

There is growing evidence for a role of HOX homeodomain proteins in normal hematopoiesis. Several HOX genes, including HOXA9 and HOXA10, are expressed in primitive hematopoietic cells, implying a role in early hematopoietic differentiation. To identify potential target genes of these two closely related transcription factors, human CD34+ umbilical cord blood cells were transduced with vectors expressing either HOXA9 or HOXA10 and analyzed with cDNA micro-arrays. Statistical analysis using significance analysis of microarrays revealed a common signature of several hundred genes, demonstrating that the transcriptomes of HOXA9 and HOXA10 largely overlap in this cellular context. Seven genes that were upregulated by both HOX proteins were validated by real-time reverse transcription polymerase chain reaction. HOXA9 and HOXA10 showed positive regulation of genes in the Wnt pathway, including Wnt10B and two Wnt receptors Frizzled 1 and Frizzled 5, an important pathway for hematopoietic stem cell (HSC) self-renewal. Other validated genes included v-ets-related gene (ERG), Iroquois 3 (IRX3), aldehyde dehydrogenase 1 (ALDH1), and very long-chain acyl-CoA synthetase homolog 1 (VLCS-H1). GenMAPP (Gene Micro Array Pathway Profiler) analysis indicated that HOXA10 repressed expression of several genes involved in heme biosynthesis and three globin genes, indicating a general suppression of erythroid differentiation. A number of genes regulated by HOXA9 and HOXA10 are expressed in normal HSC populations.

Chromatin immunoprecipitation (ChIP) scanning identifies primary glucocorticoid receptor target genes.

The global physiological effects of glucocorticoids are well established, and the framework of transcriptional regulation by the glucocorticoid receptor (GR) has been described. However, the genes directly under GR control that trigger these physiological effects are largely unknown. To address this issue in a single cell type, we identified glucocorticoid-responsive genes in A549 human lung adenocarcinoma cells by microarray analysis and quantitative real-time PCR. Reduction of GR expression by RNA interference diminished the effects of dexamethasone on all tested target genes, thus confirming the essential role of GR in glucocorticoid-regulated gene expression. To identify primary GR target genes, in which GR is a component of the transcriptional regulatory complex, we developed a strategy that uses chromatin immunoprecipitation to scan putative regulatory regions of target genes for sites occupied by specifically bound GR. We screened 11 glucocorticoid-regulated genes, and we identified GR-binding regions for eight of them (five induced and three repressed). Thus, our approach provides a means for rapid identification of primary GR target genes and glucocorticoid-response elements, which will facilitate analyses of transcriptional regulatory mechanisms and determination of hormone-regulated gene networks.

The transcriptome of the leukemogenic homeoprotein HOXA9 in human hematopoietic cells.

Hematopoietic defects in HOXA9(-/-) mice demonstrate a key role for this homeoprotein in blood cell development. Conversely, enforced HOXA9 expression is leukemogenic in mice, and HOXA9 is frequently activated in human acute myeloid leukemia (AML). Although HOXA9 is thought to function as a transcription factor, few downstream targets have been identified. We searched for early HOXA9 target genes by using a transient overexpression strategy in 3 hematopoietic cell lines (2 myeloid, 1 lymphoid). cDNA microarray analyses identified 220 genes whose expression was modulated at least 2-fold. Expression signatures in myeloid and lymphoid cells demonstrated that HOXA9 functions as both an activator and repressor of a variety of genes in cell-specific patterns suggesting that the transcriptional effects of HOXA9 are largely dependent on the cell context. Transient transcription assays and target gene expression patterns in HOXA9(-/-) marrow cells imply that we have identified direct physiologic targets. Many target genes are expressed in CD34+ stem cells or are members of gene families involved in proliferation or myeloid differentiation. Expression of 14 HOXA9 target genes correlated with high-level HOXA9 expression in primary AML. These data suggest that many genes identified in this survey may mediate the biologic effects of HOXA9 in normal and leukemic hematopoiesis.