Downstream targets of HOXB4 in early hematopoietic progenitor cells

Ectopic expression of the homeobox transcription factor HOXB4 has been shown to enhance hematopoietic stem cell (HSC) self-renewal and expansion ex vivo and in vivo. HOXB4 is considered a protein that could potentially be used for therapeutic purposes to expand HSCs. If HOXB4 is to be used for this purpose or to be used in gene therapy experiments, identifying the largely unknown downstream targets of HOXB4 in early hematopoietic progenitor cells is important. In order to investigate HOXB4 in such cells, HOXB4 was constitutively overexpressed in the early hematopoietic progenitor cell line, EML. Two genome-wide tools, RNA expression profiling using microarray technology and chromatin immunoprecipitation combined with microarray technology (ChIP-chip), were then utilized to identify the downstream targets.;RNA expression profiling revealed that 465 gene transcripts are differentially expressed in KLS (c-Kit+, Sca-1+, Lin-)-EML cells that overexpress HOXB4 (KLS-EML-HOX4) when compared to control KLS-EML cells that only overexpress GFP (KLS-EML-GFP) cells. In particular, erythroid-specific gene transcripts are observed to be highly down-regulated in KLS-EML-HOXB4 cells. ChIP-chip analysis revealed that the promoter region for 1910 genes seemed to be bound by HOXB4 in KLS-EML-HOXB4 cells. The promoter region of genes such as CD34 and the lymphoid marker B220 gene were observed to be bound by HOXB4. Further experiments suggested that HOXB4 directly down-regulates the expression of the B220 gene.;Side-by-side comparison of our ChIP-chip and RNA expression profiling data sets provided highly useful correlative information. Two genes, Gp49a and Laptm4b, were identified as candidate "stemness-related" genes in this comparative analysis. Both genes were highly ranked in both data set lists and have been shown to be preferentially expressed in long-term HSCs and down-regulated in mature bone marrow cells. These two genes have not been extensively studied and their exact roles in early hematopoietic progenitor cells are unknown. Understanding the functions of these two genes in early hematopoietic progenitor cells might potentially provide more insight into the process of HSC self-renewal.