The transcription factor HB9, encoded by the homeobox gene B9 (is
The transcription factor HB9, encoded by the homeobox gene B9 (is recurrently rearranged in young children with acute myeloid leukemia characterized by a chromosomal translocation t(7;12)-and concomitant high expression of the unrearranged, wild-type allele. (motor neuron and pancreas homeobox 1) belongs to the family of homeobox genes and is usually located on chromosome 7q36 (1). It is usually composed of three exons comprising 1206 bp and encodes the 401-amino acid transcription factor HB9. The homeobox encodes for the homeodomain, a well explained DNA-binding domain name in many transcription factors. The homeodomain is usually structured in three helices, which are involved in DNA conversation (2), and is usually highly homologous to a homeodomain consensus sequence (1). HB9 harbors a polyalanine stretch (16) and two glycine stretches (7 and 5) as additional Plau structural features, but a functional impact on DNA-binding or gene rules has not been experimentally shown yet. In mice, HB9 is usually a central mediator of cellular differentiation in pancreatic tissue and motor neurons during embryonic development (3C5). It is usually indispensable for the initiation of the dorsal pancreatic program, and hence, HB9-deficient mice show characteristic agenesis of the dorsal but not the ventral pancreatic lobe (3). Motor neuron differentiation and their proper specification also occurs in early embryonic development (embryonic day 8.5), and HB9 is specifically important to distinguish between motor neuron and interneuron identity (5). In humans, a dominating loss-of-function mutation in the gene results in sacral agenesis, concomitant anorectal, and urogenital malformations, altogether a well explained symptom complex named Currarino syndrome (6). Moreover, manifestation is usually explained in colorectal malignancy tissue and hepatocellular carcinoma (7, 8). buy 54965-21-8 Other than its role in differentiation of tissues from the endoderm and ectoderm, the function of HB9 in derivates from the mesenchyme, like hematopoietic cells, is largely unknown. Conflicting reports exist about the manifestation of in hematopoietic originate cells. Deguchi and Kehrl (9) reported manifestation in CD34-enriched and unfractionated bone marrow cells. However, we and others (10) did not observe manifestation in healthy CD34+ bone marrow cells. The only reports attributing a functional role to manifestation in hematopoiesis come from infants with acute myeloid leukemia characterized by a chromosomal translocation t(7;12)-in their leukemic cells (10C13). All patients show an aberrantly high manifestation of the non-rearranged allele in the leukemic cells (10). Of notice, a fusion mRNA transcript is usually not usually detected in great time cells of all translocation t(7;12) positive patients as a result of the genomic heterogeneity of the 7q36 breakpoint (10, 11). With a three-year event-free-survival of 0%, this leukemia entity has a depressing prognosis (14C17). We previously characterized the gene manifestation profile of translocation t(7;12) positive leukemic great time cells. Functional annotation analysis revealed that differentially expressed genes can be attributed to pathways involved in cell adhesion or closely related processes (17). Based on its high homology to other homeodomain proteins, HB9 likely functions as a transcription factor but neither its DNA-binding properties nor its target genes in hematopoietic cells have been recognized thus much. In our present work, we used global, genome-wide methods to identify both main and secondary target genes of HB9 in hematopoietic cells. buy 54965-21-8 EXPERIMENTAL PROCEDURES Cell Culture HL60 cells were produced in RPMI medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 2 mm glutamine (Invitrogen). HL60 cells transporting the plasmid were cultured in the presence of 0.5 g/ml puromycin buy 54965-21-8 (Sigma-Aldrich). All cells were incubated at 37 C in a humidified 5% CO2 incubator. A codon optimized cDNA of human and (18). HL60 cells were split the day prior electroporation, so cells are in log-phase during electroporation. For electroporation 1 106 cells were resuspended in 500 t of RPMI without supplements and mixed with 10 g of linearized vector DNA. Electroporation was carried out in an EPI 2500 electroporator (Dr. Fischer, Heidelberger, Philippines) at 400 V and 10 ms. After 24 h, growth medium was replaced and supplemented with 0.5 g/ml puromycin. Positive cells were selected for at least 4 buy 54965-21-8 weeks. Cells are.