Abstract
The c-myb proto-oncogene encodes a transcription factor, Myb, which is essential for the growth and survival of normal and malignant hematopoietic cells. We, and others, have previously shown that malignant hematopoietic cells are more dependent on c-Myb function than are normal hematopoietic cells. Based on these findings, we hypothesized that c-Myb regulates a unique set of genes in leukemic cells that are required for their growth. To identify Myb gene targets, we performed a transcriptome analysis using human myeloid leukemic cells engineered to express a conditionally active dominant negative Myb (MERT). Analysis of the microarray data derived from these experiments revealed that when Myb activity was inhibited, neuromedin U (NmU), a neuropeptide involved in energy homeostasis, decreased in expression 5 fold compared to control cells, a result that was confirmed by quantitative real-time PCR. Combined, the microarray and quantitative real-time PCR data suggested that Myb directly regulates NmU gene expression in hematopoietic cells. To address this question in the absence of a formally defined human NmU promoter, we examined the DNA sequence upstream of the predicted transcription start site (as noted in Genbank accession #NM_006681) for potential Myb transcription factor binding motifs. After scanning the DNA sequence (~2kb) upstream of the predicted transcription start site, eleven potential Myb response elements (MREs) were identified. Of these MREs, five were identified as canonical (PyAAC(G/C)G). Our search also identified potential AML1, PU.1, CBP, STAT3, and STAT5 binding motifs within the human NmU promoter region. To determine if any of the potential MREs within the NmU promoter were functional, we first completed in vitro assays using luciferase reporter constructs followed by in vivo assays using chromatin immunoprecipitation (ChIP) assays. The luciferase reporter constructs were generated after we determined the actual transcription start of human NmU by primer extension assays. Using a Fam-labeled NmU specific primer that annealed proximal to the predicted transcription start site, we observed a 20-nucleotide difference between the predicted and actual transcription start of NmU. When all eleven potential MREs within the NmU promoter were upstream of luciferase, a 6-fold increase in luciferase activity was observed compared to empty vector. We next systematically mutated the MREs to determine which one(s) Myb bound directly. Thus far, the in vitro luciferase assay has identified MREs at −446 and −626, which are proximal to NmU’s transcription start as important for Myb-mediated expression. To determine the physiologic relevance of our in vitro studies, we performed ChIP assays. When chromatin from K562 cells, a human myeloid leukemia cell line, was immunoprecipitated with anti-c-Myb, we observed the expected PCR product using primer pairs that flanked select MREs. These same results were obtained in our positive control ChIP experiment in which the chromatin was immunoprecipitated with anti-acetyl histone 4 indicating that the promoter region of NmU is poised for transcription. Further characterization of the regulation of NmU gene expression in normal and malignant hematopoietic cells may yield new clues to Myb’s role in leukemogenesis and could suggest new therapeutic targets in human leukemia cells.
Determination of the InvE Binding Site Required for Expression of IpaB of the Shigella sonnei Virulence Plasmid: Involvement of a ParB BoxA-Like Sequence