Transcriptional Regulation of the Intra-S-Phase Regulator CDC7 in Human Myeloid Leukemic Cells: A New Downstream Target of the C-Myb Protooncogene.

Abstract The c-myb proto-oncogene encodes a transcription factor, Myb, which is essential for the growth and proliferation of normal and malignant hematopoietic cells. We, and others, have previously shown that malignant hematopoietic cells are much more dependent on c-myb function than are normal hematopoeitic cells, and that transient disruption of c-myb expression causes malignant cells to undergo apoptosis while normal cells are relatively spared. Based on these findings, we hypothesized that c-myb regulates a unique set of genes in leukemic cells that are required for cell survival. 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 revealed that when Myb activity was inhibited by tamoxifen in MERT cells, CDC7, an intra-S phase regulator, decreased in expression 2.8-fold compared to untreated control cells. To verify this, we utilized real-time PCR to quantitate the expression of CDC7, and found that it decreased 5-fold in tamoxifen treated MERT cells relative to control cells. In aggregate, the microarray and real-time PCR data suggested that Myb directly regulates CDC7 gene expression in hematopoietic cells. To address this question in the absence of a formally defined human CDC7 promoter, we examined the DNA sequence upstream of the predicted transcription start site (as noted in Genbank accession # AY585721) for potential Myb transcription factor binding motifs. After scanning the DNA sequence (~3kb) upstream of the predicted transcription start site, nine potential Myb response elements (MREs) were identified. The CDC7 sequences from mouse, chimp, and yeast were also analyzed for MREs and compared to those present in the putative human CDC7 promoter to identify conserved MREs. Using this strategy, we also identified potential AML1, PU.1, CBP, STAT3, and STAT5 binding motifs within the human CDC7 promoter region. To determine if any of the potential Myb binding sites with the CDC7 promoter were actually utilized in vivo, we carried out chromatin immunoprecopitation (ChIP) assays. When the chromatin from untreated MERT cells was immunoprecipitated with anti-c-Myb, we observed one PCR product using a primer pair that flanked each conserved MRE. These same results were obtained in our positive control ChIP experiment in which the chromatin was immunoprecipitated with anti-acetyl histone 4. When Myb transactivation activity was inhibited in tamoxifen treated MERT cells, no PCR product was detected following chromatin immunoprecipitation with the anti-Myb antibody suggesting that the ChIP binding results were not due to artifact. We have just completed a primer extension assay with a Fam-labeled primer that flanked the predicted CDC7 promoter region and will use the resulting sequence data to identify the actual CDC7 transcriptional start site. We will also shortly complete identification of functional regions within the human CDC7 promoter through use of Luciferase reporter assays. Investigation of the transcriptional regulation of CDC7 in hematopoietic cells may yield new clues to Myb's role in leukemogenesis.