Intra-S-Phase Regulator cdc7 Is a Gene Target of the c-Myb Proto-Oncogene in Human Leukemias.

The c-myb proto-oncogene encodes a transcription factor, Myb, which is essential for normal hematopoiesis. Myb may also play a role in leukemogenesis but possible mechanisms remain ill defined. To gain further insights into this issue, we sought to identify Myb regulated genes in human myeloid leukemic cells utilizing a tamoxifen inducible expression system and a microarray approach. Myb function was conditionally abrogated by tamoxifen following infection of K562 cells with a bicistronic retroviral vector MIGR1 which had c-myb’s DNA binding domain (DBD) and the Drosophila engrailed protein transcription repressor domain (MERT) fused to a modified estrogen receptor that binds tamoxifen. MERT was subcloned upstream of an IRES-EGFP cassette in MIGR1 to allow for FACS purification on the basis of GFP expression. To identify Myb regulated genes, purified K562-MERT cells were exposed to 1 μM tamoxifen or ethanol (control) for three days, processed for hybridization to the microarray gene chip and analyzed by software algorithms from Incyte and Arrayex. When endogenous Myb activity was suppressed by MERT, 105 genes out of 10,000 genes on the microarray chip changed > 2-fold in expression. Of these 105 genes, 34 increased their expression >2-fold while 70 decreased their expression >2-fold. Since Myb expression is elevated in leukemic cells, we hypothesized that Myb functions in malignant hematopoietic cells to induce the expression of genes that are essential for their maintenance and survival. Therefore, we focused on those genes that decreased in expression when Myb activity was inhibited by MERT. Among the most repressed was cdc7 (2.8-fold decrease), an intra-S-phase regulator. To verify the microarray data, we utilized real-time PCR to quantitate the expression of cdc7 in our K562-MERT cells. Cdc7 expression decreased 5-fold in tamoxifen treated K562-MERT cells relative to control cells, which is consistent with the microarray data. We then performed chromatin immunoprecipitation (ChIP) experiments to determine whether cdc7 is a direct target of Myb. When the chromatin from untreated K562 and K562-MERT cells was immunoprecipitated with anti-c-Myb, we observed one PCR product using a primer pair that flanked the Myb binding sites in the promoter region of cdc7. This same result was observed in our positive control ChIP experiment in which the chromatin was immunoprecipitated with anti-acetyl histone H4, indicating that the region of the cdc7 promoter containing the Myb binding sites is poised for transcription. When Myb transactivation activity was inhibited by MERT in K562-MERT cells, no PCR product was observed following chromatin immunoprepitation with anti-c-Myb. These results strongly suggest that cdc7 is a direct gene target of c-Myb in malignant hematopoietic cells. Investigation of the transcriptional regulation of cdc7 in hematopoietic cells may yield new clues to Myb’s role in leukemogenesis.