c-Myb Plays a Role InG2/M Cell Cycle Transition by Direct Regulation of Cyclin B1 Expression in Hematopoietic Cells.
Authors: Nakata Yuji , Shetzline Susan , Sakashita Chizuko , Kalota Anna , Ptasznik Andrzej , Zhang Yi , Emerson Stephen G. , Gewirtz Alan M. 
Published: April 2020

Myb family transcription factors are found throughout the phyla, and recent studies have demonstrated that Drosophila myb, as well as plant and yeast c-myb-like transcription factors, play an important role in regulating transition though the G1/S and G2/M phases of the cell cycle. Myb’s ability to regulate passage through G2/M is due at least in part to its ability to induce Cyclin B1 expression. A recent study in human T98G ganglioblastoma cells revealed that E2F, together with B-Myb, regulated cyclin B1 expression. Though c-myb was expressed in these cells, it was not found in immunoprecipitated E2F-B-Myb protein complexes and for this reason was felt not to participate in cyclin B1 expression in these cells. Since c-myb plays such a critical role in regulating hematopoietic cell proliferation, and its role in regulating G2/M in blood cells has not previously been explored, we investigated whether c-myb was important is regulating this phase of the cell cycle using K562 and Mo7e cells, as well as PHA stimulated human T lymphocytes. In distinct contrast to findings reported for T98G cells, we now report that in normal and malignant human hematopoietic cells, c-Myb directly upregulates cyclin B1 expression. Several lines of evidence support this claim. First, cyclin B1 expression decreased in Mo7e human leukemia cells in which c-myb had been silenced with siRNA. siRNA targeted to B-myb also decreased cyclin B1 expression, while neither siRNA species decreased cdc2 or cyclin A in these cells. As expected, siRNA targeted against c-myb or B-myb impaired Mo7e cell proliferation. Simultaneous exposure to both siRNA blocked proliferation completely. Second, using an alternative strategy, an inducible dominant negative c-Myb protein also decreased cyclin B1 expression in K562 human leukemia cells. The expected consequence of this, accelerated exit from the M phase, was also observed. Third, we examined c-Myb expression in human T cells by western and Real Time PCR, pre and post PHA stimulation. c-Myb expression began to gradually increase in the G1 phase of cell cycle, continued to increase after S phase, with the maximal protein level being found in G2/M phase, and concordant with cyclin B1 expression. These results indicated a correlation between c-Myb and cyclin B1 expression but did not indicate if c-Myb regulated cyclin B1 expression directly. To address this question, several additional experiments were carried out. A CAT assay showed that overexpressing c-Myb protein could increase activity when driven by a cyclin B1 promoter construct ~5X compared to K562 control cells. Next, examination of the cyclin B1 promoter showed eight potential c-Myb binding sites. Two were canonical [5′-pyrimidine AACG/TG-3′] and located upstream of 6 others which were [5′-AACNG-3′] in type. An in vitro c-Myb binding assay revealed that c-Myb bound the canonical sites. We then performed a Chromatin Immunoprecipitation (ChIP) Assay with anti-c-Myb antibody and specifically enriched cyclin B1 promoter DNA sequences which strongly suggested that c-Myb bound the cyclin B1 promoter in vivo. A control antibody was inactive. Finally, a conditionally active c-Myb restored cyclin B1 mRNA expression in K562 human leukemia cells in presence of cycloheximide within 6 hours. Therefore, in addition to its role in regulating G1/S cell cycle transition, c-Myb also regulates cyclin B1 expression and therefore transition through the G2/M phase in human hematopoietic cells.