The Transcription Factor B-Myb Is a Key Regulator of S Phase during Endoreplication in Megakaryocytes.

During their differentiation, and uniquely within haemopoietic cells, megakaryocytes acquire a polyploid nuclear DNA content, most likely to enhance their ability to produce large numbers of platelets for subsequent release into the blood. Polyploidy is achieved through the process of endoreplication, that is, successive S-phases without concomitant mitosis. Although a number of proteins involved in the control of the cell cycle have been implicated in megakaryocytic endoreplication little is known concerning which transcriptional regulators might influence the process. Several transcription factors have been shown to regulate the expression of characteristic functional proteins during megakaryocytic differentiation, but as yet none has been linked to endoreplication. In fact, work with certain model systems for megakarocyte differentiation suggests that the regulation of megakaryocyte-specific genes might be quite separate from the control of endoreplication.

From studies on many cell types it is evident that the cell cycle regulated Myb family transcription factor B-Myb is crucial during S-phase. B-Myb expression has been described to be maximal at G1/S phase and it is tightly regulated at the level of both RNA and protein. We have examined the expression and function of B-Myb in both model cell systems for megakaryocyte differentiation and in primary murine cells. It has been described that B-Myb activity needs to be down regulated during terminal differentiation of most haemopoietic cells. However we have found that during differentiation of human megakaryoblastic cell lines induced with phorbol esters or in differentiating primary mouse megakaryocytes, B-myb RNA and protein levels remain constant. To assess the function of B-Myb in megakaryocytic cells we manipulated its level of expression. Over expression of B-Myb in the HEL megakaryoblastic cell line resulted in an increase in the number of cells entering into S-phase. When the HEL cells over expressing B-Myb were treated with phorbol ester to induce differentiation we observed an increase in the fraction of cells actively endoreplicating. On the other hand, reducing B-Myb levels through the use of siRNAs resulted in an accumulation of cells at the G1/S boundary, and through incorporation of BrdU we were able to show that this was due to decreased progression through S-phase. When similar experiments to reduce B-Myb levels were performed on HEL cells undergoing differentiation we observed the same decline in S-phase progression during endoreplicative DNA synthesis. In conclusion, our experiments are the first to identify a key transcriptional regulator of the specialised cell cycle in differentiating megakaryocytes.

Since the gene knockout for B-myb has a lethal phenotype very early in development at the blastocyst stage, future studies in primary mouse megakaryocytes will require controlled ablation of the B-myb gene. We will describe preliminary studies on a new mouse line that we have created in which the B-myb gene has been modified to enable Cre-loxP mediated deletion.