During this period, we wished to address the role of Brd4 in mitotic progression and the regulation of gene expression relevant to cell growth. The role for Brd4 in cell division has been proposed based on early embryonic lethality observed in Brd4 knock out mice and growth inhibition in Brd4 knock down cells. In line with this, it has been shown that the BRD4 gene is truncated and fused to the NUT gene in some malignant cancer. Our work began with the finding that a series of anti-tublin drugs that cause mitotic arrest perturb Brd4-chromosome interactions. In the presence of anti-tubulin drugs such as nocodazole, taxol and colchicines, the bulk of Brd4 moved from condensed chromosomes to the outer space within dividing cells. This was seen both in live cells with exogenous Brd4 fused to GFP as well as in fixed cells with endogenous Brd4 from metaphase to telophase. Interestingly, this effect was reversed when nocodazole was washed away from culture. Although Brd4 remained outside of chromosomes as long as nocodazole was present in the media, within 30 min after drug removal, Brd4 was reloaded onto chromosomes. Furthermore, after nocodazole wash-out, wild type cells expressing normal levels of Brd4 resumed mitosis and produced new daughter cells. However, cells expressing less Brd4 (due to disruption of one Brd4 allele (Brd4 +/-) were incompetent in reloading Brd4 onto chromosomes. Importantly those cells that filed to bring Brd4 back onto chromosomes were defective in the ensuing cell division. Thus, a large fraction of Brd4+/- cells did not complete mitosis, resulting in a significantly fewer number of daughter cells. These data indicate that the Brd4-chromosome interaction has an important role in normal progression of mitosis and that This interaction is required for protecting cells from adverse effects of anti-tublin drugs. Our data with chromatin immunoprecipitation assays indicate that Brd4 is recruited to the promoters of genes that regulate cell cycle progression, presumably by binding to the chromatin. Some agents including anti-tubulin drugs appear to interfere with Brd4 recruitment. It would be of importance to study genome-wide Brd4 recruitment and to identify agents that alter its pattern.
