The Bromodomain Protein Brd4 Stimulates G1 Gene Transcription and Promotes Progression to S Phase

Abstract

Brd4 is a bromodomain protein that binds to acetylated chromatin. It regulates cell growth, although the underlying mechanism has remained elusive. Brd4 has also been shown to control transcription of viral genes, whereas its role in transcription of cellular genes has not been fully elucidated. Here we addressed the role of Brd4 in cell growth and transcription using a small hairpin (sh) RNA approach. The Brd4 shRNA vector stably knocked down Brd4 protein expression by ϳ90% in NIH3T3 cells and mouse embryonic fibroblasts. Brd4 knockdown cells were growth impaired and grew more slowly than control cells. When synchronized by serum starvation and released, Brd4 knockdown cells were arrested at G 1 , whereas control cells progressed to S phase. In microarray analysis, although numerous genes were up-regulated during G 1 in control cells, many of these G 1 genes were not up-regulated in Brd4 knockdown cells. Reintroduction of Brd4 rescued expression of these G 1 genes in Brd4 knockdown cells, allowing cells to progress toward S phase. Chromatin immunoprecipitation analysis showed that Brd4 was recruited to the promoters of these G 1 genes during G 0 -G 1 progression. Furthermore, Brd4 recruitment coincided with increased binding of Cdk9, a component of P-TEFb and RNA polymerase II to these genes. Brd4 recruitment was low to absent at genes not affected by Brd4 shRNA. The results indicate that Brd4 stimulates G 1 gene expression by binding to multiple G 1 gene promoters in a cell cycle-dependent manner.Brd4 is a ubiquitously expressed 200-kDa nuclear protein that belongs to the BET family (1, 2). Proteins of this family carry two tandem bromodomains through which they interact with acetylated histones (3-6). Bromodomains are also present in other chromatin-binding proteins such as histone acetylases and chromatin remodeling factors. They also bind to acetylated histones and are involved in transcriptional regulation of many genes. Recent structural analysis indicates that the bromodomain of Brd2, a factor closely related to Brd4, forms a dimmer to bind to acetyl residues of the histone H4 tails (7). Binding of Brd4 and Brd2 to acetylated chromatin persists even during mitosis as well as meiosis when chromatin is highly condensed and transcription is interrupted (2,5,6,8).Evidence indicates that BET family proteins are multifunctional and regulate cell growth and transcription (3, 4, 9, 10). In line with this evidence, there are reports indicating that Brd4 is involved in cell growth regulation; Brd4 Ϫ/Ϫ embryos fail to grow and die early at around the time of implantation (11). Similarly, Brd4Ϫ/Ϫ embryonic stem cells do not grow in culture (12). Moreover, in some malignant cells, Brd4 is fused to the NUT gene, and the fusion protein exhibits a growth regulatory activity (13,14). In addition, overexpression of Brd4 in cultured cells is shown to alter their growth properties, in part due to the interaction of Brd4 with growth regulatory proteins such as RFC140 or Sipa1 (15, 16). The reports that Brd4 facilitates parti...

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