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 approximately 90% 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.