The bromodomain protein Brd4 stimulates G1 gene transcription and promotes progression to S phase
The bromodomain protein Brd4 stimulates G1 gene transcription and promotes progression to S phase
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DOI：
10.1074/jbc.m707603200
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发表时间：
2008-04-04
影响因子：
4.8
通讯作者：
Ozato, Keiko
中科院分区：
文献类型：
--
作者：
Mochizuki, Kazuki;Nishiyama, Akira;Ozato, Keiko
Brd4是一种含溴结构域的蛋白质，它能与乙酰化的染色质结合。它调控细胞生长，尽管其潜在机制仍不清楚。Brd4也已被证明能控制病毒基因的转录，然而它在细胞基因转录中的作用尚未完全阐明。在此我们利用小发夹RNA（shRNA）方法探讨了Brd4在细胞生长和转录中的作用。Brd4 shRNA载体在NIH3T3细胞和小鼠胚胎成纤维细胞中稳定地将Brd4蛋白表达降低了约90%。Brd4敲低的细胞生长受损，比对照细胞生长更缓慢。当通过血清饥饿使其同步化并释放后，Brd4敲低的细胞停滞在G₁期，而对照细胞则进入S期。在微阵列分析中，尽管在对照细胞的G₁期有许多基因上调，但这些G₁期基因中的许多在Brd4敲低的细胞中没有上调。重新引入Brd4可挽救Brd4敲低细胞中这些G₁期基因的表达，使细胞能够向S期进展。染色质免疫沉淀分析表明，在G₀ - G₁期进展过程中，Brd4被招募到这些G₁期基因的启动子上。此外，Brd4的招募与Cdk9（P - TEFb的一个组分）以及RNA聚合酶II对这些基因结合的增加同时发生。在不受Brd4 shRNA影响的基因上，Brd4的招募很少或不存在。结果表明，Brd4以一种细胞周期依赖的方式通过与多个G₁期基因启动子结合来刺激G₁期基因表达。
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 similar to 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.