Johnson Thomas A, Elbi Cem, Parekh Bhavin S, Hager Gordon L, John Sam
Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-5055, USA.
Mol Biol Cell. 2008 Aug;19(8):3308-22. doi: 10.1091/mbc.e08-02-0123. Epub 2008 May 28.
Brahma (BRM) and Brahma-related gene 1 (BRG1) are the ATP-dependent catalytic subunits of the SWI/SNF family of chromatin-remodeling complexes. These complexes are involved in essential processes such as cell cycle, growth, differentiation, and cancer. Using imaging approaches in a cell line that harbors tandem repeats of stably integrated copies of the steroid responsive MMTV-LTR (mouse mammary tumor virus-long terminal repeat), we show that BRG1 and BRM are recruited to the MMTV promoter in a hormone-dependent manner. The recruitment of BRG1 and BRM resulted in chromatin remodeling and decondensation of the MMTV repeat as demonstrated by an increase in the restriction enzyme accessibility and in the size of DNA fluorescence in situ hybridization (FISH) signals. This chromatin remodeling event was concomitant with an increased occupancy of RNA polymerase II and transcriptional activation at the MMTV promoter. The expression of ATPase-deficient forms of BRG1 (BRG1-K-R) or BRM (BRM-K-R) inhibited the remodeling of local and higher order MMTV chromatin structure and resulted in the attenuation of transcription. In vivo photobleaching experiments provided direct evidence that BRG1, BRG1-K-R, and BRM chromatin-remodeling complexes have distinct kinetic properties on the MMTV array, and they dynamically associate with and dissociate from MMTV chromatin in a manner dependent on hormone and a functional ATPase domain. Our data provide a kinetic and mechanistic basis for the BRG1 and BRM chromatin-remodeling complexes in regulating gene expression at a steroid hormone inducible promoter.
布拉马（BRM）和布拉马相关基因1（BRG1）是染色质重塑复合物SWI/SNF家族中依赖ATP的催化亚基。这些复合物参与细胞周期、生长、分化和癌症等重要过程。利用成像方法，在含有稳定整合的类固醇反应性MMTV-LTR（小鼠乳腺肿瘤病毒-长末端重复序列）拷贝串联重复的细胞系中，我们发现BRG1和BRM以激素依赖的方式被招募到MMTV启动子上。BRG1和BRM的招募导致了染色质重塑以及MMTV重复序列的解聚，这通过限制酶可及性的增加以及DNA荧光原位杂交（FISH）信号大小的增加得以证明。这种染色质重塑事件与RNA聚合酶II的占有率增加以及MMTV启动子处的转录激活同时发生。BRG1（BRG1-K-R）或BRM（BRM-K-R）的ATP酶缺陷形式的表达抑制了局部和高阶MMTV染色质结构的重塑，并导致转录减弱。体内光漂白实验提供了直接证据，表明BRG1、BRG1-K-R和BRM染色质重塑复合物在MMTV阵列上具有不同的动力学特性，并且它们以依赖激素和功能性ATP酶结构域的方式动态地与MMTV染色质结合和解离。我们的数据为BRG1和BRM染色质重塑复合物在类固醇激素诱导型启动子处调节基因表达提供了动力学和机制基础。
