Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden.
PLoS One. 2011;6(12):e28049. doi: 10.1371/journal.pone.0028049. Epub 2011 Dec 6.
Scaffold attachment factor A (SAF-A) participates in the regulation of gene expression by organizing chromatin into transcriptionally active domains and by interacting directly with RNA polymerase II.
Here we use co-localization, co-immunoprecipitation (co-IP) and in situ proximity ligation assay (PLA) to identify Brahma Related Gene 1 (BRG1), the ATP-driven motor of the human SWI-SNF chromatin remodeling complex, as another SAF-A interaction partner in mouse embryonic stem (mES) cells. We also employ RNA interference to investigate functional aspects of the SAF-A/BRG1 interaction.
We find that endogenous SAF-A protein interacts with endogenous BRG1 protein in mES cells, and that the interaction does not solely depend on the presence of mRNA. Moreover the interaction remains intact when cells are induced to differentiate. Functional analyses reveal that dual depletion of SAF-A and BRG1 abolishes global transcription by RNA polymerase II, while the nucleolar RNA polymerase I transcription machinery remains unaffected.
We demonstrate that SAF-A interacts with BRG1 and that both components are required for RNA Polymerase II Mediated Transcription.
支架附着因子 A（SAF-A）通过将染色质组织成转录活跃的结构域，并直接与 RNA 聚合酶 II 相互作用，参与基因表达的调控。
在这里，我们使用共定位、共免疫沉淀（co-IP）和原位邻近连接分析（PLA）来鉴定 BRG1，即人类 SWI-SNF 染色质重塑复合物的 ATP 驱动的马达，作为另一个 SAF-A 相互作用伙伴在小鼠胚胎干细胞（mES）细胞中。我们还采用 RNA 干扰技术研究 SAF-A/BRG1 相互作用的功能方面。
我们发现内源性 SAF-A 蛋白与 mES 细胞中的内源性 BRG1 蛋白相互作用，并且这种相互作用不仅仅取决于 mRNA 的存在。此外，当细胞被诱导分化时，这种相互作用仍然完整。功能分析表明，SAF-A 和 BRG1 的双重耗竭会使 RNA 聚合酶 II 介导的转录完全停止，而核仁 RNA 聚合酶 I 转录机制不受影响。
我们证明 SAF-A 与 BRG1 相互作用，并且这两个组件都是 RNA 聚合酶 II 介导的转录所必需的。
