Constitution and Function of human SWI/SNF Complex : Implication in supression of carcionogenesis

我们分离了两个相关但不同的cDNA克隆，hOsa-1和hOsa-2，这是人类SWI/SNF复合物的最大组成部分。它们具有ARID （AT-rich interaction domain）和两个与果蝇Osa高度同源的区域。共免疫沉淀研究表明，这些c端同源区域对SWI/SNF的atp依赖性酶组分BRG和BRM的相互作用很重要。在培养的细胞中，hOsa刺激类固醇激素受体的转录。缺乏ARID的突变体没有刺激转录，表明该结构域在SWI/SNF功能中的重要性。我们建立了在四环素控制下以flag标记形式表达野生型或缺失ARID突变体的HeLa细胞系。用甘油梯度沉淀法分离核提取物时，在高分子量组分中发现了推定的人类SWI/SNF成分Osa、BRG、BAF155。BAF170在野生型和突变型表达细胞提取物中分布不同。BAF170与sa-2的ARID可能存在相互作用。从高分子量沉淀物中免疫纯化的野生型和突变型Osa-2复合物的银染色显示出非常相似的模式，表明ARID的存在对复合物的形成影响很小。

We isolated two related but distinct cDNA clones, hOsa-1 and hOsa-2, the 1argest component of human SWI/SNF complex. They had ARID (AT-rich interaction domain) and two high homology region to drosophila Osa. Co-immunoprecipitation study revealed that these C-terminal homology regions were important for the interaction with BRG and BRM, ATP-dependent enzyme componens of SWI/SNF. In cultured cells hOsa stimulated transcription by steroid hormone receptors. ARID lacking mutant did not stimulate the transcription, suggesting the importance of this domain in SWI/SNF function. We established HeLa cell lines which express either wild type or a mutant missing the ARID in Flag-tagged form under the control of tetracycline administration. Putative human SWI/SNF components, Osa, BRG, BAF155, were found in the high molecular weight fractions when the nuclear extracts were separated by glycerol gradient sedimentation. Distribution of BAF170 was different between the extracts from wild type and the mutant expressing cells. There might be some interaction between BAF170 and ARID of Osa-2. Silver staining of the complexes with wild type and the mutant Osa-2 immunopurified from the high molecular weight sedimentation fractions showed quite similar pattern, suggesting the presence of ARID has little effect on the complex formation.