Human Rsc Chromatin-remodeling Complex

ATP依赖性染色质重塑复合物Rsc最初在酵母中鉴定。它有一个亚基组成类似于良好的特点SWI/SNF：2个亚基之间共享的两个复合物和至少4个其他是彼此的同源物。然而，Rsc的功能不同于SWI/SNF。Rsc是酵母有丝分裂生长所必需的，而SWI/SNF不是。Rsc突变体在细胞周期的G2/M转换期被阻滞，并且这种阻滞依赖于纺锤体检查点基因。突变体对微管不稳定药物也更敏感.这些数据表明，Rsc可能在有丝分裂中发挥作用，可能是通过稳定有丝分裂纺锤体的形成或在动粒中发挥作用。我们先前已经从人中纯化了几种ATP依赖的染色质重塑复合物，它们与酵母SWI/SNF或Rsc密切相关。通过微测序和克隆，我们现在鉴定了人SWI/SNF-B复合物的BAF 180亚基作为酵母Rsc亚基Rsc 1、Rsc 2和Rsc 4的人类同源物。它具有与三个Rsc亚基相同的一组生化基序：6个溴结构域，2个BAH区域和一个小沟结合结构域。对人、果蝇和秀丽隐杆线虫的完整基因组数据库的数据库搜索显示，BAF 180是三个Rsc亚基的最接近的同源物，表明人SWI/SNF-B是Rsc的人版本。我们将该配合物重新命名为PBAF（Polybromo-associated BAF complex）。我们随后鉴定了人PBAF复合物的许多其他亚基，并发现其中一些亚基与人SWI/SNF中的亚基相同。我们还证明，BAF 180定位于有丝分裂染色体的着丝粒，与PBAF在有丝分裂过程中的功能一致。这项工作已发表在PNAS上。作为这个项目的延续，我们已经确定了这个综合体的三个可能的新组成部分。我们已经获得了针对其中一种BAF 200的抗体。使用这种抗体，我们能够证明BAF 200确实是这种复合物的内在成分。BAF 200含有在酵母RSC复合物的组分中发现的结构域，这意味着BAF 200可能是RSC复合物家族中保守的功能亚基。我们目前正在进行siRNA敲除实验，以确定由BAF 200及其相关复合物调控的基因。

The ATP-dependent Chromatin-remodeling complex, Rsc, was originally identified in yeast. It has a subunit composition similar to the well-characterized SWI/SNF: 2 subunits are shared between the two complexes and at least 4 others are homologues of each other. However, the function of Rsc is distinct from SWI/SNF. Rsc is essential for the mitotic growth of yeast, whereas SWI/SNF is not. Rsc mutants are arrested at G2/M transition during the cell cycle, and the arrest is dependent on spindle-checkpoint genes. The mutants are also more sensitive to microtubule- destabilizing drugs. These data suggest that Rsc may play a role in mitosis, perhaps by stabilizing mitotic spindle formation or have a function at kinetochores. We have previously purified several ATP-dependent chromatin remodeling complexes from human that are closely related to either yeast SWI/SNF or Rsc. By microsequencing and cloning, we now identified the BAF180 subunit of human SWI/SNF-B complex as a human homologue of yeast Rsc subunits, Rsc1, Rsc2 and Rsc4. It has the same set of biochemical motifs as the three Rsc subunits: 6 bromodomains, 2 BAH regions, and a minor-groove binding domain. Database search of the completed genome database of human, Drosophila and C.elegans showed that BAF180 is the closest homolog of the three Rsc subunits, suggesting human SWI/SNF-B as a human version of Rsc. We renamed this complex as PBAF (for Polybromo-associated BAF complex). We subsequently identified many other subunits of human PBAF complex and found a number of them to be identical to those in human SWI/SNF. We also demonstrated that BAF180 localizes at kinetochores of mitotic chromosomes, consistent with a function for PBAF during mitosis. This work has been published in PNAS. As a continuation of this project, we have identified three possible new components of this complex. We have obtained antibody against one of them, BAF200. With this antibody, we were able to show that BAF200 is indeed an intrinsic component of this complex. BAF200 contains a domain found in a component of yeast RSC complex, implying that BAF200 may be a conserved functional subunit within RSC family of complexes. We are currently performing siRNA knockdown experiments to identify genes regulated by BAF200 and its associated complex.