Human Rsc Chromatin-remodeling Complex

atp依赖性染色质重塑复合体（Rsc）最初是在酵母中发现的。它的亚基组成与表征良好的SWI/SNF相似：两个配合物共有2个亚基，另外至少有4个亚基相互同源。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则不是。Rsc突变体在细胞周期的G2/M转变中被捕获，并且这种捕获依赖于纺锤体检查点基因。突变体对破坏微管稳定的药物也更敏感。这些数据表明Rsc可能在有丝分裂中发挥作用，可能通过稳定有丝分裂纺锤体形成或在着丝点上起作用。我们之前已经从人体内纯化了几种atp依赖性染色质重塑复合物，这些复合物与酵母SWI/SNF或Rsc密切相关。通过微测序和克隆，我们鉴定出人类SWI/SNF-B复合物的BAF180亚基与酵母Rsc亚基Rsc1、Rsc2和Rsc4具有同源性。它具有与三个Rsc亚基相同的生化基序：6个溴结构域，2个BAH区域和一个小槽结合结构域。对已完成的人类、果蝇和隐杆线虫基因组数据库进行数据库检索，发现BAF180是三个Rsc亚基最接近的同源物，提示人类SWI/SNF-B是人类版本的Rsc。我们将这个复合物重新命名为PBAF （Polybromo-associated BAF complex）。我们随后鉴定了人类PBAF复合物的许多其他亚基，并发现其中许多亚基与人类SWI/SNF中的亚基相同。我们还证实了BAF180定位于有丝分裂染色体的着丝点，这与PBAF在有丝分裂中的功能一致。这项研究发表在《美国科学院院刊》上。作为该项目的延续，我们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的抗体，BAF200。通过这种抗体，我们能够证明BAF200确实是这种复合体的内在成分。BAF200含有酵母RSC复合物成分中的一个结构域，这意味着BAF200可能是RSC复合物家族中的一个保守功能亚基。我们目前正在进行siRNA敲低实验，以鉴定受BAF200及其相关复合体调控的基因。

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.