Remodeling-independent function of the BAF Complex in T Cells and Beyond

描述（由申请人提供）：染色质重塑复合物可以利用ATP水解的能量破坏核小体并调节基因表达。BAF复合物是一种典型的哺乳动物重塑物，含有约10个亚基，其中包括重塑所必需的催化亚基Brg。我们发现，在T细胞中删除Brg会损害CD4的表达和免疫耐受。令人惊讶的是，一个催化无活性的Brg点突变体有效地挽救了这两个缺陷，这表明Brg和BAF复合物具有重塑独立功能（Remodeling Independent Function， RIF），这与体外重塑中10个BAF亚基中有6个是不可缺少的事实是一致的。RIF的分子机制是一个谜。为了解决这个问题，我们将以CD4位点为模型，确定RIF对靶基因物理结构的影响（目的1）。作为一种补充方法，我们将使用brg缺失的肿瘤细胞系来鉴定BAF亚基及其介导RIF的功能结构域，重点关注肌动蛋白和肌动蛋白相关蛋白BAF53，这是一对令人费解的亚基（Aim 2）。我们的研究挑战了十年来关于染色质建模者的教条，并且引起了普遍的兴趣，因为越来越多的证据表明组蛋白修饰酶也具有类似rif的活性，并且因为核肌动蛋白的作用已经吸引了几代生物学家，但在很大程度上回避了。

DESCRIPTION (provided by applicant): Chromatin remodeling complexes can use energy of ATP hydrolysis to disrupt nucleosomes and regulate gene expression. BAF complex is a prototypical mammalian remodeler, containing ~10 subunits including the catalytic subunit Brg essential for remodeling. We found that deleting Brg in T cells impairs CD4 expression and immune tolerance. Surprisingly, a catalytically inactive Brg point mutant effectively rescues both defects, indicating Brg and hence BAF complex possess Remodeling Independent Function (RIF), which is consistent with the fact that six of 10 BAF subunits are dispensable for remodeling in vitro. The molecular mechanism of RIF is a mystery. To attack this problem, we will determine the effects of RIF on the physical structure of target genes, using the CD4 locus as a model (Aim 1). As a complementary approach, we will use Brg-deleted tumor cell lines to identify the BAF subunits and their functional domains mediating RIF, focusing on actin and the actin-related protein BAF53, a puzzling pair of subunits (Aim 2). Our study challenges a decade-old dogma about chromatin-modelers, and is of general interest because evidence is emerging that histone modifying enzymes also possess RIF-like activities and because the roles of nuclear actin have fascinated but largely eluded generations of biologists. PUBLIC HEALTH RELEVANCE: We found that an essential transcription-regulating enzyme harbors an unconventional activity that is unrelated to the classic catalytic function but nevertheless important for transcription regulation. Here we propose to address the molecular mechanisms of this novel activity, which is of medical implications given the biological importance of this protein.