PDF(Pubmed)
Abstract：
The yeast SWR1C chromatin remodeling enzyme catalyzes the ATP-dependent exchange of nucleosomal histone H2A for the histone variant H2A.Z, a key variant involved in a multitude of nuclear functions. How the 14-subunit SWR1C engages the nucleosomal substrate remains largely unknown. Studies on the ISWI, CHD1, and SWI/SNF families of chromatin remodeling enzymes have demonstrated key roles for the nucleosomal acidic patch for remodeling activity, however a role for this nucleosomal epitope in nucleosome editing by SWR1C has not been tested. Here, we employ a variety of biochemical assays to demonstrate an essential role for the acidic patch in the H2A.Z exchange reaction. Utilizing asymmetrically assembled nucleosomes, we demonstrate that the acidic patches on each face of the nucleosome are required for SWR1C-mediated dimer exchange, suggesting SWR1C engages the nucleosome in a \'pincer-like\' conformation, engaging both patches simultaneously. Loss of a single acidic patch results in loss of high affinity nucleosome binding and nucleosomal stimulation of ATPase activity. We identify a conserved arginine-rich motif within the Swc5 subunit that binds the acidic patch and is key for dimer exchange activity. In addition, our cryoEM structure of a Swc5-nucleosome complex suggests that promoter proximal, histone H2B ubiquitylation may regulate H2A.Z deposition. Together these findings provide new insights into how SWR1C engages its nucleosomal substrate to promote efficient H2A.Z deposition.
摘要：
酵母SWR1C染色质重塑酶催化核小体组蛋白H2A与组蛋白变体H2A的ATP依赖性交换。Z,涉及多种核功能的关键变体。14亚基SWR1C如何与核小体底物接合仍然是未知的。关于ISWI的研究，CHD1和SWI/SNF家族的染色质重塑酶已经证明了核小体酸性斑块对重塑活性的关键作用，然而，这种核小体表位在SWR1C核小体编辑中的作用尚未得到测试。这里,我们采用了多种生化试验来证明酸性贴片在H2A中的重要作用。Z交换反应。利用不对称组装的核小体，我们证明了在核小体的每一面上的酸性斑块是SWR1C介导的二聚体交换所必需的，表明SWR1C以“钳状”构象接合核小体，同时接合两个补丁。单个酸性贴片的丧失导致高亲和力核小体结合的丧失和ATP酶活性的核小体刺激。我们确定了Swc5亚基中保守的富含精氨酸的基序，该基序结合酸性斑块，并且是二聚体交换活性的关键。此外,我们的Swc5核小体复合物的冷冻EM结构表明，组蛋白H2B泛素化可能调控H2A。Z沉积。这些发现共同提供了SWR1C如何参与其核小体底物以促进高效H2A的新见解。Z沉积。
