Baier Alexander S, Gioacchini Nathan, Eek Priit, Tan Song, Peterson Craig L
Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, 01605.
Medical Scientist Training Program, T.H. Chan School of Medicine, University of Massachusetts.
Res Sq. 2023 Jul 28:rs.3.rs-3050911. doi: 10.21203/rs.3.rs-3050911/v1.
The 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. Numerous 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 ubiquitinylation may regulate H2A.Z deposition. Together these findings provide new insights into how SWR1C engages its nucleosomal substrate to promote efficient H2A.Z deposition.
SWR1C染色质重塑酶催化核小体组蛋白H2A与组蛋白变体H2A.Z进行ATP依赖性交换，H2A.Z是参与多种核功能的关键变体。由14个亚基组成的SWR1C如何与核小体底物结合在很大程度上仍不清楚。对染色质重塑酶的ISWI、CHD1和SWI/SNF家族进行的大量研究已证明核小体酸性补丁在重塑活性中起关键作用，然而该核小体表位在SWR1C进行核小体编辑中的作用尚未得到验证。在此，我们采用多种生化分析方法来证明酸性补丁在H2A.Z交换反应中起重要作用。利用不对称组装的核小体，我们证明核小体每个表面上的酸性补丁是SWR1C介导的二聚体交换所必需的，这表明SWR1C以“钳状”构象与核小体结合，同时与两个补丁结合。单个酸性补丁的缺失会导致高亲和力核小体结合的丧失以及核小体对ATPase活性的刺激作用丧失。我们在Swc5亚基中鉴定出一个保守的富含精氨酸的基序，该基序与酸性补丁结合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电镜结构表明，启动子近端的组蛋白H2B泛素化可能调节H2A.Z的沉积。这些发现共同为SWR1C如何与核小体底物结合以促进高效的H2A.Z沉积提供了新的见解。
