Department of Biology, Emory University, Atlanta, GA, United States of America.
Graduate Program in Genetics and Molecular Biology, Emory University, Atlanta, GA, United States of America.
PLoS Genet. 2019 Aug 5;15(8):e1008326. doi: 10.1371/journal.pgen.1008326. eCollection 2019 Aug.
The SWR1 chromatin remodeling complex, which deposits the histone variant H2A.Z into nucleosomes, has been well characterized in yeast and animals, but its composition in plants has remained uncertain. We used the conserved SWR1 subunit ACTIN RELATED PROTEIN 6 (ARP6) as bait in tandem affinity purification experiments to isolate associated proteins from Arabidopsis thaliana. We identified all 11 subunits found in yeast SWR1 and the homologous mammalian SRCAP complexes, demonstrating that this complex is conserved in plants. We also identified several additional proteins not previously associated with SWR1, including Methyl-CpG-BINDING DOMAIN 9 (MBD9) and three members of the Alfin1-like protein family, all of which have been shown to bind modified histone tails. Since mbd9 mutant plants were phenotypically similar to arp6 mutants, we explored a potential role for MBD9 in H2A.Z deposition. We found that MBD9 is required for proper H2A.Z incorporation at thousands of discrete sites, which represent a subset of the genomic regions normally enriched with H2A.Z. We also discovered that MBD9 preferentially interacts with acetylated histone H4 peptides, as well as those carrying mono- or dimethylated H3 lysine 4, or dimethylated H3 arginine 2 or 8. Considering that MBD9-dependent H2A.Z sites show a distinct histone modification profile, we propose that MBD9 recognizes particular nucleosome modifications via its PHD- and Bromo-domains and thereby guides SWR1 to these sites for H2A.Z deposition. Our data establish the SWR1 complex as being conserved across eukaryotes and suggest that MBD9 may be involved in targeting the complex to specific genomic sites through nucleosomal interactions. The finding that MBD9 does not appear to be a core subunit of the Arabidopsis SWR1 complex, along with the synergistic phenotype of arp6;mbd9 double mutants, suggests that MBD9 also has important roles beyond H2A.Z deposition.
SWR1 染色质重塑复合物将组蛋白变体 H2A.Z 沉积到核小体中,在酵母和动物中已得到很好的描述,但在植物中的组成仍不确定。我们使用保守的 SWR1 亚基 ACTIN RELATED PROTEIN 6 (ARP6) 作为串联亲和纯化实验中的诱饵,从拟南芥中分离出相关蛋白。我们鉴定了酵母 SWR1 和同源哺乳动物 SRCAP 复合物中发现的所有 11 个亚基,证明该复合物在植物中是保守的。我们还鉴定了几个以前与 SWR1 没有关联的其他蛋白质,包括 Methyl-CpG-BINDING DOMAIN 9 (MBD9) 和三个 Alfin1 样蛋白家族成员,它们都被证明与修饰的组蛋白尾巴结合。由于 mbd9 突变体植物的表型与 arp6 突变体相似,我们探讨了 MBD9 在 H2A.Z 沉积中的潜在作用。我们发现,MBD9 是数千个离散位点正确掺入 H2A.Z 所必需的,这些位点代表了通常富含 H2A.Z 的基因组区域的一个子集。我们还发现,MBD9 优先与乙酰化组蛋白 H4 肽以及携带单甲基或二甲基化 H3 赖氨酸 4 或二甲基化 H3 精氨酸 2 或 8 的肽相互作用。考虑到 MBD9 依赖性 H2A.Z 位点显示出独特的组蛋白修饰谱,我们提出 MBD9 通过其 PHD 和溴结构域识别特定核小体修饰,从而引导 SWR1 到这些位点进行 H2A.Z 沉积。我们的数据确立了 SWR1 复合物在真核生物中的保守性,并表明 MBD9 可能通过核小体相互作用参与将复合物靶向特定基因组位点。MBD9 似乎不是拟南芥 SWR1 复合物的核心亚基,以及 arp6;mbd9 双突变体的协同表型表明,MBD9 在 H2A.Z 沉积之外还有重要作用。