National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kotlarska 2, 61137, Brno, Czech Republic.
Mendel Centre for Plant Genomics and Proteomics, Central European Institute of Technology, Masaryk University, Kamenice 5, 62500, Brno, Czech Republic.
Epigenetics Chromatin. 2023 Feb 16;16(1):6. doi: 10.1186/s13072-023-00480-z.
Structural Maintenance of Chromosomes (SMC) complexes are molecular machines driving chromatin organization at higher levels. In eukaryotes, three SMC complexes (cohesin, condensin and SMC5/6) play key roles in cohesion, condensation, replication, transcription and DNA repair. Their physical binding to DNA requires accessible chromatin.
We performed a genetic screen in fission yeast to identify novel factors required for SMC5/6 binding to DNA. We identified 79 genes of which histone acetyltransferases (HATs) were the most represented. Genetic and phenotypic analyses suggested a particularly strong functional relationship between the SMC5/6 and SAGA complexes. Furthermore, several SMC5/6 subunits physically interacted with SAGA HAT module components Gcn5 and Ada2. As Gcn5-dependent acetylation facilitates the accessibility of chromatin to DNA-repair proteins, we first analysed the formation of DNA-damage-induced SMC5/6 foci in the Δgcn5 mutant. The SMC5/6 foci formed normally in Δgcn5, suggesting SAGA-independent SMC5/6 localization to DNA-damaged sites. Next, we used Nse4-FLAG chromatin-immunoprecipitation (ChIP-seq) analysis in unchallenged cells to assess SMC5/6 distribution. A significant portion of SMC5/6 accumulated within gene regions in wild-type cells, which was reduced in Δgcn5 and Δada2 mutants. The drop in SMC5/6 levels was also observed in gcn5-E191Q acetyltransferase-dead mutant.
Our data show genetic and physical interactions between SMC5/6 and SAGA complexes. The ChIP-seq analysis suggests that SAGA HAT module targets SMC5/6 to specific gene regions and facilitates their accessibility for SMC5/6 loading.
结构维护染色体（SMC）复合物是驱动染色质组织在更高水平的分子机器。在真核生物中，三个 SMC 复合物（黏合蛋白、凝聚蛋白和 SMC5/6）在黏合、凝聚、复制、转录和 DNA 修复中发挥关键作用。它们与 DNA 的物理结合需要可及的染色质。
我们在裂殖酵母中进行了遗传筛选，以鉴定新的 SMC5/6 与 DNA 结合所需的因子。我们鉴定了 79 个基因，其中组蛋白乙酰转移酶（HATs）是最具代表性的。遗传和表型分析表明 SMC5/6 和 SAGA 复合物之间存在特别强的功能关系。此外，几个 SMC5/6 亚基与 SAGA HAT 模块组件 Gcn5 和 Ada2 相互作用。由于 Gcn5 依赖性乙酰化促进了染色质对 DNA 修复蛋白的可及性，我们首先分析了在 Δgcn5 突变体中形成的 DNA 损伤诱导的 SMC5/6 焦点。在 Δgcn5 中，SMC5/6 焦点正常形成，表明 SAGA 独立的 SMC5/6 定位到 DNA 损伤部位。接下来，我们使用 Nse4-FLAG 染色质免疫沉淀（ChIP-seq）分析在未受挑战的细胞中评估 SMC5/6 的分布。在野生型细胞中，SMC5/6 的很大一部分聚集在基因区域内，而在 Δgcn5 和 Δada2 突变体中则减少。在 gcn5-E191Q 乙酰转移酶失活突变体中也观察到 SMC5/6 水平下降。
我们的数据显示 SMC5/6 和 SAGA 复合物之间存在遗传和物理相互作用。ChIP-seq 分析表明，SAGA HAT 模块将 SMC5/6 靶向特定基因区域，并促进其可及性，以便 SMC5/6 加载。
