The SAGA histone acetyltransferase module targets SMC5/6 to specific genes

Abstract

Background 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. Results 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. Conclusion 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.

NSE5 subunit interacts with distant regions of the SMC arms in thePhyscomitrium patensSMC5/6 complex

“…To verify our Y2H results, we expressed the PpNSE5(aa190-526) and PpNSE5(aa245-526) fragments in vitro and used them in a pull-down assay (Fig. 1D; (Lelkes et al, 2023)). The Stag-PpNSE6(aa1-370) specifically precipitated PpNSE5(aa190-526), while not precipitating PpNSE5(aa245-526) (Fig.…”

“…The growth rates were determined by weighing untreated explants of mutant lines and comparing them with untreated explants of WT. Sensitivity to DNA damage was measured as described previously (Lelkes et al, 2023). Protocols for DNA isolation and rDNA copy numbers analysis were also detailed in (Lelkes et al, 2023).…”

“…Recently, we characterized the moss PpNSE6 subunit of SMC5/6 and its binding to the PpNSE5 partner (Lelkes et al, 2023). Here, we complemented this analysis with a study of the binding properties of PpNSE5.…”

“…Like with PpSMC6, we wanted to delineate the PpNSE5-binding region of PpSMC5 and used a similarly designed panel of constructs (Fig. S3A; (Lelkes et al, 2023)). We found that the Gal4BD-PpSMC5(aa280-790) headless fragment interacts with both Gal4AD-PpNSE5 and Gal4AD-PpNSE6 (Fig.…”

Characterization of the conserved features of the NSE6 subunit of the Physcomitrium patens SMC5/6 complex

“…Their cores are composed of long‐armed SMC, kleisin, and kleisin‐associated subunits. Additional factors are required to load, target, or regulate the SMC core complexes at specific chromatin sites (Litwin & Wysocki, 2018; Mahrik et al., 2023).…”