Spanning the gap: unraveling RSC dynamics in vivo

Abstract

Multiple reports over the past 2 years have provided the first complete structural analyses for the essential yeast chromatin remodeler, RSC, providing elaborate molecular details for its engagement with the nucleosome. However, there still remain gaps in resolution, particularly within the many RSC subunits that harbor histone binding domains.Solving contacts at these interfaces is crucial because they are regulated by posttranslational modifications that control remodeler binding modes and function. Modifications are dynamic in nature often corresponding to transcriptional activation states and cell cycle stage, highlighting not only a need for enriched spatial resolution but also temporal understanding of remodeler engagement with the nucleosome. Our recent work sheds light on some of those gaps by exploring the binding interface between the RSC catalytic motor protein, Sth1, and the nucleosome, in the living nucleus. Using genetically encoded photo-activatable amino acids incorporated into histones of living yeast we are able to monitor the nucleosomal binding of RSC, emphasizing the regulatory roles of histone modifications in a spatiotemporal manner. We observe that RSC prefers to bind H2B SUMOylated nucleosomes in vivo and interacts with neighboring nucleosomes via H3K14ac. Additionally, we establish that RSC is constitutively bound to the nucleosome and is not ejected during mitotic chromatin compaction but alters its binding mode as it progresses through the cell cycle. Our data offer a renewed perspective on RSC mechanics under true physiological conditions.

The role of histone acetyltransferases Gcn5 and Esa1 in recruiting the RSC complex and maintaining nucleosome-depleted regions genome-wide in Saccharomyces cerevisiae

“…Rapid RSC recruitment and histone eviction from promoters could explain why genes with FNs have more Pol II in their ORFs than SN genes. The RSC accumulation in NDRs seen in the HAT mutants but not in the tail mutants suggests that RSC can be recruited to nucleosomes using hypoacetylated (or unacetylated) tails and that subsequent acetylation might help RSC to evict histones (38,39,42,65).…”

“…S2B). It makes sense considering that acetylated H3K14, a Gcn5 target, is recognized by the Sth1 bromodomain (42,64,65).…”

Genetic Code Expansion Tools to Study Lysine Acylation

“…Site‐specific in vivo crosslinking can provide valuable mechanistic insights into the effects of PTMs on chromatin dynamics involved in processes such as transcription, replication, chromosome segregation and the response to DNA damage. [ 139 ]…”

Unnatural Amino Acid Crosslinking for Increased Spatiotemporal Resolution of Chromatin Dynamics

“…Furthermore, data suggested that these interactions were negatively regulated by importin binding to a nuclear localization sequence that also resided on the C-terminus of the protein. In a more recent application of the crosslinking approach, Jain et al used pBPA scanning to characterize the binding interface and mechanistic action of the yeast RSC remodeler complex ATPase subunit, Sth1, with the nucleosome ( Figure 3 ) [ 44 , 45 ]. In this study, pBPA proved to be an effective spatiotemporal probe that allowed for detailed insights to Sth1 binding to histones.…”

“…al. used pBPA scanning to characterize the binding interface and mechanistic action of the yeast RSC remodeler complex ATPase subunit, Sth1, with the nucleosome (Figure 3) [44,45]. In this study, pBPA proved to be an effective spatiotemporal probe that allowed for detailed insights to Sth1 binding to histones.…”