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.

Keywords: Chromatin remodelling; Genetic code expansion; Lysine acetylation; Photo-crosslinking; RSC; Sumoylation; Unnatural amino acids.

Fig. 1

a Genetic code expansion facilitates the incorporation of photo-activatable amino acids in histone proteins in living yeast. The crosslinker-containing histone is incorporated into the chromatin landscape and forms covalent adducts with binding proteins, such as the Sth1 subunit of RSC, upon irradiation. Adducts are subsequently detected by Western blot. b Crosslink-products formed between histones and Sth1 are mapped onto the structure of the nucleosome-bound RSC complex. These residues are likely in contact with the SnAC domain of Sth1, which is not resolved in the structure (image generated using pdb-file 6TDA and Chimera)

Fig. 2

Various ways in which PTMs can affect chromatin remodelers. a PTMs, such as H3K14ac, may serve as recruitment signals to direct remodelers to specific genomic loci. b Simultaneous binding of multiple histone PTMs might direct the positioning of nucleosomes, e.g. at promoters. RSC recruitment occurs prior to H3 tail acetylation. Rather than a sequestering signal, acetylation stimulates internucleosomal contacts with Sth1, communicating that the loading state has been achieved. c The presence of particular PTMs, such as H2B SUMOylation, on substrate nucleosomes may modulate the outcome of the remodeling reaction, e.g. nucleosome repositioning versus eviction. d Presence (or absence) of certain PTMs may regulate the translocation speed of the remodeler