Dataset Information

Spt6-Spn1 interaction is required for RNA Polymerase II association and precise nucleosome positioning along transcribed genes

ABSTRACT: Spt6-Spn1 is an essential histone chaperone complex that associates with RNA Polymerase II (RNAPII) and reassembles nucleosomes during gene transcription. While Spt6-Spn1 interaction is important to its histone deposition and transcription functions, a precise understanding of how Spt6-Spn1 association contributes to its functions is still limited. Here, using temperature sensitive alleles of spt6 and spn1 that disrupt their interaction, we show that Spt6-Spn1 association is broadly important for its interaction with the elongating RNAPII complex and nucleosomes. Using micrococcal nuclease (MNase)-based chromatin occupancy profiling, we further find that Spt6-Spn1 interaction is required for maintaining the preferred nucleosome positioning generated from replication-dependent histone deposition at actively transcribed genes. These analyses also reveal an overall shift of nucleosomes towards the 5’ end of genes that correlates with decreased RNAPII levels. While loss of Spt6 and Spn1 function is known to promote cryptic transcription, the nucleosome defects found in spt6 and spn1 mutants that interfere with their interaction were observed broadly across transcribed genes, further highlighting the role of DNA sequence as a mediator of cryptic transcription when nucleosomes positioning is altered. Taken together, these findings reveal Spt6-Spn1 interaction is key to its association with elongating RNAPII and for its ability to precisely organize nucleosomes across transcription units.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE284959 | GEO | 2025/04/09

REPOSITORIES: GEO

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Project description:Spt6 is a multifunctional histone chaperone involved in the maintenance of chromatin structure during elongation by RNA polymerase II (Pol II). Spt6 has a tandem SH2 (tSH2) domain within its C-terminus that recognizes Pol II CTD peptides phosphorylated on Ser2, Ser5 or Try1 in vitro. Deleting the tSH2 domain, however, only has a partial effect on Spt6 occupancy in vivo, suggesting that more complex mechanisms are involved in the Spt6 recruitment. Our results show that the Ser2 kinases Bur1 and Ctk1, but not the Ser5 kinase Kin28, cooperate in recruiting Spt6, genome-wide. Interestingly, the Ser2 kinases promote the association of Spt6 in early transcribed regions and not toward the 3' end of genes, where phosphorylated Ser2 reaches its maximum level. Additionally, our results uncover an unexpected role for histone deacetylases (Rpd3 and Hos2) in promoting Spt6 interaction with elongating Pol II. Finally, our data suggest that phosphorylation of the Pol II CTD on Tyr1 promotes the association of Spt6 with the 3' end of transcribed genes, independently of Ser2 phosphorylation. Collectively, our results show that a complex network of interactions, involving the Spt6 tSH2 domain, CTD phosphorylation and histone deacetylases, coordinate the recruitment of Spt6 to transcribed genes in vivo. We examined the genome-wide distribution (using ChIP-chip) of Spt6. Spt6 occupancy was also assayed in mutants for CTD Serine 2 and Serine 5 kinases and in mutants for histone deacetylases. ChIPs were performed with a Myc-tagged version of Spt6. Most ChIPs (in Cy5) were hybridyzed against a control ChIP sample from an isogenic non-tagged strain (in Cy3). In the ChIP experiments with the spt6-202del mutant, non immunoprecipitated DNA (input) was used as the control. In addition to Spt6 ChIPs, the project includes RNAPII (Rpb3) ChIP-chip datasets, where an anti-Rpb3 antibody was used to ChIP RNAPII and non immunoprecipitated DNA (input) was used as the control. All ChIP-chip experiments were done in duplicates. Each microarray was normalized using the Lima Loess and replicates were combined using a weighted average method as previously described (Pokholok et al., 2005).

Dataset Information

Spt6-Spn1 interaction is required for RNA Polymerase II association and precise nucleosome positioning along transcribed genes

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets