Essential histone chaperones collaborate to regulate transcription and chromatin integrity

Abstract

Histone chaperones are critical for controlling chromatin integrity during transcription, DNA replication, and DNA repair. Three conserved and essential chaperones, Spt6, Spn1/Iws1, and FACT, associate with elongating RNA polymerase II and interact with each other physically and/or functionally; however, there is little understanding of their individual functions or their relationships with each other. In this study, we selected for suppressors of a temperature-sensitive spt6 mutation that disrupts the Spt6-Spn1 physical interaction and that also causes both transcription and chromatin defects. This selection identified novel mutations in FACT. Surprisingly, suppression by FACT did not restore the Spt6-Spn1 interaction, based on coimmunoprecipitation, ChIP, and mass spectrometry experiments. Furthermore, suppression by FACT bypassed the complete loss of Spn1. Interestingly, the FACT suppressor mutations cluster along the FACT-nucleosome interface, suggesting that they alter FACT-nucleosome interactions. In agreement with this observation, we showed that the spt6 mutation that disrupts the Spt6-Spn1 interaction caused an elevated level of FACT association with chromatin, while the FACT suppressors reduced the level of FACT-chromatin association, thereby restoring a normal Spt6-FACT balance on chromatin. Taken together, these studies reveal previously unknown regulation between histone chaperones that is critical for their essential in vivo functions.

The interaction between the Spt6-tSH2 domain and Rpb1 affects multiple functions of RNA Polymerase II

“…The NTD of Spt6 binds nucleosomes and Spn1, supporting a complex relationship among the histone chaperone activities of Spt6, Spn1 and FACT ( 3 , 40 ), so inefficient recruitment of Spt6 to transcription complexes could lead to changes in chromatin due to a simple reduction in available chaperone capacity (Figure 9D ). Comparison of the changes in nucleosome occupancy and positioning in interface mutants with those caused by a FACT mutant supported this interpretation for some genes but also revealed distinct properties for the Spt6-tSH2:Rpb1 interaction.…”

“…We therefore compared its effects with those of interface mutants as a partial probe of how reduced Spt6 recruitment alone might affect transcript levels. The functions of Spn1 and its interaction with Spt6 display complex genetic interactions with the histone chaperone FACT ( 40 ), so to attempt to determine the contribution of a generic reduction in global chaperone activity to the phenotypes of interface mutants we also tested the effects of two FACT alleles that cause distinct but overlapping defects in chromatin architecture ( pob3 Q308K and spt16 – 11 ; 15 , 41 ).…”

“…The NTD of Spt6 binds to nucleosomes and also to Spn1, these interactions are competitive with one another in vitro , and the function of the Spt6:Spn1 interface is important for balancing the activities of Spn1 and FACT in vivo ( 3 , 42 , 40 ; see Figure 1A ). The Spt6-tSH2:Rpb1 interface could affect chromatin management by altering the localization or orientation of the Spt6-NTD:Spn1 module, or it could affect nucleosome integrity before, during, or after transcription and therefore dictate the need for cooperation among the histone chaperone activities of Spt6, Spn1, and FACT.…”

Targeted protein degradation reveals a direct role of SPT6 in RNAPII elongation and termination

“…To maintain epigenetic information during transcription, histone chaperones travel with RNAPII and ensure the correct re-assembly of nucleosomes with DNA during transcription elongation ( Eitoku et al., 2008 ; Viktorovskaya et al., 2021 ). SPT6 is a RNAPII-associated histone chaperone that is conserved in eukaryotes ( Andrulis et al., 2000 ; Eitoku et al., 2008 ; Kaplan et al., 2000 ; Ni et al., 2008 ).…”

Genome-wide mapping of G-quadruplex structures with CUT&Tag

“…G4 profiles on gene-coding regions showed a strong peak around the transcription start sites (TSS), while largely absent in the gene body despite the presence of PQS (Supplementary Figure 2B). Efficient chromatin maintenance during RNA Polymerase passage, relying on Spt6, FACT and other histone chaperones is known to maintain dense nucleosome occupancy in gene bodies (48), thus likely disfavoring formation of G4. Promoters of non-expressed genes, exhibiting neither H3K4me3 or H3K27me3 showed the lowest G4 signal (Supplementary Figure 2D).…”