Two secured FACT recruitment mechanisms are essential for heterochromatin maintenance

Abstract

FACT (facilitate chromatin transcription) is involved in heterochromatic silencing, but its mechanisms and function remain unclear. We reveal that the Spt16 recruitment mechanism operates in two distinct ways in heterochromatin. First, Pob3 mediates Spt16 recruitment onto the heterochromatin through its Spt16 dimerization and tandem PH domains. Without Pob3, Spt16 recruitment is partially reduced, exhibiting a silencing defect and impaired H2A/H2B organization. Second, heterochromatin protein 1 (HP1)/Swi6 mediates Spt16 recruitment onto the heterochromatin by physical interaction of the Swi6 chromo-shadow domain (CSD) and Spt16 peptidase-like domains. Several CSD mutants are tested for Spt16 binding activity, and the charged loop connecting b1 and b2 is critical for Spt16 binding and heterochromatic silencing. Loss of these pathways causes a severe defect in H3K9 methylation and HP1/Swi6 localization in the pericentromeric region, exhibiting transcriptional silencing defects and disordered heterochromatin. Our findings suggest that FACT and HP1/Swi6 work intimately to regulate heterochromatin organization.

The histone chaperone FACT facilitates heterochromatin spreading by regulating histone turnover and H3K9 methylation states

“…This suggests that FACT protects heterochromatin from illegitimate transcription and likely transcription-induced histone turnover. A recent study revealed that FACT is recruited to heterochromatin via an interaction with the chromoshadow domain of Swi6 ( Takahata et al, 2021 ). It is thus tempting to speculate that this interaction keeps FACT in the vicinity of heterochromatin and together with other silencing factors creates a physical barrier for RNAPII and histone replacement, which would promote the H3K9me3 state.…”

Opposing Roles of FACT for Euchromatin and Heterochromatin in Yeast

“…ChIP analysis of Spt16 exhibited that the binding level of Spt16 to the heterochromatic region in the pob3∆ strain was decreased to half that of the wild type, suggesting the existence of a Pob3-independent recruitment mechanism of Spt16 onto heterochromatin. Genetic analysis also revealed that pob3∆swi6∆ double disruption exhibited an additive silencing defect compared with that shown in each of the pob3∆ and swi6∆ single mutant strains [115]. Therefore, we assumed that the recruitment of Spt16 onto heterochromatin is partially dependent on Swi6.…”

“…Genetic analysis and ChIP-qPCR showed that fission yeast strains lacking pob3 + (pob3∆) had comparable levels of histone H3K9 methylation and Swi6 localization in the heterochromatic region to those of the wild type strain, but with high levels of heterochromatic expression of ncRNAs. Phenotypic analysis of the pob3∆ strain indicated that heterochromatic silencing was defective in heterochromatin without significant loss of levels of histone H3K9 methylation and HP1/Swi6 binding [114,115]. ChIP analysis of Spt16 exhibited that the binding level of Spt16 to the heterochromatic region in the pob3∆ strain was decreased to half that of the wild type, suggesting the existence of a Pob3-independent recruitment mechanism of Spt16 onto heterochromatin.…”

Leaving histone unturned for epigenetic inheritance

“…FACT associates with Swi6 and this association requires the nuclear rim protein Amo1 and the RNA processing Rix1 complex (RIXC) complex. FACT, Amo1, and RIXC are all required for heterochromatin maintenance, but dispensable for heterochromatin establishment [79–82]. The tethering of heterochromatin to Amo1 at the nuclear periphery may create a specialized domain with a higher concentration of silencing proteins, which aids the recruitment of FACT to suppress histone turnover [80].…”