Forkhead Transcription Factors Establish Origin Timing and Long-Range Clustering in S. cerevisiae

Abstract

SUMMARY The replication of eukaryotic chromosomes is organized temporally and spatially within the nucleus through epigenetic regulation of replication origin function. The characteristic initiation timing of specific origins is thought to reflect their chromatin environment or sub-nuclear positioning, however the mechanism remains obscure. Here we show that the yeast Forkhead transcription factors, Fkh1 and Fkh2, are global determinants of replication origin timing. Forkhead regulation of origin timing is independent of local levels or changes of transcription. Instead, we show that Fkh1 and Fkh2 are required for the clustering of early origins and their association with the key initiation factor Cdc45 in G1-phase, suggesting that Fkh1 and Fkh2 selectively recruit origins to emergent replication factories. Fkh1 and Fkh2 bind Fkh-activated origins, and interact physically with ORC, providing a plausible mechanism to cluster origins. These findings add a new dimension to our understanding of the epigenetic basis for differential origin regulation and its connection to chromosomal domain organization.

Nucleosome occupancy as a novel chromatin parameter for replication origin functions

“…2C). It was also recently reported that Forkhead transcription factors, Fkh1 and Fkh2, bind near origins, and their presence can dictate origin timing (Knott et al 2012). We found no correlation between origins with altered timing in Δfkh1fkh2 with nucleosome occupancy (P = 0.194, χ 2 test), suggesting that Fkh1 and Fkh2 affect origin properties independently of nucleosome occupancy (Fig.…”

“…For example, Fkh1 and Fkh2 (Knott et al 2012), and the proximity to telomeres and centromeres are known to affect origin properties, but nucleosome occupancy does not significantly correlate with these parameters. Indeed, although orc1-161 abolishes major changes in nucleosome occupancy at origins between G2/M and G1 and greatly disrupts proper origin usage profiles, changes in nucleosome occupancy do not perfectly correlate with the changes in origin activities.…”

“…(C) Nucleosome occupancy correlated with ORC binding properties for 66 origins, which were previously classified as DNA-dependent (red), chromatin-dependent (blue), or weak (green) (Hoggard et al 2013). (D) Nucleosome occupancy correlated with origins where activation depended on Forkhead proteins (Knott et al 2012). Origins that decrease (blue), increase (green), or are unchanged (red) in activity in the fkh1fkh2 mutant are shown.…”

“…We correlated H3 occupancy with 66 origins that were classified based on differences in ORC binding properties (Hoggard et al 2013) and used the Fisher's exact test to calculate statistical significance due to the small sample size of origins. A study reported 350 origins that were Fkh1 and Fkh2 dependent (Knott et al 2012), which was used in our analysis. Mcm2-7 ChIP-seq and ORC footprinting data sets (Belsky et al 2015) were used for analysis of pre-RC binding.…”

Conservation of replication timing reveals global and local regulation of replication origin activity

“…Recently the proximal binding of Forkhead transcription factors (Fkh1 and Fkh2) has been implicated in determining early origin activation time (Knott et al 2012). We find that of the 24 origins that replicate early in S. cerevisiae, but later in at least one other sensu stricto species, only seven are reported to be associated with Fkh1 and/or Fkh2 in S. cerevisiae (Supplemental Table S4; Venters et al 2011).…”