Broadly Applicable Control Approaches Improve Accuracy of ChIP-Seq Data.

Chromatin ImmunoPrecipitation (ChIP) is a widely used method for the analysis of protein-DNA interactions in vivo; however, ChIP has pitfalls, particularly false-positive signal enrichment that permeates the data. We have developed a new approach to control for non-specific enrichment in ChIP that involves the expression of a non-genome-binding protein targeted in the IP alongside the experimental target protein due to the sharing of epitope tags. ChIP of the protein provides a "sensor" for non-specific enrichment that can be used for the normalization of the experimental data, thereby correcting for non-specific signals and improving data quality as validated against known binding sites for several proteins that we tested, including Fkh1, Orc1, Mcm4, and Sir2. We also tested a DNA-binding mutant approach and showed that, when feasible, ChIP of a site-specific DNA-binding mutant of the target protein is likely an ideal control. These methods vastly improve our ChIP-seq results in S. cerevisiae and should be applicable in other systems.

Dbf4 Zn-Finger Motif Is Specifically Required for Stimulation of Ctf19-Activated Origins in Saccharomyces cerevisiae.

Eukaryotic genomes are replicated in spatiotemporal patterns that are stereotypical for individual genomes and developmental profiles. In the model system Saccharomyces cerevisiae, two primary mechanisms determine the preferential activation of replication origins during early S phase, thereby largely defining the consequent replication profiles of these cells. Both mechanisms are thought to act through specific recruitment of a rate-limiting initiation factor, Dbf4-dependent kinase (DDK), to a subset of licensed replication origins. Fkh1/2 is responsible for stimulation of most early-firing origins, except for centromere (CEN)-proximal origins that recruit DDK via the kinetochore protein Ctf19, which is required for their early firing. The C-terminus of Dbf4 has been implicated in its recruitment to origins via both the Fkh1/2 and Ctf19 mechanisms. Here, we show that the Zn-finger motif within the C-terminus is specifically required for Dbf4 recruitment to CENs to stimulate CEN-proximal/Ctf19-dependent origins, whereas stimulation of origins via the Fkh1/2 pathway remains largely intact. These findings re-open the question of exactly how Fkh1/2 and DDK act together to stimulate replication origin initiation.

Rpd3 regulates single-copy origins independently of the rDNA array by opposing Fkh1-mediated origin stimulation.

Eukaryotic chromosomes are organized into structural and functional domains with characteristic replication timings, which are thought to contribute to epigenetic programming and genome stability. Differential replication timing results from epigenetic mechanisms that positively and negatively regulate the competition for limiting replication initiation factors. Histone deacetylase Sir2 negatively regulates initiation of the multicopy (∼150) rDNA origins, while Rpd3 histone deacetylase negatively regulates firing of single-copy origins. However, Rpd3's effect on single-copy origins might derive indirectly from a positive function for Rpd3 in rDNA origin firing shifting the competitive balance. Our quantitative experiments support the idea that origins compete for limiting factors; however, our results show that Rpd3's effect on single-copy origin is independent of rDNA copy-number and of Sir2's effects on rDNA origin firing. Whereas RPD3 deletion and SIR2 deletion alter the early S phase dynamics of single-copy and rDNA origin firings in opposite fashion, unexpectedly only RPD3 deletion suppresses overall rDNA origin efficiency across S phase. Increased origin activation in rpd3Δ requires Fkh1/2, suggesting that Rpd3 opposes Fkh1/2-origin stimulation, which involves recruitment of Dbf4-dependent kinase (DDK). Indeed, Fkh1 binding increases at Rpd3-regulated origins in rpd3Δ cells in G1, supporting a mechanism whereby Rpd3 influences initiation timing of single-copy origins directly through modulation of Fkh1-origin binding. Genetic suppression of a DBF4 hypomorphic mutation by RPD3 deletion further supports the conclusion that Rpd3 impedes DDK recruitment by Fkh1, revealing a mechanism of Rpd3 in origin regulation.

Dynamic relocalization of replication origins by Fkh1 requires execution of DDK function and Cdc45 loading at origins.

Chromosomal DNA elements are organized into spatial domains within the eukaryotic nucleus. Sites undergoing DNA replication, high-level transcription, and repair of double-strand breaks coalesce into foci, although the significance and mechanisms giving rise to these dynamic structures are poorly understood. In S. cerevisiae, replication origins occupy characteristic subnuclear localizations that anticipate their initiation timing during S phase. Here, we link localization of replication origins in G1 phase with Fkh1 activity, which is required for their early replication timing. Using a Fkh1-dependent origin relocalization assay, we determine that execution of Dbf4-dependent kinase function, including Cdc45 loading, results in dynamic relocalization of a replication origin from the nuclear periphery to the interior in G1 phase. Origin mobility increases substantially with Fkh1-driven relocalization. These findings provide novel molecular insight into the mechanisms that govern dynamics and spatial organization of DNA replication origins and possibly other functional DNA elements.