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.

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

Eukaryotic genomes are replicated in spatiotemporal patterns that are stereotypical for individual genomes and developmental profiles. In the model system , 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.

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.

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 , replication origins occupy characteristic subnuclear localizations that anticipate their initiation timing during S phase.