Post-translational histone modifications (PTMs) play important roles in regulating various DNA-templated cellular functions including transcription, DNA damage repair, DNA replication and telomeric silencing. Histone H3 Threonine 45 phosphorylation (H3T45ph) is a modification that is enriched during S-phase of the cell cycle in Saccharomyces cerevisiae and is carried out by the kinase Cdc7-Dbf4 (DDK) in vivo and in vitro. To understand the function of this mark we identified the genomic H3T45ph locations by performing chromatin immunoprecipitation followed by high throughput sequencing (ChIP-Seq) of wild-type budding yeast cell chromatin. Remarkably, this PTM is observed not only at origins of DNA replication, but is also uniquely distributed on the promoters of highly transcribed RNA polymerase I, II and III-regulated genes dedicated to protein synthesis and glycolysis. Here we show that H3T45 phosphorylation, mediated by Cdc7-Dbf4, is required for polymerase recruitment and full expression of ribosome protein (RP) and tRNA genes as well as for the regulation of cohesin loading. Collectively, we identify an unexpected gene regulatory function for the DNA-replication kinase Cdc7-Dbf4 and identify a novel histone modification regulating the protein biosynthesis gene machinery.