Cas5 was originally identified in a genetic screen for transcription factor mutants that are hypersensitive to the echinocandin caspofungin, and microarray analysis implicated Cas5 in the regulation of genes important for cell wall integrity in response to cell wall stress22. However, the role of Cas5 under basal conditions remained elusive. To further understand Cas5 function under basal conditions, we sought to explore the impact of Cas5 on regulation of global transcriptional profiles in rich medium. To do so, we used chromatin immunoprecipitation (ChIP) of RNA polymerase II (PolII) coupled to sequencing (ChIP-Seq) with a wild-type strain and cas5Δ/cas5Δ mutant. RNA PolII occupancy has been found to serve as a more precise measure of transcriptional regulation than steady state transcript levels, given that transcript levels depend on RNA turnover in addition to transcription factor activity and RNA PolII-mediated transcription30. As RNA PolII recruitment corresponds to transcriptional activity, an increase in RNA PolII occupancy indicates upregulation of gene expression, whereas a decrease in RNA PolII occupancy indicates downregulation30, 31. Further, strong correlation in RNA PolII binding between our biological replicates supported the reproducibility of this method (Supplementary Fig. 1). We identified 329 genes that had increased RNA PolII occupancy, and 275 genes that had reduced PolII occupancy in the cas5Δ/cas5Δ mutant as compared with the wild-type strain under standard growth conditions (Supplementary Data 1). To identify the physiological roles of Cas5, we subjected these gene sets to pathway analysis using Gene Ontology (GO). The sets that had reduced RNA PolII occupancy upon deletion of CAS5 were significantly enriched in genes with functions in diverse processes, including metabolic processes and interaction with host (Fig. 1a and Supplementary Data 1). In contrast, the gene set that had increased RNA PolII occupancy in a cas5Δ/cas5Δ mutant was enriched in genes with functions associated with rRNA processing, respiration, and amino acid biogenesis (Fig. 1a and Supplementary Data 1). We also employed Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis as a secondary approach to identify genes and pathways affected by deletion of CAS5. Consistent with our GO term analysis, many genes with RNA PolII-binding profiles that were affected by Cas5 were associated with ribosome biogenesis and metabolic pathways (Fig. 1b and Supplementary Data 1). KEGG analysis also identified a signature of differentially bound genes with functions in meiosis, cell cycle, and DNA replication (Fig. 1c and Supplementary Data 1). Thus, Cas5 governs diverse biological responses under basal conditions.Fig. 1Cas5 has a profound impact on RNA PolII binding at genes implicated in cell wall and cell cycle processes under both basal and cell wall stress conditions. a Heat maps showing the genes with increased (red) and decreased (blue) RNA PolII binding in a cas5∆/cas5∆ mutant relative to wildtype. Enriched GO processes are indicated, and were clustered using the DAVID Gene Functional Classification Tool. b Bar chart showing the number of genes differentially bound by PolII (DBGs differentially bound genes), with increased binding in red and decreased binding in blue, in a cas5∆/cas5∆ mutant relative to wildtype based on their assigned KEGG pathways. KEGG pathways with four or more genes assigned are shown. c Bar chart showing the number of genes differentially bound by PolII, with increased binding in red and decreased binding in blue, in a cas5∆/cas5∆ mutant relative to wildtype belonging to select KEGG pathways involved in cell cycle and related processes. d, e Bar charts showing the number of differentially bound genes involved in the indicated physiological pathways upon caspofungin treatment, with increased binding in red and decreased binding in blue, in a wild-type strain in response to caspofungin. KEGG pathways were grouped according to the ratio of genes with increased PolII binding to genes with decreased PolII binding in response to caspofungin, with d enriched for genes with increased PolII binding and e enriched for genes with decreased PolII binding. KEGG pathways with five or more genes assigned are shown. f Venn diagram depicting number of genes differentially bound by RNA PolII in response to caspofungin in the wild-type reference (increased, green; decreased, yellow) and cas5Δ/cas5Δ mutant (increased, red; decreased, blue) strains. See Supplementary Data 1–4 for full data sets