HyCCAPP as a tool to characterize promoter DNA-protein interactions in Saccharomyces cerevisiae

• Published in: Genomics (San Diego, Calif.) Calif.), 2016-06, Vol.107 (6), p.267-273
• Main Authors: Guillen-Ahlers, Hector, Rao, Prahlad K., Levenstein, Mark E., Kennedy-Darling, Julia, Perumalla, Danu S., Jadhav, Avinash Y.L., Glenn, Jeremy P., Ludwig-Kubinski, Amy, Drigalenko, Eugene, Montoya, Maria J., Göring, Harald H., Anderson, Corianna D., Scalf, Mark, Gildersleeve, Heidi I.S., Cole, Regina, Greene, Alexandra M., Oduro, Akua K., Lazarova, Katarina, Cesnik, Anthony J., Barfknecht, Jared, Cirillo, Lisa A., Gasch, Audrey P., Shortreed, Michael R., Smith, Lloyd M., Olivier, Michael
• Format: Article
• Language: English
• Subjects: Chromatin; Chromatin - genetics; Chromatin immunoprecipitation; Chromatin Immunoprecipitation - methods; DNA-Binding Proteins - genetics; DNA-protein interactions; ENO2; Galactokinase - genetics; Phosphopyruvate Hydratase - genetics; Promoter Regions, Genetic; Protein Binding - genetics; Proteomics; Proteomics - methods; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins - genetics; Yeast
• ISSN: 0888-7543; 1089-8646
• DOI: 10.1016/j.ygeno.2016.05.002

Currently available methods for interrogating DNA-protein interactions at individual genomic loci have significant limitations, and make it difficult to work with unmodified cells or examine single-copy regions without specific antibodies. In this study, we describe a physiological application of the Hybridization Capture of Chromatin-Associated Proteins for Proteomics (HyCCAPP) methodology we have developed. Both novel and known locus-specific DNA-protein interactions were identified at the ENO2 and GAL1 promoter regions of Saccharomyces cerevisiae, and revealed subgroups of proteins present in significantly different levels at the loci in cells grown on glucose versus galactose as the carbon source. Results were validated using chromatin immunoprecipitation. Overall, our analysis demonstrates that HyCCAPP is an effective and flexible technology that does not require specific antibodies nor prior knowledge of locally occurring DNA-protein interactions and can now be used to identify changes in protein interactions at target regions in the genome in response to physiological challenges. •An unbiased method to study DNA-protein interactions in vivo is proposed.•Sequence-specific hybridization of crosslinked chromatin fragments is described.•Whole genome sequencing validates the specificity of the process.•Novel DNA-protein interactions at single-copy regions are identified.