title: HyCCAPP as a tool to characterize promoter DNA-protein interactions in Saccharomyces cerevisiae.
abstract: 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.
authors.affiliation: Department of Genetics, Texas Biomedical Research Institute, San Antonio, TX 78227, USA; Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
authors.forename: Hector
authors.initials: H
authors.lastname: Guillen-Ahlers
authors.affiliation: Department of Genetics, Texas Biomedical Research Institute, San Antonio, TX 78227, USA.
authors.forename: Prahlad K
authors.initials: PK
authors.lastname: Rao
authors.affiliation: Department of Chemistry, University of Wisconsin, Madison, WI 53706, USA.
authors.forename: Mark E
authors.initials: ME
authors.lastname: Levenstein
authors.affiliation: Department of Chemistry, University of Wisconsin, Madison, WI 53706, USA.
authors.forename: Julia
authors.initials: J
authors.lastname: Kennedy-Darling
authors.affiliation: Department of Genetics, Texas Biomedical Research Institute, San Antonio, TX 78227, USA.
authors.forename: Danu S
authors.initials: DS
authors.lastname: Perumalla
authors.affiliation: Department of Genetics, Texas Biomedical Research Institute, San Antonio, TX 78227, USA.
authors.forename: Avinash Y L
authors.initials: AY
authors.lastname: Jadhav
authors.affiliation: Department of Genetics, Texas Biomedical Research Institute, San Antonio, TX 78227, USA.
authors.forename: Jeremy P
authors.initials: JP
authors.lastname: Glenn
authors.affiliation: Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
authors.forename: Amy
authors.initials: A
authors.lastname: Ludwig-Kubinski
authors.affiliation: Department of Genetics, Texas Biomedical Research Institute, San Antonio, TX 78227, USA.
authors.forename: Eugene
authors.initials: E
authors.lastname: Drigalenko
authors.affiliation: Department of Genetics, Texas Biomedical Research Institute, San Antonio, TX 78227, USA.
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Montoya
authors.affiliation: Department of Genetics, Texas Biomedical Research Institute, San Antonio, TX 78227, USA.
authors.forename: Harald H
authors.initials: HH
authors.lastname: Göring
authors.affiliation: Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
authors.forename: Corianna D
authors.initials: CD
authors.lastname: Anderson
authors.affiliation: Department of Chemistry, University of Wisconsin, Madison, WI 53706, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Scalf
authors.affiliation: Department of Genetics, Texas Biomedical Research Institute, San Antonio, TX 78227, USA.
authors.forename: Heidi I S
authors.initials: HI
authors.lastname: Gildersleeve
authors.affiliation: Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
authors.forename: Regina
authors.initials: R
authors.lastname: Cole
authors.affiliation: Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
authors.forename: Alexandra M
authors.initials: AM
authors.lastname: Greene
authors.affiliation: Department of Cell Biology, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
authors.forename: Akua K
authors.initials: AK
authors.lastname: Oduro
authors.affiliation: Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
authors.forename: Katarina
authors.initials: K
authors.lastname: Lazarova
authors.affiliation: Department of Chemistry, University of Wisconsin, Madison, WI 53706, USA.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Cesnik
authors.affiliation: Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
authors.forename: Jared
authors.initials: J
authors.lastname: Barfknecht
authors.affiliation: Department of Cell Biology, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Cirillo
authors.affiliation: Department of Genetics, University of Wisconsin, Madison, WI 53706, USA.
authors.forename: Audrey P
authors.initials: AP
authors.lastname: Gasch
authors.affiliation: Department of Chemistry, University of Wisconsin, Madison, WI 53706, USA.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Shortreed
authors.affiliation: Department of Chemistry, University of Wisconsin, Madison, WI 53706, USA.
authors.forename: Lloyd M
authors.initials: LM
authors.lastname: Smith
authors.affiliation: Department of Genetics, Texas Biomedical Research Institute, San Antonio, TX 78227, USA; Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA. Electronic address: molivier@txbiomed.org.
authors.forename: Michael
authors.initials: M
authors.lastname: Olivier
mesh_terms: D002843:Chromatin / Q000235:genetics; D047369:Chromatin Immunoprecipitation / Q000379:methods; D004268:DNA-Binding Proteins / Q000235:genetics*; D005686:Galactokinase / Q000235:genetics*; D010751:Phosphopyruvate Hydratase / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000235:genetics; D040901:Proteomics / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*
chemical_list: D002843:Chromatin; D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; C524219:GAL1 protein, S cerevisiae; D005686:Galactokinase; D010751:Phosphopyruvate Hydratase; C516797:enolase 2, S cerevisiae
keywords: Chromatin; Chromatin immunoprecipitation; DNA-protein interactions; ENO2; Proteomics; Yeast