Sequence-specific capture of protein-DNA complexes for mass spectrometric protein identification. PLoS One 2011;6(10):e26217
Date
10/27/2011Pubmed ID
22028835Pubmed Central ID
PMC3197616DOI
10.1371/journal.pone.0026217Scopus ID
2-s2.0-80054776386 (requires institutional sign-in at Scopus site) 24 CitationsAbstract
The regulation of gene transcription is fundamental to the existence of complex multicellular organisms such as humans. Although it is widely recognized that much of gene regulation is controlled by gene-specific protein-DNA interactions, there presently exists little in the way of tools to identify proteins that interact with the genome at locations of interest. We have developed a novel strategy to address this problem, which we refer to as GENECAPP, for Global ExoNuclease-based Enrichment of Chromatin-Associated Proteins for Proteomics. In this approach, formaldehyde cross-linking is employed to covalently link DNA to its associated proteins; subsequent fragmentation of the DNA, followed by exonuclease digestion, produces a single-stranded region of the DNA that enables sequence-specific hybridization capture of the protein-DNA complex on a solid support. Mass spectrometric (MS) analysis of the captured proteins is then used for their identification and/or quantification. We show here the development and optimization of GENECAPP for an in vitro model system, comprised of the murine insulin-like growth factor-binding protein 1 (IGFBP1) promoter region and FoxO1, a member of the forkhead rhabdomyosarcoma (FoxO) subfamily of transcription factors, which binds specifically to the IGFBP1 promoter. This novel strategy provides a powerful tool for studies of protein-DNA and protein-protein interactions.
Author List
Wu CH, Chen S, Shortreed MR, Kreitinger GM, Yuan Y, Frey BL, Zhang Y, Mirza S, Cirillo LA, Olivier M, Smith LMAuthor
Lisa A. Cirillo PhD Assistant Dean, Associate Professor in the Cell Biology, Neurobiology and Anatomy department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AnimalsBase Sequence
Chromatin
Cross-Linking Reagents
DNA
Escherichia coli
Exodeoxyribonucleases
Forkhead Box Protein O1
Forkhead Transcription Factors
Formaldehyde
Insulin-Like Growth Factor Binding Protein 1
Mass Spectrometry
Mice
Nucleic Acid Hybridization
Oligonucleotides
Polymerase Chain Reaction
Promoter Regions, Genetic
Proteomics
Substrate Specificity
