Sequence-specific capture of protein-DNA complexes for mass spectrometric protein identification.
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.
Department of Chemistry, University of Wisconsin, Madison, Wisconsin, United States of America.
Cheng-Hsien
CH
Wu
Siyuan
S
Chen
Michael R
MR
Shortreed
Gloria M
GM
Kreitinger
Yuan
Y
Yuan
Brian L
BL
Frey
Yi
Y
Zhang
Shama
S
Mirza
Lisa A
LA
Cirillo
Michael
M
Olivier
Lloyd M
LM
Smith
D000818:Animals; D001483:Base Sequence; D002843:Chromatin / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005090:Exodeoxyribonucleases / Q000378:metabolism; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors / Q000737:chemistry* / Q000378:metabolism*; D005557:Formaldehyde / Q000494:pharmacology; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000737:chemistry* / Q000378:metabolism*; D013058:Mass Spectrometry; D051379:Mice; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D040901:Proteomics / Q000379:methods*; D013379:Substrate Specificity
D002843:Chromatin; D003432:Cross-Linking Reagents; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; C467200:Foxo1 protein, mouse; D018970:Insulin-Like Growth Factor Binding Protein 1; D009841:Oligonucleotides; D005557:Formaldehyde; D004247:DNA; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III