The successful completion of the human genome and model organism sequences has ushered in a new era in biological research, with attention now focused on understanding the way in which genome sequence information is expressed and controlled. The focus of this proposed Wisconsin Center of Excellence in Genomics Science is to facilitate understanding of the complex and integrated regulatory mechanisms affecting gene transcription by developing novel technology for the comprehensive characterization and quantitative analysis of proteins interacting with DMA. This new technology will help provide for a genomewide functional interpretation of the underlying mechanisms by which gene transcriptional regulation is altered during biological processes, development, disease, and in response to physiological, pharmacological, or environmental stressors. The development of chromatin immunoprecipitation approaches has allowed identification of the specific DMA sequences bound by proteins of interest. We propose to reverse this strategy and develop an entirely novel technology that will use oligonucleotide capture to pull down DNA sequences ot interest, and mass spectrometry to identify and characterize the proteins and protein complexes bound and associated with particular DNA regions. This new approach will create an invaluable tool for deciphering the critical control processes regulating an essential biological function. The proposed interdisciplinary and multi-institutional Center of Excellence in Genomics Science combines specific expertise at the Medical College of Wisconsin, the University of Wisconsin Madison, and Marquette University. Technological developments in four specific areas will be pursued to develop this new approach: (1) cross-linking of proteins to DNA and fragmentation of chromatin;(2) capture of the protein-DNA complexes in a DNA sequence-specific manner;(3) mass spectrometry analysis to identify and quantify bound proteins, determine posttranslational modifications, and characterize protein complex stoichipmetry;and (4) informatics to develop tools enabling the global analysis of the relationship between changes in protein-DNA interactions and gene expression. The Center will use carefully selected biological systems of increasing complexity from three species (yeast, mouse, human) to develop and test the technology in an integrated genome-wide analysis platform that includes efficient data management and analysis tools. As part of the Center mission, we will combine our technology development efforts with an interdisciplinary training program for students and fellows designed to train qualified scientists experienced in cutting-edge genomics technology. Data, technology, and software will be widely disseminated by multiple mechanisms including licensing and commercialization activities.

Public Health Relevance

The development of this novel technology to comprehensively identify and characterize genpmic protein- DNA interactions will help in the interpretation of a core function of the genome, gene transcription. The tools and technologies developed as part of the CEGS will be broadly available to the scientific community, and will help decipher crucial molecular mechanisms regulating the transcriptional levels of all genes across the genome, and how these regulatory mechanisms are altered in disease. This knowledge will revolutionize our understanding of genome biology, and impact how genomic information will be used in clinical medicine.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Specialized Center (P50)
Project #
7P50HG004952-05
Application #
8528664
Study Section
Special Emphasis Panel (ZHG1-HGR-N (J1))
Program Officer
Gatlin, Christine L
Project Start
2009-08-12
Project End
2014-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
5
Fiscal Year
2013
Total Cost
$2,574,664
Indirect Cost
$744,395
Name
Texas Biomedical Research Institute
Department
Type
DUNS #
007936834
City
San Antonio
State
TX
Country
United States
Zip Code
78245
Dai, Yunxiang; Kennedy-Darling, Julia; Shortreed, Michael R et al. (2017) Multiplexed Sequence-Specific Capture of Chromatin and Mass Spectrometric Discovery of Associated Proteins. Anal Chem 89:7841-7846
Aguilar-Hernández, Victor; Kim, Do-Young; Stankey, Robert J et al. (2017) Mass Spectrometric Analyses Reveal a Central Role for Ubiquitylation in Remodeling the Arabidopsis Proteome during Photomorphogenesis. Mol Plant 10:846-865
Buxton, Katherine E; Kennedy-Darling, Julia; Shortreed, Michael R et al. (2017) Elucidating Protein-DNA Interactions in Human Alphoid Chromatin via Hybridization Capture and Mass Spectrometry. J Proteome Res 16:3433-3442
Guillen-Ahlers, Hector; Rao, Prahlad K; Levenstein, Mark E et al. (2016) HyCCAPP as a tool to characterize promoter DNA-protein interactions in Saccharomyces cerevisiae. Genomics 107:267-73
Cesnik, Anthony J; Shortreed, Michael R; Sheynkman, Gloria M et al. (2016) Human Proteomic Variation Revealed by Combining RNA-Seq Proteogenomics and Global Post-Translational Modification (G-PTM) Search Strategy. J Proteome Res 15:800-8
Hoffman, Elizabeth A; Frey, Brian L; Smith, Lloyd M et al. (2015) Formaldehyde crosslinking: a tool for the study of chromatin complexes. J Biol Chem 290:26404-11
Shortreed, Michael R; Wenger, Craig D; Frey, Brian L et al. (2015) Global Identification of Protein Post-translational Modifications in a Single-Pass Database Search. J Proteome Res 14:4714-20
Guillen-Ahlers, Hector; Shortreed, Michael R; Smith, Lloyd M et al. (2014) Advanced methods for the analysis of chromatin-associated proteins. Physiol Genomics 46:441-7
Sheynkman, Gloria M; Shortreed, Michael R; Frey, Brian L et al. (2014) Large-scale mass spectrometric detection of variant peptides resulting from nonsynonymous nucleotide differences. J Proteome Res 13:228-40
Kennedy-Darling, Julia; Guillen-Ahlers, Hector; Shortreed, Michael R et al. (2014) Discovery of Chromatin-Associated Proteins via Sequence-Specific Capture and Mass Spectrometric Protein Identification in Saccharomyces cerevisiae. J Proteome Res 13:3810-25

Showing the most recent 10 out of 36 publications