Wisconsin Center of Excellence in Genomics Science

Summary

Principal Investigator: Michael Olivier
Abstract: DESCRIPTION (provided by applicant): 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.
Funding Period: 2009-08-12 - 2015-06-30
more information: NIH RePORT

Top Publications

  1. pmc Affinity purification of the Arabidopsis 26 S proteasome reveals a diverse array of plant proteolytic complexes
    Adam J Book
    Department of Genetics, University of Wisconsin, Madison, Wisconsin 53706, USA
    J Biol Chem 285:25554-69. 2010
  2. pmc Current analysis platforms and methods for detecting copy number variation
    Wenli Li
    Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA
    Physiol Genomics 45:1-16. 2013
  3. pmc Quantitative proteomics reveals factors regulating RNA biology as dynamic targets of stress-induced SUMOylation in Arabidopsis
    Marcus J Miller
    Department of Genetics, 425 G Henry Mall, University of Wisconsin Madison, Madison, Wisconsin 53706, USA
    Mol Cell Proteomics 12:449-63. 2013
  4. pmc Characterization and quantification of intact 26S proteasome proteins by real-time measurement of intrinsic fluorescence prior to top-down mass spectrometry
    Jason D Russell
    Department of Chemistry, University of Wisconsin Madison, Madison, Wisconsin, United States of America
    PLoS ONE 8:e58157. 2013
  5. pmc Discovery and mass spectrometric analysis of novel splice-junction peptides using RNA-Seq
    Gloria M Sheynkman
    Department of Chemistry, University of Wisconsin Madison, 1101 University Ave, Madison, Wisconsin 53706, USA
    Mol Cell Proteomics 12:2341-53. 2013
  6. pmc Advanced proteomic analyses yield a deep catalog of ubiquitylation targets in Arabidopsis
    Do Young Kim
    Department of Genetics, University of Wisconsin, Madison, Wisconsin 53706, USA
    Plant Cell 25:1523-40. 2013
  7. pmc Variant discovery in targeted resequencing using whole genome amplified DNA
    Amit R Indap
    Department of Biology, Boston College, Chestnut Hill, MA, USA
    BMC Genomics 14:468. 2013
  8. pmc Chemical derivatization of peptide carboxyl groups for highly efficient electron transfer dissociation
    Brian L Frey
    Department of Chemistry, University of Wisconsin Madison, 1101 University Avenue, Madison, WI, 53706, USA
    J Am Soc Mass Spectrom 24:1710-21. 2013
  9. pmc Large-scale mass spectrometric detection of variant peptides resulting from nonsynonymous nucleotide differences
    Gloria M Sheynkman
    Department of Chemistry, University of Wisconsin Madison, 1101 University Avenue, Madison, Wisconsin 53706, United States
    J Proteome Res 13:228-40. 2014
  10. pmc Defining flexible vs. inherent promoter architectures: the importance of dynamics and environmental considerations
    Dana J Huebert
    Program in Cellular and Molecular Biology University of Wisconsin Madison Madison, WI USA
    Nucleus 3:399-403. 2012

Detail Information

Publications23

  1. pmc Affinity purification of the Arabidopsis 26 S proteasome reveals a diverse array of plant proteolytic complexes
    Adam J Book
    Department of Genetics, University of Wisconsin, Madison, Wisconsin 53706, USA
    J Biol Chem 285:25554-69. 2010
    ..Taken together, it appears that a diverse and highly dynamic population of proteasomes is assembled in plants, which may expand the target specificity and functions of intracellular proteolysis...
  2. pmc Current analysis platforms and methods for detecting copy number variation
    Wenli Li
    Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA
    Physiol Genomics 45:1-16. 2013
    ..In this article, we evaluate a suite of commonly used and recently developed approaches to uncovering genome-wide CNVs and discuss the relative merits of each...
  3. pmc Quantitative proteomics reveals factors regulating RNA biology as dynamic targets of stress-induced SUMOylation in Arabidopsis
    Marcus J Miller
    Department of Genetics, 425 G Henry Mall, University of Wisconsin Madison, Madison, Wisconsin 53706, USA
    Mol Cell Proteomics 12:449-63. 2013
    ..Collectively, our quantitative data emphasize the importance of SUMO to RNA-related processes protecting plants from adverse environments...
  4. pmc Characterization and quantification of intact 26S proteasome proteins by real-time measurement of intrinsic fluorescence prior to top-down mass spectrometry
    Jason D Russell
    Department of Chemistry, University of Wisconsin Madison, Madison, Wisconsin, United States of America
    PLoS ONE 8:e58157. 2013
    ..In addition, our approach permitted the unambiguous identification and quantification both isoforms of the proteasome-associated protein DSS1...
  5. pmc Discovery and mass spectrometric analysis of novel splice-junction peptides using RNA-Seq
    Gloria M Sheynkman
    Department of Chemistry, University of Wisconsin Madison, 1101 University Ave, Madison, Wisconsin 53706, USA
    Mol Cell Proteomics 12:2341-53. 2013
    ..To our knowledge this is the first example of using sample-specific RNA-Seq data to create a splice-junction database and discover new peptides resulting from alternative splicing. ..
  6. pmc Advanced proteomic analyses yield a deep catalog of ubiquitylation targets in Arabidopsis
    Do Young Kim
    Department of Genetics, University of Wisconsin, Madison, Wisconsin 53706, USA
    Plant Cell 25:1523-40. 2013
    ....
  7. pmc Variant discovery in targeted resequencing using whole genome amplified DNA
    Amit R Indap
    Department of Biology, Boston College, Chestnut Hill, MA, USA
    BMC Genomics 14:468. 2013
    ....
  8. pmc Chemical derivatization of peptide carboxyl groups for highly efficient electron transfer dissociation
    Brian L Frey
    Department of Chemistry, University of Wisconsin Madison, 1101 University Avenue, Madison, WI, 53706, USA
    J Am Soc Mass Spectrom 24:1710-21. 2013
    ..This carboxyl derivatization strategy greatly improves proteome coverage obtained from ETD-MS/MS of tryptic digests, and we anticipate that it will also enhance identification and localization of post-translational modifications. ..
  9. pmc Large-scale mass spectrometric detection of variant peptides resulting from nonsynonymous nucleotide differences
    Gloria M Sheynkman
    Department of Chemistry, University of Wisconsin Madison, 1101 University Avenue, Madison, Wisconsin 53706, United States
    J Proteome Res 13:228-40. 2014
    ..The SAP peptides included 192 allelic pairs; the relative expression levels of the two alleles were evaluated for 51 of those pairs and were found to be comparable in all cases. ..
  10. pmc Defining flexible vs. inherent promoter architectures: the importance of dynamics and environmental considerations
    Dana J Huebert
    Program in Cellular and Molecular Biology University of Wisconsin Madison Madison, WI USA
    Nucleus 3:399-403. 2012
    ..Our results argue that, while some aspects of promoter architecture are fixed across environments, the level to which promoters are "open" or "covered" by nucleosomes depends on the conditions investigated...
  11. pmc Absolute quantification of prion protein (90-231) using stable isotope-labeled chymotryptic peptide standards in a LC-MRM AQUA workflow
    Robert Sturm
    Department of Chemistry, University of Wisconsin, Madison, WI 53705, USA
    J Am Soc Mass Spectrom 23:1522-33. 2012
    ..To the authors' knowledge, this is the first report on the use of a non-tryptic peptide in a LC-MRM AQUA workflow...
  12. pmc Instant spectral assignment for advanced decision tree-driven mass spectrometry
    Derek J Bailey
    Department of Chemistry, University of Wisconsin, Madison, WI 53706, USA
    Proc Natl Acad Sci U S A 109:8411-6. 2012
    ..The decision tree logic enabled by inSeq promises to circumvent problems with the conventional data-dependent acquisition paradigm and provides a direct route to streamlined and expedient targeted protein analysis...
  13. pmc Alkylating tryptic peptides to enhance electrospray ionization mass spectrometry analysis
    Suzanne E Kulevich
    Department of Chemistry, University of Wisconsin Madison, 1101 University Avenue, Madison, Wisconsin 53706, United States
    Anal Chem 82:10135-42. 2010
    ..Increased chromatographic retention and ionization efficiency is observed for the alkylated Oct4 peptide compared to its unmodified form...
  14. pmc Parallel detection of intrinsic fluorescence from peptides and proteins for quantification during mass spectrometric analysis
    Jason D Russell
    Department of Chemistry, University of Wisconsin Madison, Madison, Wisconsin 53706, United States
    Anal Chem 83:2187-93. 2011
    ..Chromatographically separated intact proteins analyzed using UV-IF prior to top-down mass spectrometry demonstrated sensitive detection of proteins as large as 77 kDa...
  15. pmc Characterization and diagnostic value of amino acid side chain neutral losses following electron-transfer dissociation
    Qiangwei Xia
    Department of Chemistry, University of Wisconsin Madison, Madison, WI 53706, USA
    J Am Soc Mass Spectrom 22:255-64. 2011
    ....
  16. pmc To understand the whole, you must know the parts: unraveling the roles of protein-DNA interactions in genome regulation
    Lloyd M Smith
    Department of Chemistry, University of Wisconsin, 1101 University Avenue, Madison, WI 53706, USA
    Analyst 136:3060-5. 2011
    ..Developing a new technology to provide this information constitutes a "Grand Challenge" for Analytical Chemistry. In this brief article we outline the nature of this challenge, and propose one strategy to address it...
  17. pmc Sequential abundant ion fragmentation analysis (SAIFA): an alternative approach for phosphopeptide identification using an ion trap mass spectrometer
    Marla Chesnik
    Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA
    Anal Biochem 418:197-203. 2011
    ..To the best of our knowledge, this is the first report to use SAIFA for phosphopeptide identification...
  18. pmc Sequence-specific capture of protein-DNA complexes for mass spectrometric protein identification
    Cheng Hsien Wu
    Department of Chemistry, University of Wisconsin, Madison, Wisconsin, United States of America
    PLoS ONE 6:e26217. 2011
    ..This novel strategy provides a powerful tool for studies of protein-DNA and protein-protein interactions...
  19. pmc Dynamic changes in nucleosome occupancy are not predictive of gene expression dynamics but are linked to transcription and chromatin regulators
    Dana J Huebert
    Program in Cellular and Molecular Biology, University of Wisconsin Madison, Madison, Wisconsin, USA
    Mol Cell Biol 32:1645-53. 2012
    ..Together, these results highlight the complexities of stress-dependent chromatin changes and their effects on gene expression...
  20. pmc RNA-Mediated Gene Assembly from DNA Arrays
    Cheng Hsien Wu
    Department of Chemistry, University of Wisconsin Madison, 1101 University Ave, Madison, WI 53706 USA http smith chem wisc edu
    Angew Chem Int Ed Engl 51:4628-32. 2012
    ..These RNA molecules self-assemble into the desired full-length transcript by hybridization and ligation, which is then converted into double-stranded DNA by reverse transcriptase polymerase chain reaction...
  21. pmc Methylation of yeast ribosomal protein S2 is elevated during stationary phase growth conditions
    Daniel T Ladror
    Department of Chemistry, University of Wisconsin Madison, 1101 University Avenue, Madison, WI 53706, USA
    Biochem Biophys Res Commun 445:535-41. 2014
    ..This suggests that yeast in the stationary phase increase methylation of RPS2 in order to regulate ribosomal synthesis. These results demonstrate the utility of mass spectrometry for quantifying dynamic changes in ribosomal PTMs. ..