“The regulation of gene transcription is fundamental to the existence of complex multicellular organisms such as humans. This process dictates which genes are expressed in which tissues, and controls how various cell types grow, differentiate, and respond to their environments. Although the deciphering of the human genome sequence has given us the “source code” for life, we still know far too little about the mechanisms that control which sets of genes are active in which tissues, and how their expression is regulated. It is clear, however, that much of this system depends upon the sequence-specific interactions of regulatory proteins with particular genetic loci. To be able to unravel the details of these interactions on a genome-wide basis, it is necessary to know what proteins are bound to the DNA where in the genome, and to be able to monitor how those proteins change over time and in response to external stimuli.”
“While technologies already exist for the genome-wide analysis of gene transcription and DNA methylation, there is a desperate need for new technologies that enable the comprehensive parallel analysis of all protein-DNA interactions (including histones) without prior knowledge or assumptions (Smith, Shortreed, and Olivier, Analyst. 2011 Aug 7; 136(15):3060-5.)
The Wisconsin Center of Excellence in Genomics Science is inventing novel technologies for the comprehensive characterization and quantitative analysis of proteins interacting with DNA. In this approach, a specific DNA fragment is captured in a sequence-specific manner, allowing the isolation and subsequent characterization of all proteins bound to that region. As for chromatin immunoprecipitation, formaldehyde may be used to crosslink proteins and DNA in vivo, locking into place the protein-DNA interactions which are present at that time. The chromatin is then fragmented, either by physical means such as sonication, or by restriction enzyme digestion. Next, an exonuclease removes one of the two strands of the DNA duplexes protruding from the complex, leaving behind a free single-stranded region suitable for DNA 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 refer to this methodology as GENECAPP, for Global ExoNuclease-based Enrichment of Chromatin-Associated Proteins for Proteomics.
The technology development efforts have resulted in initial protocols that can be found in the Protocols and Methodology section (Resources). These efforts have also been described in our recent publications.
The methodologies are being applied, at this initial stage, to carefully selected biological systems that have been well-studied, and thus provide a gold standard reference for our new GENECAPP approach. Our initial work has focused on two biological systems:
- The mouse IGFBP1 promoter region. This in vitro system allows the examination of a single transcription factor FoxO1, in conjunction with nucleosomes for early technology development. The model has been described in detail in our recent publication (Wu et al., PloS One. 2011; 6 (10) :e26217).
- The Upstream Activator Sequence UASGal between the Gal1 and Gal10 genes of S. cerevisiae. This well-studied region allows the analysis of chomatin in vivo using the GENECAPP approach. Binding proteins for this region have been well characterized under different growth conditions, and the model has been chosen to serve as a reference system to test the ability of GENECAPP to identify known DNA-rotein interactions. Results from our efforts using the GENECAPP technology in this system are still in progress.
In addition to the efforts of the Wisconsin CEGS outlined above, technologies and methods developed by the Wisconsin CEGS have been incorporated into multiple other research projects. Several of these projects are now funded independently, illustrating the broad impact this technology development effort is having even at this early stage.
Additional Projects Utilizing Technologies Developed by the Wisconsin CEGS:
- Regulation of Angiogenesis and Renin Expression in Rats
- Impact of HCMV proteins on viral replication and cellular signaling pathways
- microRNA Ribonucleoprotein Complex Components and Regulation of Target Protein Expression
- Using Mass Spectrometry to Identify Pathways Regulated by SH2B3 during Cardiac Remodeling
- An Integrated Approach to Understanding Host-Pathogen Interactions
- Identification of Transcription Elongation Complex Composition at Specific Genetic Loci in Escherichia coli