It is well established that DNA-binding factors modulate transcription in eukaryotes. It is thus of interest to know how and where these factors interact with promoter DNA. To date, such information has largely been obtained from investigations with protein extracts and nucleosome-free DNA templates. Future advances in our understanding of transcriptional processes will require new tools for evaluating the validity of in vitro results and determining how transcription complexes operate in vivo. The overall goal of this research is to develop a novel method for characterizing in vivo interactions between nuclear transcription factors and their binding sites in cellular promoters. The strategy relies on the ability of formaldehyde to rapidly and efficiently cross-link proteins with their DNA-binding sites in vivo. Fixed chromatin is then isolated mechanically or enzymatically-cleaved into smaller fragments, and immuno-enriched for the covalently-linked protein and DNA complex of interest. Cross-links in complexes are reversible at 65C, thus yielding individual protein and DNA components for Western blot or PCR analysis, respectively. Although untested as yet with living cells, preliminary results with a basic/leucine-zipper transcription factor and plant chromatin in vitro suggest that the proposed methodology is technically feasible. A successful outcome to this work will result in a significant paradigm shift in our ability to probe the molecular mechanisms of gene expression. Important biological questions that may be addressed by this work include: 1) identifying in vivo promoter-binding sites for a transcription factor, 2) isolating new genes that are regulated by a transcription factor, 3) analyzing changes in the DNA-binding activity of a factor at a known promoter site, and 4) identifying which member of a multi-gene family of factors binds to a common target site in vivo.
This research is expected to provide new tools for understanding how genes are regulated in cells. As genes underlie fundamental events in an organism's development, homeostasis, and defense, we envision that work here will advance our understanding of how multicellular organisms orchestrate these processes.
