Technologies for making genome-wide observations

Microarray profiling of phage-display selections for rapid mapping of transcription factor-DNA interactions

One of the major challenges in characterizing transcriptional networks is to validate the large number of computational predictions of transcription factor binding sites. In order to address this challenge, we have developed a phage-display system for identification of proteins that recognize computationally predicted regulatory elements. Our approach relies on in vitro selection of a diverse population of T7-phage for binding to immobilized DNA containing predicted binding sites. These phage libraries are constructed from genomic fragments encoding peptides between 50-500 amino-acids in length. Unlike traditional phage-display, which requires many rounds of selections in order to enrich the library for subsequent sequencing of clones, we are able to in effect ‘sequence’ the entire library by amplification of inserts and hybridization to a microarray containing all open reading frames. We have shown that this is a powerful technology for rapidly identifying diverse transcription factor classes that recognize known binding sites. We have also used this system to identify a novel trans-factor that recognizes one of our strongest computational predictions in yeast (PAC element: GCGATGAG). This accomplishment is particularly significant since previous biochemical and one-hybrid attempts in multiple laboratories had failed to identify this factor. A manuscript describing the technology and its applications to the identification of yeast transcription factors was recently published in PLoS Genetics (Freckleton et al., 2009, 5(4):e1000449).

Global protein occupancy profiling of bacterial chromosomes

In order to comprehensively model transcriptional regulation, we need experimental observations beyond transcriptional output. In particular, we need to simultaneously monitor the interactions of all protein regulators with DNA at high spatial and temporal resolution. We have developed such a technology that reveals global in vivo protein occupancy across an entire bacterial chromosome at the resolution of individual binding sites. To achieve this, we have developed a biochemical separation procedure that allows us to selectively enrich minimal protein-DNA complexes from in vivo formaldehyde cross-linked samples, and to use a high-density tiling array to quantify the abundances of the enriched sequences genome-wide. We achieve the biochemical separation through a modification of phenol/chloroform extractions, where protein-DNA complexes are trapped at the interface between the aqueous and organic phases. Through subsequent molecular manipulations, labeling, and hybridizations, we can now quantify occupancy at the spatial resolution of ~30 base-pairs, with extremely high signal-to-noise. Application of this technology to E. coli reveals thousands of protein occupancy peaks which are largely confined within and in the close proximity to non-coding regulatory regions. Strikingly, we also observe extensive (>1 kilobase) protein occupancy domains (EPODs), the majority of which are localized to transcriptionally-silent loci dominated by conserved hypothetical ORFs. These regions are highly enriched in both predicted and experimentally determined binding sites of nucleoid proteins, and exhibit extreme biophysical characteristics such as high intrinsic curvature. Our observations implicate these transcriptionally-silent EPODs as the elusive organizing centers, long proposed to topologically isolate chromosomal domains. A manuscript describing the technology and its initial application to large-scale chromosomal occupancy was recently published in Molecular Cell (Vora et al., 2009, 35(2):247-53). This technology will allow us to make simultaneous high-resolution measurements of transcriptional output and protein-DNA interaction on a genomic scale. These observations will allow comprehensive and unbiased systems-level modeling of transcriptional regulation in prokaryotes.

Related publications

Global discovery of adaptive mutations
Nature Methods. 2009 Aug; 6(8):581-3 PDF
Goodarzi H, Hottes AK, Tavazoie S

Global protein occupancy landscape of a bacterial genome
Molecular Cell. 2009 Jul 31;35(2):247-53 PDF
Vora T, Hottes AK, Tavazoie S

Microarray profiling of phage-display selections for rapid mapping of transcription factor-DNA interactions.
PLoS Genet. 2009 Apr;5(4):e1000449. Epub 2009 Apr 10. PDF
Freckleton G, Lippman SI, Broach JR, Tavazoie S