Microarray Profiling of Phage-Display Selections for Rapid Mapping of Transcription Factor–DNA Interactions

Modern computational methods are revealing putative transcription-factor (TF) binding sites at an extraordinary rate. However, the major challenge in studying transcriptional networks is to map these regulatory element predictions to the protein transcription factors that bind them. We have developed a microarray-based profiling of phage-display selection (MaPS) strategy that allows rapid and global survey of an organism's proteome for sequence-specific interactions with such putative DNA regulatory elements. Application to a variety of known yeast TF binding sites successfully identified the cognate TF from the background of a complex whole-proteome library. These factors contain DNA-binding domains from diverse families, including Myb, TEA, MADS box, and C2H2 zinc-finger. Using MaPS, we identified Dot6 as a trans-active partner of the long-predicted orphan yeast element Polymerase A & C (PAC). MaPS technology should enable rapid and proteome-scale study of bi-molecular interactions within transcriptional networks

MaPS identifies <i>RAP1</i> as the gene whose product interacts with the Rap1 binding site.

<p>The yeast genomic DNA phage display library was selected for three rounds against a double-stranded oligonucleotide and PCR products of an upstream region containing Rap1 binding sites. The selected population of phage were profiled through microarray hybridization. Displayed is the distribution of the mean percentile rank for five independent such selections performed. The ORF corresponding to Rap1 had the highest mean percentile rank out of a total of 6242 ORFs queried on the array.</p

The Dot6 protein is a sequence-specific PAC element binding factor.

<p>Gel shift assay was performed with Dot6 DNA-binding domain and DNA containing PAC elements. Purified recombinant GST-Dot6 was incubated with 50 fM biotinylated probe containing 4 copies of the PAC element (bPAC4). Unbiotinylated competitor (PAC4) had identical sequence to the probe, or mutations in each copy of the PAC element (XPAC4).</p

Dot6 binds to PAC-containing promoters in vivo.

<p>Chromatin immunoprecipitation was performed on extracts of strain Y3648 (TAP-Dot6) and B4741 (untagged) as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000449#s4" target="_blank">Materials and Methods</a>. Quantification of these data provided the percent of input DNA for the promoters of each of the indicated genes recovered in the immunoprecipitate. Shown are the fold enrichment of those values relative to the percent input DNA for the <i>ACT1</i> promoter recovered in the same immunoprecipitate. PCR quantification was performed in triplicate with less than 20% variation among replicates of individual samples.</p

The Rap1 DNA-binding domain is enriched in a sequence-specific and salt-dependent manner.

<p>A phage display library was affinity selected against indicated quantities of double-stranded DNA containing Rap1 binding sites under indicated salt conditions. Results from PCR of input phage library (input) or after second round of selection are shown. The intensity of a band is proportional to its abundance in the library. Lanes designated RAP1 indicate results of specific PCR against a single phage with the <i>RAP1</i> DNA-binding domain known to be in the library and the red arrows mark the expected size of this clone. Gel-isolation and sequencing of the selected bands at this location confirmed that they correspond to this clone. The blue arrows point out the PCR products corresponding to the <i>MCM1</i> DNA-binding domain (lower band) and <i>MCM1</i> ORF (upper band). The remaining bands show variable enrichment as a function of salt concentration and likely represent non-specific enrichment during the selection.</p

An overview of Microarray profiling of phage-display selection technology.

<p>(A) 1–3 kb fragments of yeast genomic DNA are cloned into T7 bacteriophage to create a translational fusion between the capsid protein and the peptide sequence encoded by the insert. (B) The library of phage are exposed to immobilized target DNA molecules and non-binding phage are washed away. Bound phage are eluted, amplified in liquid culture, and the process is repeated over multiple rounds. The sequence content of the enriched phage population is determined by PCR amplification of the inserts, labeling, and hybridization to a yeast ORF microarray.</p

Bacterial fitness in chronic wounds appears to be mediated by the capacity for high-density growth, not virulence or biofilm functions.

While much is known about acute infection pathogenesis, the understanding of chronic infections has lagged. Here we sought to identify the genes and functions that mediate fitness of the pathogen Pseudomonas aeruginosa in chronic wound infections, and to better understand the selective environment in wounds. We found that clinical isolates from chronic human wounds were frequently defective in virulence functions and biofilm formation, and that many virulence and biofilm formation genes were not required for bacterial fitness in experimental mouse wounds. In contrast, genes involved in anaerobic growth, some metabolic and energy pathways, and membrane integrity were critical. Consistent with these findings, the fitness characteristics of some wound impaired-mutants could be represented by anaerobic, oxidative, and membrane-stress conditions ex vivo, and more comprehensively by high-density bacterial growth conditions, in the absence of a host. These data shed light on the bacterial functions needed in chronic wound infections, the nature of stresses applied to bacteria at chronic infection sites, and suggest therapeutic targets that might compromise wound infection pathogenesis