Linking DNA-binding proteins to their recognition sequences by using protein microarrays

Abstract

Analyses of whole-genome sequences and experimental data sets have revealed a large number of DNA sequence motifs that are conserved in many species and may be functional. However, methods of sufficient scale to explore the roles of these elements are lacking. We describe the use of protein arrays to identify proteins that bind to DNA sequences of interest. A microarray of 282 known and potential yeast transcription factors was produced and probed with oligonucleotides of evolutionarily conserved sequences that are potentially functional. Transcription factors that bound to specific DNA sequences were identified. One previously uncharacterized DNA-binding protein, Yjl103, was characterized in detail. We defined the binding site for this protein and identified a number of its target genes, many of which are involved in stress response and oxidative phosphorylation. Protein microarrays offer a high-throughput method for determining DNA-protein interactions.

Fig. 1.

Probing the transcription factor microarray. (A) Probes were made by extending a universal primer labeled at its 5′ end with a fluorophore on an oligonucleotide template containing conserved sequence motifs. Because the length of the sequence motifs varies and we kept the length of the oligonucleotide probes constant, three or four copies of a motif are present in each probe. (B) Rap1 protein binds to a probe containing Rap1-binding sites. Each protein depicted on the right-hand and left-hand sides was spotted six times on the nitrocellulose surface and probed with an oligonucleotide containing three Rap1-binding sites (ACACCCAT/GCA) (labeled with Cy3, shown in green) and a probe containing three Rap1-binding sites with two nucleotide changes (ACACttAT/GCA) (labeled with Cy5, shown in red). Probing with reciprocally labeled probes is depicted below C. (C) Yeast transcription factor microarrays probed with fluorescent DNA probes. The GST-fused transcription factors purified from yeast (see Materials and Methods) were spotted (in quadruplicate) on each slide and probed with Cy5-labeled anti-GST (Left) or a pair of probes (Right). Examples of specific DNA binding are enlarged at the right. Yjl103c binds specifically to P3A but not P3B.

Fig. 2.

EMSA of seven proteins that showed specific DNA binding on protein microarrays. Only one probe of each probe pair (Left) binds specifically to the protein. There are two or three base pair differences in each motif in each pair of probes. P30A binds to both Hms1 and Rds2. P38A is used as a control to show that binding is specific to P30A. See Materials and Methods for details.

Fig. 3.

EMSA of Yjl103c. (A and B) A constant concentration of Yjl103c (5 μM) incubated with increasing amounts of labeled probes P3A (wild-type binding site) (A) and P3B (mutant binding site) (B), respectively. Probe concentrations increase from 60 to 600 pM. (C) Constant concentration of probes P3A and P3B (250 pM) with increasing amounts of Yjl103c. Protein increases from 0.7 μM to 8.5 μM. (D) Competition with unlabeled DNA: Increasing amounts of cold competitor DNA are added to the reaction with constant concentration of Yjl103c (1.6 μM) and labeled probe P3A (250 pM). Cold competitor is added at effective excess of labeled probe of 10-fold, 50-fold, 100-fold, and 800-fold.

Fig. 4.

Yjl103c binds to CGGN₈CGG and CGGN₉CGG. Oligonucleotides containing variations of the putative binding site of Yjl103c were used to probe the transcription factor microarrays. Binding intensity, relative to the wild-type probe, is plotted on the right (average of three to five independent probings with each sequence).

Fig. 5.

ChIP assay for Yjl103 binding. Chromatin was crosslinked to proteins, Yjl103 tagged with a 13-myc epitope was precipitated with anti-myc antibody, and the precipitated DNA was released from protein and detected by PCR (as described in Materials and Methods) using primers specific for sequences upstream of the indicated 19 genes (query promoter) and primers specific for the GAL4 promoter (control promoter) that amplify a 150-bp fragment.