A gateway-compatible yeast one-hybrid system

Abstract

Since the advent of microarrays, vast amounts of gene expression data have been generated. However, these microarray data fail to reveal the transcription regulatory mechanisms that underlie differential gene expression, because the identity of the responsible transcription factors (TFs) often cannot be directly inferred from such data sets. Regulatory TFs activate or repress transcription of their target genes by binding to cis-regulatory elements that are frequently located in a gene's promoter. To understand the mechanisms underlying differential gene expression, it is necessary to identify physical interactions between regulatory TFs and their target genes. We developed a Gateway-compatible yeast one-hybrid (Y1H) system that enables the rapid, large-scale identification of protein-DNA interactions using both small (i.e., DNA elements of interest) and large (i.e., gene promoters) DNA fragments. We used four well-characterized Caenorhabditis elegans promoters as DNA baits to test the functionality of this Y1H system. We could detect approximately 40% of previously reported TF-promoter interactions. By performing screens using two complementary libraries, we found novel potentially interacting TFs for each promoter. We recapitulated several of the Y1H-based protein-DNA interactions using luciferase reporter assays in mammalian cells. Taken together, the Gateway-compatible Y1H system will allow the high-throughput identification of protein-DNA interactions and may be a valuable tool to decipher transcription regulatory networks.

Figure 1

Creating DNAbait::reporter constructs by multisite LR cloning. (A) Gateway-cloned ORFs encoding the Y1H reporters His3 and β-galactosidase (β-Gal) were BP cloned into pDONR201 to generate HIS3 and lacZ Entry clones. DNA baits were BP cloned into pDONR-P4-P1R to generate DNA bait Entry clones. (B) The DNA baits were fused to HIS3 and lacZ by a multisite Gateway LR reaction using the DNA bait Entry clones, the Y1H reporter entry clones, and the Y1H Destination vectors to generate DNAbait::HIS3 and DNAbait::lacZ Destination clones.

Figure 2

A Gateway-compatible Y1H system. (A) “Non-Gateway” Y1H control assay. As a DNA bait, six copies of the murine consensus p53-binding site (p53BS) fused to the HIS3 reporter gene was used with Gal4 AD alone (-) or with AD-p53DB as prey. (B) As a DNA bait, six copies of the murine consensus p53-binding site (p53BS) fused by a multisite Gateway reaction to the HIS3 reporter gene was used with Gal4 AD alone (-) or with AD-p53DB. Here, the p53BS bait is flanked by AttB4 and AttB1 Gateway recombination sites. Cells were plated on Sc-His,-Ura media with 0, 20, or 40 mM 3AT as indicated.

Figure 4

TF-promoter interactions detected by the Gateway-compatible Y1H system. DNAbait::reporter strains were transformed with Gateway Destination plasmids containing either full-length TF-encoding ORFs, or the part of these ORFs that is predicted to encode the DB, fused to Gal4 AD. An empty Gateway vector containing only Gal4 AD was used as a negative control (-). The resulting yeast transformants were grown on Sc-His,-Ura,-Trp media (upper row), Sc-His,-Ura,-Trp, containing 40 mM 3AT (middle row), or spotted on YEPD plates containing a nitrocellulose filter for β-Gal assays (lower row).

Figure 3

Self-activation by C. elegans promoters. (A) The Y1H Destination plasmids containing the Gateway-cloned promoters P_fog-3 or P_hlh-8 were integrated into the genome of YM4271 yeast. Twenty-four double integrant colonies were picked and spotted either onto (1) Sc-His,-Ura media plus increasing amounts of 3AT to determine self-activation of the HIS3 reporter, and onto (2) YEPD plates containing a nitrocellulose filter for a β-Gal assay. Circles indicate colonies that were selected for downstream Y1H experiments. (B) To determine if individual colonies exhibit phenotypic variations after clonal expansion, three P_fog-3 colonies, varying in their extent of self-activation (low = 1, moderate = 2, and high = 3), were picked and restreaked on Sc-His,-Ura media after which they were replica-plated onto selective 20 mM 3AT-containing media.

Figure 6

Graphic representation of the protein-promoter interactions detected. Proteins in green were detected by targeted Y1H experiments, proteins in red were identified by AD-cDNA library screens, and proteins in blue by AD-TF minilibrary screens. The TF family is indicated between parentheses under the respective ORF name. Homeo = homeodomain, Myb = Myb-like DNA binding, ZF = zinc finger, NHR = nuclear hormone receptor type zinc finger, RPEL = RPEL repeat.

Figure 5

Examples of retest of P_hlh-8 and P_fog-3 positives. (A) P_hlh-8 retest. (B) P_fog-3 retest. Double integrant yeast cells were transformed with an empty vector (containing Gal4 AD) and PCR products containing sequences that encode potential interaction partners as described previously (Walhout and Vidal 2001). Transformants were grown on Sc-His,Ura,-Trp plates with 40 mM 3AT or spotted on YEPD plates containing a nitrocellulose filter for β-Gal assays. Controls: 1 = no DNA, 2 = circular (uncut) empty AD vector, 3 = linear (cut) AD vector.

Figure 7

Validation of Y1H-based protein-DNA interactions. Results of a representative experiment validating Y1H TF-P_fog-3 interactions by transient transfections and luciferase assays are shown. Negative controls include an empty GST-tag-containing vector and GST-php-3. Values are means ± SEM of three replicates. (^*) P < 0.05 vs. negative controls.