The goal of enhanced yeast one-hybrid assays is the identify the set of transcription factors that bind to a DNA sequence of interest. The advantage of this technique is that enhanced yeast one-hybrid assays can evaluate more than 1000 transcription factors in a single experiment. Conversely, chromatin immunoprecipitation evaluates only one transcription factor at a time.
This method constitutes a key tool for functional genomics to identify the repertoire of transcription factors that bind to gene promoters and enhancers and to identify altered transcription factor binding to non-coding variants. Demonstrating the procedure will be Dr.Xing Liu a postdoc from my laboratory. Thaw the yeast glycerol stock plates with the TF prey array on ice.
Resuspend the yeast using a 12 channel pipette and proceed to the next step within 1 to 3 minutes. To spot the yeast into Sc minus tryptophan rectangular plates, use a high density array robot to select multiwell 96 plates as the source, 96 agar plates as the target, and 96 long pin pads. Pin pads are not reusable and should be discarded.
Select the Spot Many program to make two copies per 96 well plate. Do not use the recycle or revisit options to avoid back-contamination of the frozen stocks. Also, select the option to swirl up and down in the source to mix the yeast.
Follow instructions for where and when to place the plates and allow the robot to spot the yeast into Sc minus tryptophan rectangular plates. Bag the spotted array and incubate it agar side up at 30 degrees Celsius for 2 to 3 days. To generate 384 colony arrays in Sc minus tryptophan rectangular plates, select 96 agar plates as the source, 384 agar plate as the target, and 96 short pin pads.
Then select the 1:4 array single source program. In this way, four 96 colony plates will be consolidated into one 384 colony plate. Do not use the recycle or revisit options to avoid contamination between different plates.
Bag the plates and incubate the spotted 384 colony array agar side up at 30 degrees Celsius for 2 days. To generate 1536 colony arrays in Sc minus tryptophan rectangular plates, select 384 agar plates as the source, the 1536 agar plates as the target, and 384 short pin pads. Then select the 1:4 assay single source program.
The goal is to copy each colony into four colonies to obtain quadruplicates. Use the recycle and revisit options as this involves copying each colony 4 times. Follow the robot's instructions and allow the robot to spot the yeast into the plates before bagging and incubating the spotted 1536 colony array agar side up at 30 degrees Celsius for 3 days.
To amplify the 1536 colony array in Sc minus tryptophan rectangular plates, select 1536 agar plates as the source, 1536 agar plates as the target, and 1536 short pin pads. Select the Replicate Many program to replicate 3 to 4 copies. Use the recycle and revisit option but throw out the pad when switching to a different plate of the array to avoid cross-contamination.
Allow the robot to amplify the 1536 colony array in Sc minus tryptophan rectangular plates. Finally, bag the plates and incubate the spotted 1536 colony array agar side up at 30 degrees Celsius for 3 days to use for mating steps. To prepare the DNA bait strain lawns for mating, spot the DNA yeast bait strains on an Sc minus uracil and histidine plate and grow for 3 days at 30 degrees Celsius.
Streak the yeast into a 15 centimeter Sc minus uracil and histidine plate using a sterile toothpick so that each plate fits 12 to 16 different strains. Incubate the plate one day at 30 degrees Celsius. The next day, streak the yeast into a 15 centimeter Sc minus uracil and histidine plate using a sterile toothpick so that each plate fits 4 different strains.
Incubate the plate for one day at 30 degrees Celsius. Following incubation, scrape the yeast using a sterile toothpick, making sure not to scrape any agar. Add the yeast into a 1.5 milliliter tube with 500 microliters of sterile water.
Now add 10 to 15 sterile glass beads and the yeast suspension onto a YAPD rectangular plate. Shake thoroughly in all directions for one minute to ensure the yeast is spread throughout the plate. Invert the plate immediately, and tap so that the beads go to the lid.
Remove and recycle the beads. Bag the plates and incubate them agar side down for 1 to 2 days at 30 degrees Celsius. Then, proceed to the mating step.
To mate the yeast DNA bait and TF array strains, transfer the TF array to a YAPD rectangular plate with the robot. Select the 1536 agar plate as the source and the target, and the 1536 short pin pad. Select the Replicate Many program.
Select 4 source plates and the recycle and revisit options. Each TF array plate can be used to transfer 3 to 4 YAPD plates. The TF array plates used to mating must be 2 to 3 days old, but not more as mating may be inefficient.
To transfer the lawn of a DNA bait strain to the YAPD plates already containing the TF array, select the 1536 agar plate as the source and the target and the 1536 short pin pad. Select the Replicate Many program. Use a random offset in the source with a radius of approximately 0.6 millimeters to avoid taking yeast from the same spot and select Mix on Target to facilitate contact between yeast strains.
Use the lawn containing the DNA bait strains as the source, and the YAPD plates containing the spotted TF array as the target before bagging the plates and incubating them agar side up at 30 degrees Celsius for one day. For selection of diploid yeast, select the 1536 agar plate as the source and the target, and select the 1536 short pin pad. Select the Replicate program.
Select Mix on Source and on Target. Allow the robot to transfer the mated yeast from the YAPD plates to the Sc minus uracil and tryptophan plates before bagging the plates and incubating them agar side up at 30 degrees Celsius for 2 to 3 days. Now, select the 1536 agar plates as the source and the target and the 1536 short pin pad.
Select the Replicate program. Transfer the diploid yeast from the Sc minus uracil and tryptophan plates to the readout rectangular 3-AT and X-gal containing plates using the robot. Bag the plates and incubate them agar side up at 30 degrees Celsius for up to 7 days.
For DNA bait strains with high background reporter activity, take pictures on days 2, 3, and 4. Otherwise, take pictures at days 4 and 7. Keep the TF array plates at room temperature and copy again after 7 days for a new round of screening.
To illustrate the type of results that can be obtained using enhanced yeast one-hybrid assays, the promoter regions of the CCL15 and IL17F genes were screened against an array of 1086 human TFs. The CCL15 promoter is an example of a non-autoactive DNA bait, where interactions, even weak ones, can be easily detected. The IL17F promoter is an example of an autoactive DNA bait with uneven background reporter activity where some interactions can be detected, while for several TFs, it is uncertain whether the reporter activity is higher than background.
There are several problems that can occur when performing enhanced yeast one-hybrid assays. In this case, colonies are too small and failed to transfer. Typically, approximately 95%of TF prey colonies display normal growth.
In this example, there is no yeast growth in a portion of the plate. This issue is generally related to a failure in the mating step if the 1536 pin pad fails to make contact with the yeast in the DNA bait strain lawn, in the TF array, or in the mating plate. In these two examples, no interactions are detected.
This issue is often related to either unintended inactivating mutations in the reporter genes, in particular LacZ, or too high autoactivity that mask interactions. In this case, the plate presents random blue spots which is often related to bacterial contamination. The most important thing to consider is proper plate preparation.
Making sure that all components are added and that agar height is uniform as all subsequent steps depend on this. Although most reagents are not hazardous, it is important to wear gloves at all times to avoid contamination of the samples and plates. As with any DNA binding assay, it is important to validate the interactions identified using functional assays such as reporter assays, transcription factor knockdown or knockout, followed by measuring expression of the target gene.
This method has allowed the identification of transcription factors that regulate genes involved in particular biological processes as well as transcription factors with alternate binding to non-coding disease-associated variants.
