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Summary

Here, we present an enhanced yeast one-hybrid screening protocol to identify the transcription factors (TFs) that can bind to a human DNA region of interest. This method uses a high-throughput screening pipeline that can interrogate the binding of >1,000 TFs in a single experiment.

Abstract

Identifying the sets of transcription factors (TFs) that regulate each human gene is a daunting task that requires integrating numerous experimental and computational approaches. One such method is the yeast one-hybrid (Y1H) assay, in which interactions between TFs and DNA regions are tested in the milieu of the yeast nucleus using reporter genes. Y1H assays involve two components: a ‘DNA-bait’ (e.g., promoters, enhancers, silencers, etc.) and a ‘TF-prey,’ which can be screened for reporter gene activation. Most published protocols for performing Y1H screens are based on transforming TF-prey libraries or arrays into DNA-bait yeast strains. Here, we describe a pipeline, called enhanced Y1H (eY1H) assays, where TF-DNA interactions are interrogated by mating DNA-bait strains with an arrayed collection of TF-prey strains using a high density array (HDA) robotic platform that allows screening in a 1,536 colony format. This allows for a dramatic increase in throughput (60 DNA-bait sequences against >1,000 TFs takes two weeks per researcher) and reproducibility. We illustrate the different types of expected results by testing human promoter sequences against an array of 1,086 human TFs, as well as examples of issues that can arise during screens and how to troubleshoot them.

Introduction

A central problem in the biomedical field is determining the mechanisms by which each human gene is regulated. Transcription is the first step in controlling gene expression levels, and it is regulated by sets of transcription factors (TFs) that are unique to each gene. Given that humans encode for >1,500 TFs^1,2, identifying the complete set of TFs that control the expression of each gene remains an open challenge. 

Two types of methods can be used to map TF-DNA interactions: TF-centered and DNA-centered methods³ (Figure 1A). In TF-centered methods, a TF of interest is probed for binding to genomic DNA regions or to determine its DNA binding specificity. These methods include chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing, protein binding microarrays, and SELEX^4,5,6. In DNA-centered methods, a DNA sequence of interest is probed to determine the set of TFs that bind to the DNA sequence. The most widely applied of such methods is yeast one-hybrid (Y1H) assays, in which interactions between TFs and DNA regions are tested in the milieu of the yeast nucleus using reporter genes^7,8,9. 

Y1H assays involve two components: a ‘DNA-bait’ (e.g., promoters, enhancers, silencers, etc.) and a ‘TF-prey,’ which can be screened for reporter gene activation^9,10 (Figure 1B). The DNA-bait is cloned upstream of two reporter genes (LacZ and HIS3) and both DNA-bait::reporter constructs are integrated into the yeast genome to generate chromatinized ‘DNA-bait strains.’ The TF-prey, encoded in a plasmid that expresses a TF fused to the activation domain (AD) of the yeast Gal4 TF, is introduced into the DNA-bait strain to fish for TF-DNA interactions. If the TF-prey binds to the DNA-bait sequence, then the AD present in the TF-prey will lead to the activation of both reporter genes. As a result, cells with a positive interaction can be selected for growth on plates lacking histidine, as well as overcoming a competitive inhibitor, 3-Amino-1,2,4-triazole (3-AT), and visualized as blue colonies in the presence of X-gal. Because the potent yeast Gal4 AD is used, Y1H assays can detect interactions involving transcriptional activators as well as repressors. In addition, given that TF-preys are expressed from a strong yeast promoter (ADH1), interactions can be detected even for TFs that have low endogenous expression levels, which are challenging to detect by ChIP^11,12.

Most published protocols for performing Y1H assays are based on introducing TF-preys into the yeast DNA-bait strains by transforming pooled TF-prey libraries followed by selection, colony picking, and sequencing to identify the interacting TF, or by transforming individual clones^8,9. These are time-consuming protocols, limiting the number of DNA sequences that can be tested per researcher. A recent improvement of Y1H assays, called enhanced Y1H (eY1H), has dramatically increased the screening throughput by using a high density array (HDA) robotic platform to mate yeast DNA-bait strains with a collection of yeast strains each expressing a different TF-prey^10,13 (Figure 1C). These screens employ a 1,536 colony format allowing to test most human TFs in quadruplicate using only three plates. Further, given that TF-DNA interactions are tested in a pairwise manner, this approach allows for comparing interactions between DNA-baits (such as two noncoding single nucleotide variants) and between different TFs or TF variants^11,12,14.

Using eY1H assays, we have delineated the largest human and Caenorhabditis elegans DNA-centered TF-DNA interactions networks to-date. In particular, we have identified 2,230 interactions between 246 human developmental enhancers and 283 TFs¹². Further, we have employed eY1H assays to uncover altered TF binding to 109 single nucleotide noncoding variants associated with genetic diseases such as developmental malformation, cancer, and neurological disorders. More recently, we used eY1H to delineate a network comprising 21,714 interactions between 2,576 C. elegans gene promoters and 366 TFs¹¹. This network was instrumental to uncover the functional role of dozens of C. elegans TFs.

The protocols to generate DNA-bait stains and evaluate the levels of background reporter activity have been reported elsewhere^15,16,17. Here, we describe an eY1H pipeline that can be used to screen any human genomic DNA region against an array of 1,086 human TFs. Once a yeast DNA-bait strain is generated and a TF-prey array is spotted onto the corresponding plates, the entire protocol can be performed in two weeks (Table 1). More importantly, the protocol can be parallelized so that a single researcher can screen 60 DNA-bait sequences simultaneously. To demonstrate the protocol, we screened the promoters of two cytokine genes CCL15 and IL17F. In addition, we show results from failed screens to illustrate the types of problems that may arise when performing eY1H assays and how to troubleshoot them.

Protocol

1. Preparations Sc −U −H plates (150 mm Petri dishes)  NOTE: These plates will be used for growing the DNA-bait yeast strains.  Dissolve the drop-out mix, yeast nitrogen base (YNB), adenine hemisulfate, and ammonium sulfate in 920 mL of water, and pH to 5.9 with 5 M NaOH (approximately 1 mL per liter of media; see Table 2 for composition). Pour into a 2 L flask and add a stir bar. In a second 2 L flask, add the agar to 950 mL of water (do not add a stir bar as it will cause the agar to boil over in the autoclave).  Autoclave for 40 min at 15 psi on a liquid cycle.  Immediately pour the contents of the first flask, including the stir bar, into the agar containing flask. Add the glucose, mix well on a stir plate, and cool to 55 °C in a water bath.  Add the leucine and the tryptophan to the media. Mix well on a stir plate and pour into 150 mm sterile Petri dishes (~80 mL per dish). Dry for 3–5 days at room temperature, wrap in plastic bags, and store at room temperature for up to 6 months.  YAPD rectangular plates NOTE: These plates will be used for growing the lawn for the DNA-bait strain and for mating with the TF array collection. Dissolve powders (see Table 3 for composition), except for agar, in 950 mL of water in a 2 L flask and add a stir bar. In a second 2 L flask, add the agar to 950 mL of water (do not add a stir bar as it will cause the agar to boil over in the autoclave). Autoclave for 40 min at 15 psi on a liquid cycle. Immediately pour the contents of the first flask, including the stir bar, into the agar containing flask. Add the glucose, mix well on a stir plate and cool to 55 °C. Pour media into the rectangular plates (see Table of Materials; ~70 mL per plate) using a peristaltic pump (5 mL/sec) and a 6 mm tubing. Dry for 1 day at room temperature, wrap in plastic bags, and store in the cold room for up to 6 months.  NOTE: Although the suggested media volume is 70 mL per plate, 50–80 mL per plate can be used. The three critical issues to consider when pouring plates are 1) that they are leveled so that the agar media has the same thickness throughout the plate (use a leveled table or surface for plate pouring and do not pour in stacks of more than seven plates), 2) to ensure the absence of bubbles in the agar media (bubbles should be popped using a sterile needle), and 3) drying the plates for only one day and wrapping the plates in plastic bags to avoid failures in pinning yeast. Sc −Trp and Sc −U −Trp rectangular plates  NOTE: These plates will be used for growing the TF array collection (Sc −Trp) and to select diploid yeast after mating (Sc −U −Trp). Dissolve the drop-out mix, YNB, adenine hemisulfate, and ammonium sulfate in 920 mL of water, and pH to 5.9 with NaOH 5M (approximately 1 mL per liter of media) (see Table 4 for composition). Pour into a 2 L flask and add a stir bar.  In a second 2 L flask, add the agar to 950 mL of water (do not add a stir bar as it will cause the agar to boil over in the autoclave).  Autoclave for 40 min at 15 psi on a liquid cycle.  Immediately pour the contents of the first flask, including the stir bar, into the agar containing flask. Add the glucose, mix well on a stir plate, and cool to 55 °C.  Add the leucine, histidine, and uracil (omit the uracil for the Sc −U −Trp plates).  Mix well on a stir plate and pour into rectangular plates (~70 mL per plate) using a peristaltic pump (5 mL/sec) and 6 mm tubing. Dry for 1 day at room temperature, wrap the plates in plastic bags, and store in the cold room for up to 3 months.  Sc −U −H −Trp + 3AT + X-gal rectangular plates ​NOTE: These plates will be used as readout plates for eY1H assays. Dissolve the drop-out mix, YNB, adenine hemisulfate, and ammonium sulfate in 850 mL of water (see Table 5 for composition). Do not pH. Pour into a 2 L flask and add a stir bar.  In a second 2 L flask, add the agar to 850 mL of water (do not add a stir bar as it will cause the agar to boil over in the autoclave).  Autoclave for 40 min at 15 psi on a liquid cycle. Prepare 10x BU salts (1L) by combining 900 mL of water, 70 g of Na2HPO4∙7H2O, and 34.5 g of NaH2PO4∙H2O. Mix using a stir bar to dissolve powders and adjust the pH to 7.0 using 5 M NaOH. Add water to bring to 1 L and autoclave. Prepare the X-gal solution by adding 3.5 g of X-gal powder to a 50 mL plastic tube containing 42.5 mL of dimethyl formamide. Add X-gal powder to dimethyl formamide to dissolve more easily (this takes 30 min). Keep stock solution in the dark (either use opaque 50 ml tube or cover in foil). Store at -20 °C. Immediately pour the contents of the first flask, including the stir bar, into the agar containing flask. Add the glucose and the 10x BU salts (see Table 5 for composition), mix well on a stir plate, and cool to 55 °C.  Add the leucine, 3AT, and X-gal (see Table 5 for composition).  Mix well on a stir plate and pour into the rectangular plates (~70 mL per plate) using a peristaltic pump (5 mL/sec) and a 6 mm tubing. Dry for 1 day at room temperature, wrap in plastic bags, and store in the cold room covered in aluminum foil (3AT and X-gal are light sensitive) for up to 1 month.  2. Spotting a TF array Thawing the TF-prey array Thaw the yeast glycerol stock plates with the TF-prey array on ice.  ​NOTE: TF-prey arrays can be generated as previously published10,12,13,18. Resuspend the yeast using a 12-channel pipette within 1–3 min before the next step.  Spotting the yeast into Sc −Trp rectangular plates In the HDA robot (see Table of Materials), select multi-well 96 plates as source, 96 agar plates as target, and 96 long pin pads.  ​NOTE: Pin pads are not reusable and should be discarded. Select the Replicate 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.  Select the option to swirl up and down in the source to mix the yeast. Bag the spotted array and incubate agar-side up at 30 °C for 2–3 days.  Generating 384 colony arrays in Sc −Trp rectangular plates In the robot, select 96 agar plates as source, 384 agar plate as target, and 96 short pin pads.  Select the 1:4 Array program. In this way, four 96 colony plates (each containing a different TF) 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 °C for 2 days. Generating 1,536 colony arrays in Sc −Trp rectangular plates NOTE: This will result in arrays containing four colonies for each TF-prey. In the robot, select 384 agar plates as source, the 1,536 agar plates as target, and 384 short pin pads.  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 it involves copying four times each colony.  Bag the plates and incubate the spotted 1536-colony array agar-side up at 30 °C for 3 days. Amplifying the 1,536 colony array in Sc −Trp rectangular plates In the robot, select 1,536 agar plates as source, 1,536 agar plates as target, and 1,536 short pin pads.  Select the Replicate Many program to replicate 3–4 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.  Bag the plates and incubate the spotted 1536-colony array agar-side up at 30 °C for 3 days to use for mating steps (see below). After that, keep the plates at room temperature and copy again after 7 days for a new round of screening. 3. eY1H screen Preparing DNA-bait strain lawns for mating Spot the yeast DNA-bait strains on a Sc −U −H plate and grow for 3 days at 30 °C.  Streak the yeast into a 15 cm Sc −U −H plate using a sterile toothpick, so that each plate fits 12–16 different strains. Incubate one day at 30 °C. Streak the yeast into a 15 cm Sc −U −H plate using a sterile toothpick, so that each plate fits 4 different strains. Incubate for one day at 30 °C. Scrape the yeast using a sterile toothpick, making sure not to scrape any agar and add into a 1.5 tube with 500 µL of sterile water.  Add 10–15 sterile glass beads onto a YAPD rectangular plate. Add the yeast suspension onto the plate and shake thoroughly in all directions for 1 min to ensure the yeast is spread through all 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 agar-side down for 1–2 days at 30 °C. Then proceed to the mating step. Mating of yeast DNA-bait and TF array strains Transfer the TF array to a YAPD rectangular plate with the robot. Select the 1,536 agar plate as source and target, and the 1,536 short pin pad. Select the Replicate Many program. Each TF array plate can be used to transfer to 3–4 YAPD plates (depending on the number of plates in the array). The TF array plates used for mating must be 2–3 days old but not more as mating may be inefficient. Transfer the lawn of a DNA-bait strain to the YAPD plates already containing the TF array with the robot. Select the 1,536 agar plate as source and target, and the 1,536 short pin pad. Select the Replicate Many program. Use a random offset in the source with a radius of ~0.6 mm to avoid taking yeast from the same spot, and mix on target to facilitate contact between yeast strains. Use the lawn containing the DNA-bait strains (section 3.1) as source, and the YAPD plates containing the TF array spotted in step 3.2.1 as target. Bag the plates and incubate agar-side up at 30 °C for 1 day. Selection of diploid yeast Transfer the mated yeast from the YAPD plates to Sc −U −Trp plates with the robot. Select the 1,536 agar plate as source and target, and the 1,536 short pin pad. Select the Replicate program. Mix on source and on target. Bag the plates and incubate agar-side up at 30 °C for 2–3 days (longer incubation leads to high background reporter activity). Transfer to readout plates Transfer the diploid yeast from the Sc −U −Trp plates to the readout rectangular plates Sc −U −H −Trp + 5mM 3AT + 0.4 mM X-gal using the robot. Select the 1,536 agar plates as source and target, and the 1,536 short pin pad. Select the Replicate program. Bag the plates and incubate agar-side up at 30 °C for up to 7 days. Imaging of readout plates 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. Positive interactions are identified by growth and blue color of the yeast colonies and can be manually determined, or it can be determined using image analysis software.

Representative Results

Three main factors should be considered when analyzing results from eY1H assays: the background reporter activity of the DNA-bait strain, the strength of the reporter activity corresponding to TF-DNA interactions, and the number of positive colonies. The background reporter activity (i.e., autoactivity) of the DNA-bait strain refers to the overall growth and color of the yeast colonies in the readout plate, even in the absence of a TF-prey. Ideally, non-autoactive strains show a background white or light brown color, wit…

Discussion

The robotic eY1H mating screening approach described here greatly increases the throughput to identify the set of TFs that bind to a DNA region of interest, compared to previous library screening or arrayed screening approaches based on transformation. Further, the TF-DNA interactions detected by eY1H assays are highly reproducible as 90% of interactions detected are positive for all four colonies tested per TF, and 90% of interactions retest in an independent screen of the same yeast DNA-bait strain^10<…

Materials

3-Amino-1,2,4-triazole (3AT) ~95 % TLC	Sigma	A8056-100G	Competitive inhibitor for products of HIS3 gene
Adenine sulfate (hemisulfate), dihydrate	US Biologicals	A0865	Required for proper yeast growth
Agar High Gel Strength – Bacteriological grade	American International Chemical	AGHGUP	Nutritive media for yeast growth
Ammonium Sulfate	US Biologicals	A1450	Nitrogen source in synthetic yeast media
D+ Glucose Anhydrous	US Biologicals	G3050	Required for yeast growth
Drop-Out Mix minus His, Leu, Tryp and Uracil, adenine rich w/o yeast nitrogen base	US Biologicals	D9540-02	Synthetic complete media required for yeast growth
edge Multiparameter pH Meter	Hanna Instruments	HI2020-01	To measure pH of selective media
Flat Toothpicks 750ct	Diamond		To streak yeasts on petridishes
Glass Beads	Walter Stern	100C	To spreak yeast when making lawns
Glycerol ≥99%	Millipore Sigma	G9012-1L	Required to make frozen yeast stocks
L-Histidine	US Biologicals	H5100	For yeast growth selection in selective media
L-Leucine	US Biologicals	L2020-05	For yeast growth selection in selective media
L-Tryptophan	Sigma	T-0254	For yeast growth selection in selective media
N,N-Dimethylformamide	Sigma	319937-1L	To make X-gal solution
Omnipense Elite	Wheaton	W375030-A	For dispensing accurate volumes of media into Singer plates
Peptone, Bacteriological	American International Chemical	PEBAUP	Protein source required for yeast growth
Petri Dish, 150×15 mm	VWR	10753-950	For growing yeast baits for screening
PlusPlates	Singer Instruments	PLU-003	To make rectangular agar plates to use with Singer Robot
Precision Low Temperature BOD Refrigerated Incubator	ThermoFisher Scientific	PR205745R	To incubate yeast plates at constant temperature
RePads 1,536 short	Singer Instruments	REP-005	To transfer the TF-prey array, mate yeast, and transfer yeast to diploid selection and readout plates
RePads 384 short	Singer Instruments	REP-004	To transfer TF-prey array from 384 to 1,536 colony format
RePads 96 long	Singer Instruments	REP-001	To transfer TF-prey array from glycerol stock to agar plate
RePads 96 short	Singer Instruments	REP-002	To transfer TF-prey array from 96 to 384 colony format
Singer HDA RoToR robot	Singer Instruments		For transfering yeast in high-throughput manner
Sodium Hydroxide (Pellets/Certified ACS)	Fisher	S318-1	For adjusting pH of selective media
Sodium Phosphate dibasic heptahydrate	Santa Cruz Biotechnology	sc-203402C	Required for LacZ reporter activity on X-gal
Sodium Phosphate monobasic monohydrate	Santa Cruz Biotechnology	sc-202342B	Required for LacZ reporter activity on X-gal
Uracil	Sigma	U0750-100G	For yeast growth selection in selective media
X-gal (5-Bromo-4-chloro-3-indoxyl-beta-Dgalactopyranoside)	Gold Biotechnology	X4281C100	β-galactosidase turns colorless X-gal blue to detect protein-DNA interaction
Yeast Extract	US Biologicals	Y2010	Nutritious medium for growth and propagation of yeast
Yeast Nitrogen Base (powder) w/o AA, carbohydrate and w/o AS	US Biologicals	Y2030	Required for vigorous yeast growth