Enhanced Yeast One-hybrid Screens To Identify Transcription Factor Binding To Human DNA Sequences

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

Protocol

1. Preparations

1. Sc −U −H plates (150 mm Petri dishes) 
   NOTE: These plates will be used for growing the DNA-bait yeast strains. 
   1. 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.
   2. 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). 
   3. Autoclave for 40 min at ....

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

List of materials used in this article

Name	Company	Catalog Number	Comments
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 750 ct	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 mm x15 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