This work was supported by the National Institutes of Health [R35-GM128625 to J.I.F.B.].&#160;

1. Preparations
<ol>
	<li>Sc &#8722;U &#8722;H plates (150 mm Petri dishes)&#160; 
	NOTE: These plates will be used for growing the DNA-bait yeast strains.&#160;
	<ol>
		<li>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.</li>
		<li>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).&#160;</li>
		<li>Autoclave for 40 min at 15 psi on a liquid cycle.&#160;</li>
		<li>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 &#176;C in a water bath.&#160;</li>
		<li>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&#8211;5 days at room temperature, wrap in plastic bags, and store at room temperature for up to 6 months.&#160;</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>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.</li>
		<li>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).</li>
		<li>Autoclave for 40 min at 15 psi on a liquid cycle.</li>
		<li>Immediately pour the contents of the first flask, including the stir bar, into the agar containing flask.</li>
		<li>Add the glucose, mix well on a stir plate and cool to 55 &#176;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.&#160; 
		NOTE: Although the suggested media volume is 70 mL per plate, 50&#8211;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.</li>
	</ol>
	</li>
	<li>Sc &#8722;Trp and Sc &#8722;U &#8722;Trp rectangular plates&#160; 
	NOTE: These plates will be used for growing the TF array collection (Sc &#8722;Trp) and to select diploid yeast after mating (Sc &#8722;U &#8722;Trp).
	<ol>
		<li>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.&#160;</li>
		<li>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).&#160;</li>
		<li>Autoclave for 40 min at 15 psi on a liquid cycle.&#160;</li>
		<li>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 &#176;C.&#160;</li>
		<li>Add the leucine, histidine, and uracil (omit the uracil for the Sc &#8722;U &#8722;Trp plates).&#160;</li>
		<li>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.&#160;</li>
	</ol>
	</li>
	<li>Sc &#8722;U &#8722;H &#8722;Trp + 3AT + X-gal rectangular plates 
	&#8203;NOTE: These plates will be used as readout plates for eY1H assays.
	<ol>
		<li>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.&#160;</li>
		<li>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).&#160;</li>
		<li>Autoclave for 40 min at 15 psi on a liquid cycle.</li>
		<li>Prepare 10x BU salts (1L) by combining 900 mL of water, 70 g of Na2HPO4&#8729;7H2O, and 34.5 g of NaH2PO4&#8729;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.</li>
		<li>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 &#176;C.</li>
		<li>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 &#176;C.&#160;</li>
		<li>Add the leucine, 3AT, and X-gal (see Table 5 for composition).&#160;</li>
		<li>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.&#160;</li>
	</ol>
	</li>
</ol>
2. Spotting a TF array
<ol>
	<li>Thawing the TF-prey array
	<ol>
		<li>Thaw the yeast glycerol stock plates with the TF-prey array on ice.&#160; 
		&#8203;NOTE: TF-prey arrays can be generated as previously published10,12,13,18.</li>
		<li>Resuspend the yeast using a 12-channel pipette within 1&#8211;3 min before the next step.&#160;</li>
	</ol>
	</li>
	<li>Spotting the yeast into Sc &#8722;Trp rectangular plates
	<ol>
		<li>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.&#160; 
		&#8203;NOTE: Pin pads are not reusable and should be discarded.</li>
		<li>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.&#160;</li>
		<li>Select the option to swirl up and down in the source to mix the yeast.</li>
		<li>Bag the spotted array and incubate agar-side up at 30 &#176;C for 2&#8211;3 days.&#160;</li>
	</ol>
	</li>
	<li>Generating 384 colony arrays in Sc &#8722;Trp rectangular plates
	<ol>
		<li>In the robot, select 96 agar plates as source, 384 agar plate as target, and 96 short pin pads.&#160;</li>
		<li>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.</li>
		<li>Bag the plates and incubate the spotted 384-colony array agar-side up at 30 &#176;C for 2 days.</li>
	</ol>
	</li>
	<li>Generating 1,536 colony arrays in Sc &#8722;Trp rectangular plates 
	NOTE: This will result in arrays containing four colonies for each TF-prey.
	<ol>
		<li>In the robot, select 384 agar plates as source, the 1,536 agar plates as target, and 384 short pin pads.&#160;</li>
		<li>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.&#160;</li>
		<li>Bag the plates and incubate the spotted 1536-colony array agar-side up at 30 &#176;C for 3 days.</li>
	</ol>
	</li>
	<li>Amplifying the 1,536 colony array in Sc &#8722;Trp rectangular plates
	<ol>
		<li>In the robot, select 1,536 agar plates as source, 1,536 agar plates as target, and 1,536 short pin pads.&#160;</li>
		<li>Select the Replicate Many program to replicate 3&#8211;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.&#160;</li>
		<li>Bag the plates and incubate the spotted 1536-colony array agar-side up at 30 &#176;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.</li>
	</ol>
	</li>
</ol>
3. eY1H screen
<ol>
	<li>Preparing DNA-bait strain lawns for mating
	<ol>
		<li>Spot the yeast DNA-bait strains on a Sc &#8722;U &#8722;H plate and grow for 3 days at 30 &#176;C.&#160;</li>
		<li>Streak the yeast into a 15 cm Sc &#8722;U &#8722;H plate using a sterile toothpick, so that each plate fits 12&#8211;16 different strains. Incubate one day at 30 &#176;C.</li>
		<li>Streak the yeast into a 15 cm Sc &#8722;U &#8722;H plate using a sterile toothpick, so that each plate fits 4 different strains. Incubate for one day at 30 &#176;C.</li>
		<li>Scrape the yeast using a sterile toothpick, making sure not to scrape any agar and add into a 1.5 tube with 500 &#181;L of sterile water.&#160;</li>
		<li>Add 10&#8211;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.</li>
		<li>Invert the plate immediately and tap so that the beads go to the lid. Remove and recycle the beads.&#160;</li>
		<li>Bag the plates and incubate agar-side down for 1&#8211;2 days at 30 &#176;C. Then proceed to the mating step.</li>
	</ol>
	</li>
	<li>Mating of yeast DNA-bait and TF array strains
	<ol>
		<li>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&#8211;4 YAPD plates (depending on the number of plates in the array). The TF array plates used for mating must be 2&#8211;3 days old but not more as mating may be inefficient.</li>
		<li>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.</li>
		<li>Bag the plates and incubate agar-side up at 30 &#176;C for 1 day.</li>
	</ol>
	</li>
	<li>Selection of diploid yeast
	<ol>
		<li>Transfer the mated yeast from the YAPD plates to Sc &#8722;U &#8722;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.</li>
		<li>Bag the plates and incubate agar-side up at 30 &#176;C for 2&#8211;3 days (longer incubation leads to high background reporter activity).</li>
	</ol>
	</li>
	<li>Transfer to readout plates
	<ol>
		<li>Transfer the diploid yeast from the Sc &#8722;U &#8722;Trp plates to the readout rectangular plates Sc &#8722;U &#8722;H &#8722;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.</li>
		<li>Bag the plates and incubate agar-side up at 30 &#176;C for up to 7 days.</li>
	</ol>
	</li>
	<li>Imaging of readout plates
	<ol>
		<li>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.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.&#160;

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 strain10<...

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 &#62;1,500 TFs1,2, identifying the complete set of TFs that control the expression of each gene remains an open challenge.&#160;
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 SELEX4,5,6. 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 genes7,8,9.&#160;
Y1H assays involve two components: a &#8216;DNA-bait&#8217; (e.g., promoters, enhancers, silencers, etc.) and a &#8216;TF-prey,&#8217; which can be screened for reporter gene activation9,10 (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 &#8216;DNA-bait strains.&#8217; 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 ChIP11,12.
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 clones8,9. 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-prey10,13 (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 variants11,12,14.
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 elsewhere15,16,17. 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.

<table><tbody><tr><td>3-Amino-1,2,4-triazole (3AT) ~95 % TLC</td><td>Sigma</td><td>A8056-100G</td><td>Competitive inhibitor for products of HIS3 gene</td></tr><tr><td>Adenine sulfate (hemisulfate), dihydrate</td><td>US Biologicals</td><td>A0865</td><td>Required for proper yeast growth</td></tr><tr><td>Agar High Gel Strength - Bacteriological grade</td><td>American International Chemical</td><td>AGHGUP</td><td>Nutritive media for yeast growth</td></tr><tr><td>Ammonium Sulfate</td><td>US Biologicals</td><td>A1450</td><td>Nitrogen source in synthetic yeast media</td></tr><tr><td>D+ Glucose Anhydrous</td><td>US Biologicals</td><td>G3050</td><td>Required for yeast growth</td></tr><tr><td>Drop-Out Mix minus His, Leu, Tryp and Uracil, adenine rich w/o yeast nitrogen base</td><td>US Biologicals</td><td>D9540-02</td><td>Synthetic complete media required for yeast growth</td></tr><tr><td>edge Multiparameter pH Meter</td><td>Hanna Instruments</td><td>HI2020-01</td><td>To measure pH of selective media</td></tr><tr><td>Flat Toothpicks 750 ct</td><td>Diamond</td><td></td><td>To streak yeasts on petridishes</td></tr><tr><td>Glass Beads</td><td>Walter Stern</td><td>100C</td><td>To spreak yeast when making lawns</td></tr><tr><td>Glycerol &amp;ge;99%</td><td>Millipore Sigma</td><td>G9012-1L</td><td>Required to make frozen yeast stocks</td></tr><tr><td>L-Histidine</td><td>US Biologicals</td><td>H5100</td><td>For yeast growth selection in selective media</td></tr><tr><td>L-Leucine</td><td>US Biologicals</td><td>L2020-05</td><td>For yeast growth selection in selective media</td></tr><tr><td>L-Tryptophan</td><td>Sigma</td><td>T-0254</td><td>For yeast growth selection in selective media</td></tr><tr><td>N,N-Dimethylformamide</td><td>Sigma</td><td>319937-1L</td><td>To make X-gal solution</td></tr><tr><td>Omnipense Elite</td><td>Wheaton</td><td>W375030-A</td><td>For dispensing accurate volumes of media into Singer plates</td></tr><tr><td>Peptone, Bacteriological</td><td>American International Chemical</td><td>PEBAUP</td><td>Protein source required for yeast growth</td></tr><tr><td>Petri Dish, 150 mm x15 mm</td><td>VWR</td><td>10753-950</td><td>For growing yeast baits for screening</td></tr><tr><td>PlusPlates</td><td>Singer Instruments</td><td>PLU-003</td><td>To make rectangular agar plates to use with Singer Robot</td></tr><tr><td>Precision Low Temperature BOD Refrigerated Incubator</td><td>ThermoFisher Scientific</td><td>PR205745R</td><td>To incubate yeast plates at constant temperature</td></tr><tr><td>RePads 1,536 short</td><td>Singer Instruments</td><td>REP-005</td><td>To transfer the TF-prey array, mate yeast, and transfer yeast to diploid selection and readout plates</td></tr><tr><td>RePads 384 short</td><td>Singer Instruments</td><td>REP-004</td><td>To transfer TF-prey array from 384 to 1,536 colony format</td></tr><tr><td>RePads 96 long</td><td>Singer Instruments</td><td>REP-001</td><td>To transfer TF-prey array from glycerol stock to agar plate</td></tr><tr><td>RePads 96 short</td><td>Singer Instruments</td><td>REP-002</td><td>To transfer TF-prey array from 96 to 384 colony format</td></tr><tr><td>Singer HDA RoToR robot</td><td>Singer Instruments</td><td></td><td>For transfering yeast in high-throughput manner</td></tr><tr><td>Sodium Hydroxide (Pellets/Certified ACS)</td><td>Fisher</td><td>S318-1</td><td>For adjusting pH of selective media</td></tr><tr><td>Sodium Phosphate dibasic heptahydrate</td><td>Santa Cruz Biotechnology</td><td>sc-203402C</td><td>Required for LacZ reporter activity on X-gal&amp;nbsp;</td></tr><tr><td>Sodium Phosphate monobasic monohydrate</td><td>Santa Cruz Biotechnology</td><td>sc-202342B</td><td>Required for LacZ reporter activity on X-gal&amp;nbsp;</td></tr><tr><td>Uracil</td><td>Sigma</td><td>U0750-100G</td><td>For yeast growth selection in selective media</td></tr><tr><td>X-gal (5-Bromo-4-chloro-3-indoxyl-beta-Dgalactopyranoside)</td><td>Gold Biotechnology</td><td>X4281C100</td><td>&amp;beta;-galactosidase turns colorless X-gal blue to detect protein-DNA interaction</td></tr><tr><td>Yeast Extract</td><td>US Biologicals</td><td>Y2010</td><td>Nutritious medium for growth and propagation of yeast</td></tr><tr><td>Yeast Nitrogen Base (powder) w/o AA, carbohydrate and w/o AS&amp;nbsp;</td><td>US Biologicals</td><td>Y2030</td><td>Required for vigorous yeast growth</td></tr></tbody></table>

enhanced yeast one-hybrid screens to identify transcription factor binding to human dna sequences

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 &#8216;DNA-bait&#8217; (e.g., promoters, enhancers, silencers, etc.) and a &#8216;TF-prey,&#8217; 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 &#62;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.

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...

Watch this Scientific Journal Video about Enhanced Yeast One-hybrid Screens To Identify Transcription Factor Binding To Human DNA Sequences at JoVE.com

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

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 &#62;1,000 TFs in a single experiment.

Identifying the sets of transcription factors (TFs) that regulate each human gene is a daunting task that requires integrating numerous experimental and ...

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Genetics

7.8K Views.  Boston University. 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.

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

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between proteins such as transcription factors (TFs) and their DNA target. The method presented here utilizes the underlying concept of the Y1H but is modified further to study and validate protein complexes binding to their target DNA. Hence, it is referred to as the modified yeast one-hybrid (Y1.5H) assay. This assay is cost effective and can be easily performed in a regular laboratory setting. Albeit using a heterologous system, the described method could be a valuable tool to test and validate the heteromeric protein complex binding to their DNA target(s) for functional genomics in any system of study, especially plant genomics.

The modified yeast one-hybrid assay described here is an extension of the classical yeast one-hybrid (Y1H) assay to study and validate the heteromeric protein complex-DNA interaction in a heterologous system for any functional genomics study.

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between ...

Biochemistry

Informatic Analysis of Sequence Data from Batch Yeast 2-Hybrid Screens

We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing high-throughput short-read DNA sequencing. The resulting sequence datasets can not only track what genes in a population that are enriched during selection for positive yeast 2-hybrid interactions, but also give detailed information about the relevant subdomains of proteins sufficient for interaction. Here, we describe a full suite of stand-alone software programs that allow non-experts to perform all the bioinformatics and statistical steps to process and analyze DNA sequence fastq files from a batch yeast 2-hybrid assay. The processing steps covered by these software include: 1) mapping and counting sequence reads corresponding to each candidate protein encoded within a yeast 2-hybrid prey library; 2) a statistical analysis program that evaluates the enrichment profiles; and 3) tools to examine the translational frame and position within the coding region of each enriched plasmid that encodes the interacting proteins of interest.

Deep sequencing of yeast populations selected for positive yeast 2-hybrid interactions potentially yields a wealth of information about interacting partner proteins. Here, we describe the operation of specific bioinformatics tools and customized updated software to analyze sequence data from such screens.

We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing ...

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the discovery of high-affinity interactors that are highly enriched in the library of interacting candidates. In a traditional format, the yeast 2-hybrid assay can yield too many colonies to analyze when conducted at low stringency where low affinity interactors might be found. Moreover, without a comprehensive and complete interrogation of the same library against different bait plasmids, a comparative analysis cannot be achieved. Although some of these problems can be addressed using arrayed prey libraries, the cost and infrastructure required to operate such screens can be prohibitive. As an alternative, we have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing a strategy termed DEEPN (Dynamic Enrichment for Evaluation of Protein Networks), which incorporates high-throughput DNA sequencing and computation to follow the evolution of a population of plasmids that encode interacting partners. Here, we describe customized reagents and protocols that allow a DEEPN screen to be executed easily and cost-effectively.

Batch processing of yeast 2-hybrid screens allows for direct comparison of the interaction profiles of multiple bait proteins with a highly complex set of prey fusion proteins. Here, we describe refined methods, new reagents, and how to implement their use for such screens.

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the ...

Yeast As a Chassis for Developing Functional Assays to Study Human P53

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of different functional assays to study the impacts of 1) binding site [i.e., response element (RE)] sequence variants on P53 transactivation specificity or 2) TP53 mutations, co-expressed cofactors, or small molecules on P53 transactivation activity. Different basic and translational research applications have been developed. Experimentally, these approaches exploit two major advantages of the yeast model. On one hand, the ease of genome editing enables quick construction of qualitative or quantitative reporter systems by exploiting isogenic strains that differ only at the level of a specific P53-RE to investigate sequence-specificity of P53-dependent transactivation. On the other hand, the availability of regulated systems for ectopic P53 expression allows the evaluation of transactivation in a wide range of protein expression. Reviewed in this report are extensively used systems that are based on color reporter genes, luciferase, and the growth of yeast to illustrate their main methodological steps and to critically assess their predictive power. Moreover, the extreme versatility of these approaches can be easily exploited to study different TFs including P63 and P73, which are other members of TP53 gene family.

Presented here are four protocols to construct and exploit yeast Saccharomyces cerevisiae reporter strains to study human P53 transactivation potential, impacts of its various cancer-associated mutations, co-expressed interacting proteins, and the effects of specific small molecules.

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of ...

Cancer Research

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

Immunology and Infection

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Biology

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype.

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay EMSA and DNA-affinity Precipitation Assay DAPA

We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical ...

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

Summary

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Chapters in this video

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies

Informatic Analysis of Sequence Data from Batch Yeast 2-Hybrid Screens

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

Yeast As a Chassis for Developing Functional Assays to Study Human P53

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

High-throughput Yeast Plasmid Overexpression Screen

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)