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 strain10,12,19. More importantly, TF-DNA interactions detected by eY1H validate at a 40%&#8211;70% rate when tested in human reporter assays12,20, in primary human cells (unpublished results), and in C. elegans knockout animals11. This is a similar validation rate to that observed for ChIP-seq data21.
Although interactions identified by eY1H are highly reproducible when retesting the same yeast DNA-bait strain, testing different yeast strains for the same DNA-bait sometimes produces different, although overlapping, sets of TF-DNA interactions. This is usually due to differences in background reporter activity between strains. In addition, testing fragments of a DNA sequence results in the detection of more TF-DNA interactions than testing the full sequence, in particular when overlapping fragments are tested. This may be related with the assay being more efficient in identifying interactions that are close to the reporter minimal promoters, and because testing overlapping fragments reduces the chances that a binding site may be occluded by yeast nucleosomes. Thus, for small scale projects, it is recommended that overlapping 0.5&#8211;1 kb fragments of a regulatory region are tested and that two independent strains are screened for each DNA-bait sequence8.
There are several critical steps in the eY1H screening protocol to avoid some of the issues presented in Figure 3. First, although most media ingredients are stable for several months (except for 3AT and X-gal), a lack of proper colony growth likely indicates that at least one of the ingredients may have lost activity and should be replaced. Second, it is important to prepare the rectangular plates so that the agar is leveled and so that they do not dry for more than one day to avoid failure in pinning when using the robotic platform. Finally, it is key to use the robotic platform programs as indicated in the protocol (revisit, recycle, mixing, etc.) for the yeast to be transferred effectively, for mating to be efficient, and to avoid cross contamination between yeast clones.
The examples we selected to illustrate the use of eY1H screens correspond to human gene promoters. However, other regulatory regions can also be tested including enhancers and silencers. For example, we have used eY1H assays to evaluate TF binding to human developmental enhancers and to C. elegans first introns12,22. In addition, given that interactions are tested in a pairwise manner, eY1H assays can be used to compare interactions between non-coding variants, and between TF coding sequence variants. For example, using eY1H assays we identified altered TF binding to 109 noncoding variants associated with different genetic diseases, and also differential interactions profiles for 58 TF missense mutations12,14. Although this protocol focuses on evaluating TF binding to human regulatory regions, DNA regions from other species can also be tested provided that a TF-prey is available or can be generated. Indeed, TF-prey arrays have been generated for C. elegans10, Drosophila melanogaster23, Mus musculus24, Arabidopsis thaliana25,26, and Zea mays27. Thus, with increasingly available resources, eY1H assays may be applied to additional systems.&#160;
Although eY1H assays have been instrumental to identify the repertoire of TFs that bind to different regulatory regions in human and other species, they are not free of caveats8,11,12,19,20,25. One of the limitations is that interactions are tested in the milieu of the yeast nucleus and, although the DNA-baits are chromatinized, the chromatin structure in yeast may not reflect the chromatin structure in the species from where the DNA-bait originated and will not reflect cell type differences observed in vivo. Thus, interactions identified by eY1H assays must be validated in reporter or other functional assays. Of note, we and others have found TF-DNA interactions detected by eY1H validate at a 40%&#8211;70% rate in functional assays11,12,20,23. Another limitation of eY1H assays is that it cannot detect interactions involving TFs that require post-translational modifications absent in yeast to bind to DNA, TFs that are not properly folded in yeast when fused to the AD, and TFs that are missing from the array8. In addition, in the current format, eY1H assays do not detect interactions involving heterodimeric TFs, as each yeast colony in the TF array expresses a single TF-prey. Thus, further improvements in the assay will increase the breadth of TFs that can be tested and expand the capabilities of eY1H assays to identify novel TF-DNA interactions

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 border="0" cellpadding="0" cellspacing="0" style="width:1195px;" width="1195">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:555px;">3-Amino-1,2,4-triazole (3AT) ~95 % TLC</td>
			<td style="width:223px;">Sigma</td>
			<td style="width:103px;">A8056-100G</td>
			<td style="width:315px;">Competitive inhibitor for products of HIS3 gene</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adenine sulfate (hemisulfate), dihydrate</td>
			<td>US Biologicals</td>
			<td>A0865</td>
			<td>Required for proper yeast growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar High Gel Strength - Bacteriological grade</td>
			<td>American International Chemical</td>
			<td>AGHGUP</td>
			<td>Nutritive media for yeast growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium Sulfate</td>
			<td>US Biologicals</td>
			<td>A1450</td>
			<td>Nitrogen source in synthetic yeast media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D+ Glucose Anhydrous</td>
			<td>US Biologicals</td>
			<td>G3050</td>
			<td>Required for yeast growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;width:555px;">edge Multiparameter pH Meter</td>
			<td style="width:223px;">Hanna Instruments</td>
			<td>HI2020-01</td>
			<td>To measure pH of selective media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flat Toothpicks 750ct</td>
			<td style="width:223px;">Diamond</td>
			<td style="width:103px;"></td>
			<td>To streak yeasts on petridishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:555px;">Glass Beads</td>
			<td style="width:223px;">Walter Stern</td>
			<td style="width:103px;">100C</td>
			<td>To spreak yeast when making lawns</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol &#8805;99%</td>
			<td>Millipore Sigma</td>
			<td>G9012-1L</td>
			<td>Required to make frozen yeast stocks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Histidine</td>
			<td>US Biologicals</td>
			<td>H5100</td>
			<td>For yeast growth selection in selective media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Leucine</td>
			<td>US Biologicals</td>
			<td>L2020-05</td>
			<td>For yeast growth selection in selective media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Tryptophan</td>
			<td>Sigma</td>
			<td>T-0254</td>
			<td>For yeast growth selection in selective media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N,N-Dimethylformamide</td>
			<td>Sigma</td>
			<td>319937-1L</td>
			<td>To make X-gal solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Omnipense Elite</td>
			<td style="width:223px;">Wheaton</td>
			<td>W375030-A</td>
			<td>For dispensing accurate volumes of media into Singer plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peptone, Bacteriological</td>
			<td>American International Chemical</td>
			<td>PEBAUP</td>
			<td>Protein source required for yeast growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri Dish, 150x15 mm</td>
			<td>VWR</td>
			<td>10753-950</td>
			<td>For growing yeast baits for screening</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:555px;">PlusPlates</td>
			<td style="width:223px;">Singer Instruments</td>
			<td style="width:103px;">PLU-003</td>
			<td>To make rectangular agar plates to use with Singer Robot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:555px;">Precision Low Temperature BOD Refrigerated Incubator</td>
			<td style="width:223px;">ThermoFisher Scientific</td>
			<td>PR205745R</td>
			<td>To incubate yeast plates at constant temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:555px;">RePads 1,536 short</td>
			<td style="width:223px;">Singer Instruments</td>
			<td style="width:103px;">REP-005</td>
			<td>To transfer the TF-prey array, mate yeast, and transfer yeast to diploid selection and readout plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:555px;">RePads 384 short</td>
			<td style="width:223px;">Singer Instruments</td>
			<td style="width:103px;">REP-004</td>
			<td>To transfer TF-prey array from 384 to 1,536 colony format</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:555px;">RePads 96 long</td>
			<td style="width:223px;">Singer Instruments</td>
			<td style="width:103px;">REP-001</td>
			<td>To transfer TF-prey array from glycerol stock to agar plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:555px;">RePads 96 short</td>
			<td style="width:223px;">Singer Instruments</td>
			<td style="width:103px;">REP-002</td>
			<td>To transfer TF-prey array from 96 to 384 colony format</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:555px;">Singer HDA RoToR robot</td>
			<td style="width:223px;">Singer Instruments</td>
			<td style="width:103px;"></td>
			<td>For transfering yeast in high-throughput manner</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Hydroxide (Pellets/Certified ACS)</td>
			<td>Fisher</td>
			<td>S318-1</td>
			<td>For adjusting pH of selective media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Phosphate dibasic heptahydrate</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-203402C</td>
			<td>Required for LacZ reporter activity on X-gal&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Phosphate monobasic monohydrate</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-202342B</td>
			<td>Required for LacZ reporter activity on X-gal&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Uracil</td>
			<td>Sigma</td>
			<td>U0750-100G</td>
			<td>For yeast growth selection in selective media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X-gal (5-Bromo-4-chloro-3-indoxyl-beta-Dgalactopyranoside)</td>
			<td>Gold Biotechnology</td>
			<td>X4281C100</td>
			<td>&#946;-galactosidase turns colorless X-gal blue to detect protein-DNA interaction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast Extract</td>
			<td>US Biologicals</td>
			<td>Y2010</td>
			<td>Nutritious medium for growth and propagation of yeast</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast Nitrogen Base (powder) w/o AA, carbohydrate and w/o AS&#160;</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, with colonies for positive interactions being bigger and blue. Autoactive DNA-bait strains show yeast growth in media lacking histidine and a blue color in the presence of X-gal for all colonies in the plate, which is likely related to the binding of yeast transcriptional activators to the DNA-bait8. The strength of the reporter activity corresponding to the TF-DNA interactions detected (i.e., the size of the colony and intensity of blue) depends on many parameters such as the affinity, the TF expression level in yeast, the number of binding sites and distance to the yeast minimal promoters located upstream of the reporter genes, and the background reporter activity of the DNA-bait strain. For example, a weak interaction may be easily detected in a low background bait but may be difficult to detect in an autoactive or uneven background bait. It is also important to note that reporter activity levels in yeast do not necessarily correlate with the regulatory activity in human cells, as the chromatin structure, nucleosome positioning, and distance effects are different between yeast and human. Further, interactions in human are likely to be influenced by the binding of other TFs and cofactors or may be masked by functionally redundant TFs8. Finally, interactions are considered positive in eY1H assays when at least two of the four colonies show reporter expression above background levels. However, we have observed that ~90% of interactions identified result from all four colonies corresponding to a TF being positive10,12,19.
To illustrate the type of results that can be obtained using eY1H assays we screened the promoter regions (2 kb upstream of the transcription start sites) of the CCL15 and IL17F genes, against an array of 1,086 human TFs (Figure 2). The CCL15 promoter is an example of a non-autoactive DNA-bait where interactions, even weak ones, can be easily detected (Figure 2A). 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 (Figure 2B).
Problems that can be encountered when performing eY1H assays
Although the screening eY1H protocol is straight-forward and robust, several problems can be encountered during the screen:
1) Colonies are too small and fail to transfer (Figure 3A): Although it is expected that some yeast expressing exogenous TFs may display slow growth given that yeast gene expression may be dysregulated, typically ~95% of TF-prey colonies display normal growth. If more than 10% of colonies fail to grow, the most frequent causes are problems with the media or with the yeast transfer. Suboptimal growth is frequently related to one of the media components losing activity (e.g., uracil, histidine, or leucine), which can be solved by preparing fresh media with fresh stock solutions. Alternatively, this may also be related to a pinning offset that affects colony transfer. In this case, verify that the 1,536 pads pin the center of the yeast colonies in the source plates.&#160;
2) No yeast growth in a portion of the plate (Figure 3B): This issue is generally related with a failure in the mating step if the 1,536 pin pad fails to make contact with the yeast in the DNA-bait strain lawn, the TF array, or in the mating plate. In almost every case, this is due to uneven agar media level during plate pouring or due to excessive drying of the plate.&#160;
3) No interactions detected (Figure 3C and D): This issue is often related to either unintended inactivating mutations in the reporter genes, in particular LacZ (Figure 3C), or to high autoactivity that mask interactions (Figure 3D). To troubleshoot this problem, it is recommended to screen another independently obtained strain corresponding to the same DNA-bait.&#160;
4) The plate presents random blue spots (Figure 3E): This issue is often related to bacterial contamination. To solve this issue, streak the yeast to obtain individual colonies, and repeat the screen.
The above are the most frequent problems encountered when performing eY1H assays. Should other problems arise, preparing new media, confirming that appropriate settings for the HDA robot were used, and testing multiple strains per DNA-bait would likely solve most issues.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59192/59192fig1.jpg" /> 
Figure 1: Outline of eY1H assay screens. (A) Comparison between TF-centered and DNA-centered methods to identify protein-DNA interactions. (B) Schematics of eY1H assays. A DNA sequence of interest (promoter, enhancer, silencer, etc.) cloned upstream of the HIS3 and LacZ reporter genes is integrated into the yeast genome. The resulting DNA-bait strain is mated to a collection of yeast strains harboring TFs fused to the Gal4 activation domain (AD). Positive interactions are determined by the yeast&#8217;s ability to grow without histidine and overcoming competitive inhibitor 3-AT, and turn blue in the presence of X-gal. (C) Pipeline for eY1H screens. A lawn of a yeast DNA-bait strain grown in a YAPD plate is mated in a YAPD plate to a 1,536 colony array expressing TFs fused to AD grown on a Sc &#8722;Trp plate. After one day, the yeast is transferred to a Sc &#8722;U &#8722;Trp to select for diploid yeast. After a 2&#8211;3 day incubation, the yeast is transferred to a Sc &#8722;U &#8722;H &#8722;Trp + 3AT + X-gal plate (readout plate) to identify protein-DNA interactions. Each interaction is tested in quadruplicate.&#160; <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59192/59192fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59192/59192fig2.jpg" /> 
Figure 2: Examples of eY1H readout plates. (A) Interactions involving the promoter of CCL15, a non-autoactive bait. Background reporter activity for this bait is low (reduced growth in the absence of histidine and absence of blue color for non-interacting TFs). (B) Interactions involving the promoter of IL17F, an autoactive bait. Background reporter activity for this bait is high (growth in the absence of histidine and background blue color throughout the plate) and uneven, making it challenging to identify protein-DNA interactions. Strong, medium, and weak interactions are squared in red, orange, and yellow, respectively. The HGNC names of the interacting TFs are shown. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59192/59192fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59192/59192fig3.jpg" /> 
Figure 3: Problems in eY1H screens. (A) TF-prey array where multiple colonies failed to grow. (B) Readout plate where colonies in the lower left corner have failed to transfer. (C) Non-autoactive DNA-bait strain that does not display positive interactions. (D) Highly autoactive DNA-bait strain that does not display positive interactions. (E) Readout plate displaying multiple blue colonies due to contamination. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59192/59192fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Spotting a TF array</td>
		</tr>
		<tr>
			<td>Day 1</td>
			<td>Spot yeast into Sc &#8211;Trp plate in 96 colony format (2.1 and 2.2)</td>
		</tr>
		<tr>
			<td>Day 4</td>
			<td>Generate 384 colony TF array (2.3)</td>
		</tr>
		<tr>
			<td>Day 6</td>
			<td>Generate 1,536 colony TF array (2.4)</td>
		</tr>
		<tr>
			<td>Day 9</td>
			<td>Amplify 1,536 colony TF array (2.5)</td>
		</tr>
		<tr>
			<td colspan="2">Preparing DNA-bait strain lawns for mating</td>
		</tr>
		<tr>
			<td>Day 6</td>
			<td>Spot the yeast DNA-bait strains (3.1.1)</td>
		</tr>
		<tr>
			<td>Day 9</td>
			<td>Streak yeast, 12-16 strains per plate (3.1.2)</td>
		</tr>
		<tr>
			<td>Day 10</td>
			<td>Streak yeast, 4 strains per plate (3.1.3)</td>
		</tr>
		<tr>
			<td>Day 11</td>
			<td>Prepare DNA-bait lawns (3.1.4 and 3.1.5)</td>
		</tr>
		<tr>
			<td colspan="2">Mating of yeast DNA-bait and TF array strains</td>
		</tr>
		<tr>
			<td>Day 12</td>
			<td>Mating in YAPD plates (3.2)</td>
		</tr>
		<tr>
			<td colspan="2">Selection of diploid yeast</td>
		</tr>
		<tr>
			<td>Day 13</td>
			<td>Selection of diploid yeast in Sc &#8211;U&#8211;Trp plates (3.3)</td>
		</tr>
		<tr>
			<td colspan="2">Transfer to readout plates</td>
		</tr>
		<tr>
			<td>Day 15</td>
			<td>Transfer diploid yeast to readout plates (3.4)</td>
		</tr>
		<tr>
			<td colspan="2">Imaging of readout plates</td>
		</tr>
		<tr>
			<td>Days 17-22</td>
			<td>Image acquisition of readout plates depending on background (3.5)</td>
		</tr>
	</tbody>
</table>
Table 1: TFF Array.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>REAGENT</td>
			<td>QUANTITY (for 2 L)</td>
		</tr>
		<tr>
			<td>Drop-out mix synthetic minus histidine, leucine, tryptophan and uracil, adenine, rich (2 g) w/o yeast nitrogen base</td>
			<td>2.6 g</td>
		</tr>
		<tr>
			<td>Yeast nitrogen base without amino acids and without ammonium sulfate (YNB)</td>
			<td>3.4 g</td>
		</tr>
		<tr>
			<td>Adenine hemisulfate dihydrate</td>
			<td>160 mg</td>
		</tr>
		<tr>
			<td>Ammonium sulfate</td>
			<td>10 g</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>35 g</td>
		</tr>
		<tr>
			<td>Glucose (40%, w/v) in water, sterile</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Leucine (100 mM), sterile filtered</td>
			<td>16 mL</td>
		</tr>
		<tr>
			<td>Tryptophan (40 mM), sterile filtered</td>
			<td>16 mL</td>
		</tr>
	</tbody>
</table>
Table 2: Reagent List I.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>REAGENT</td>
			<td>QUANTITY (for 2 L)</td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>40 g</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>20 g</td>
		</tr>
		<tr>
			<td>Adenine hemisulfate dihydrate</td>
			<td>0.32 g</td>
		</tr>
		<tr>
			<td>Glucose (40%, w/v) in water, sterile</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>35 g</td>
		</tr>
	</tbody>
</table>
Table 3: Reagent List II.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>REAGENT</td>
			<td>QUANTITY (for 2 L)</td>
		</tr>
		<tr>
			<td>Drop-out mix synthetic minus histidine, leucine, tryptophan and uracil, adenine rich (2 g) w/o yeast nitrogen base</td>
			<td>2.6 g</td>
		</tr>
		<tr>
			<td>Yeast nitrogen base without amino acids and without ammonium sulfate (YNB)</td>
			<td>3.4 g</td>
		</tr>
		<tr>
			<td>Adenine hemisulfate dihydrate</td>
			<td>160 mg</td>
		</tr>
		<tr>
			<td>Ammonium sulfate</td>
			<td>10 g</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>35 g</td>
		</tr>
		<tr>
			<td>Glucose (40%, w/v) in water, sterile</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Leucine (100 mM), sterile filtered</td>
			<td>16 mL</td>
		</tr>
		<tr>
			<td>Histidine (100 mM), sterile filtered</td>
			<td>16 mL</td>
		</tr>
		<tr>
			<td>Uracil (20 mM), sterile filtered (omit for Sc &#8211;U &#8211;Trp plates)</td>
			<td>16 mL</td>
		</tr>
	</tbody>
</table>
Table 4:&#160;Reagent List III.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>REAGENT</td>
			<td>QUANTITY (for 2 L)</td>
		</tr>
		<tr>
			<td>Drop-out mix synthetic minus histidine, leucine, tryptophan and uracil, adenine rich (2 g) w/o yeast nitrogen base</td>
			<td>2.6 g</td>
		</tr>
		<tr>
			<td>Yeast nitrogen base without amino acids and without ammonium sulfate (YNB)</td>
			<td>3.4 g</td>
		</tr>
		<tr>
			<td>Adenine hemisulfate dihydrate</td>
			<td>160 mg</td>
		</tr>
		<tr>
			<td>Ammonium sulfate</td>
			<td>10 g</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>35 g</td>
		</tr>
		<tr>
			<td>Glucose (40%, w/v) in water, sterile</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>10x BU salts</td>
			<td>200 mL</td>
		</tr>
		<tr>
			<td>Leucine (100 mM), sterile filtered</td>
			<td>16 mL</td>
		</tr>
		<tr>
			<td>3AT (2 M), sterile filtered</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>X-gal (80 mg/mL) in DMF</td>
			<td>4 mL</td>
		</tr>
	</tbody>
</table>
Table 5:&#160;Reagent List IV.

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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Nanyang Technological University

Research

JoVE Journal

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

Methyl-binding DNA capture Sequencing for Patient Tissues

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable development and differentiation of different tissue types. Dysregulation of this process is often the hallmark of various diseases like cancer. Here, we outline one of the recent sequencing techniques, Methyl-Binding DNA Capture sequencing (MBDCap-seq), used to quantify methylation in various normal and disease tissues for large patient cohorts. We describe a detailed protocol of this affinity enrichment approach along with a bioinformatics pipeline to achieve optimal quantification. This technique has been used to sequence hundreds of patients across various cancer types as a part of the 1,000 methylome project (Cancer Methylome System).

Here we present a protocol to investigate genome wide DNA methylation in large scale clinical patient screening studies using the Methyl-Binding DNA Capture sequencing (MBDCap-seq or MBD-seq) technology and the subsequent bioinformatics analysis pipeline.

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable ...

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m7GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4FH), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m7GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.

Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve random mutagenesis, error-prone PCR is a convenient and efficient strategy for generating a diverse pool of mutants (i.e., a mutant library). Error-prone PCR is the method of choice when a researcher seeks to mutate a pre-defined region, such as the coding region of a gene while leaving other genomic regions unaffected. After the mutant library is amplified by error-prone PCR, it must be cloned into a suitable plasmid. The size of the library generated by error-prone PCR is constrained by the efficiency of the cloning step. However, in the fission yeast, Schizosaccharomyces pombe, the cloning step can be replaced by the use of a highly efficient one-step fusion PCR to generate constructs for transformation. Mutants of desired phenotypes may then be selected using appropriate reporters. Here, we describe this strategy in detail, taking as an example, a reporter inserted at centromeric heterochromatin.

This article describes a detailed methodology for random mutagenesis of a target gene in fission yeast. As an example, we target rpt4+, which encodes a subunit of the 19S proteasome, and screen for mutations that destabilize heterochromatin.

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve ...

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 ...

Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and host cell proteins on viral DNA synthesis. Here we address the lack of a cell-based, high-coverage, and high-resolution assay that is capable of defining retroviral reverse transcription intermediates within the physiological context of virus infection. The described method captures the 3'-termini of nascent complementary DNA (cDNA) molecules within HIV-1 infected cells at single nucleotide resolution. The protocol involves harvesting of whole cell DNA, targeted enrichment of viral DNA via hybrid capture, adaptor ligation, size fractionation by gel purification, PCR amplification, deep sequencing, and data analysis. A key step is the efficient and unbiased ligation of adaptor molecules to open 3'-DNA termini. Application of the described method determines the abundance of reverse transcripts of each particular length in a given sample. It also provides information about the (internal) sequence variation in reverse transcripts and thereby any potential mutations. In general, the assay is suitable for any questions relating to DNA 3'-extension, provided that the template sequence is known.

Here we present a deep sequencing approach that provides an unbiased determination of nascent 3&#39;-termini as well as mutational profiles of single-stranded DNA molecules. The main application is the characterization of nascent retroviral complementary DNAs (cDNAs), the intermediates generated during the process of retroviral reverse transcription.

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and ...

Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational steps are used to regulate specific genes. Sequence-specific nucleic acid-binding proteins target specific sequences to control spatial or temporal gene expression. The binding sites in nucleic acids are typically characterized by mutational analysis. However, numerous proteins of interest have no known binding site for such characterization. Here we describe an approach to identify previously unknown binding sites for RNA-binding proteins. It involves iterative selection and amplification of sequences starting with a randomized sequence pool. Following several rounds of these steps-transcription, binding, and amplification-the enriched sequences are sequenced to identify a preferred binding site(s). Success of this approach is monitored using in vitro binding assays. Subsequently, in vitro and in vivo functional assays can be used to assess the biological relevance of the selected sequences. This approach allows identification and characterization of a previously unknown binding site(s) for any RNA-binding protein for which an assay to separate protein-bound and unbound RNAs exists.

Sequence specificity is critical for gene regulation. Regulatory proteins that recognize specific sequences are important for gene regulation. Defining functional binding sites for such proteins is a challenging biological problem. An iterative approach for identification of a binding site for an RNA-binding protein is described here and is applicable to all RNA-binding proteins.

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational ...

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic screening using chemical mutagens is a useful strategy for mapping mutant phenotypes to genes among Drosophila and other model organisms that share conserved cellular pathways with humans. If the mutated gene of interest is not lethal in early developmental stages of flies, a climbing assay can be conducted to screen for phenotypic indicators of decreased brain functioning, such as low climbing rates. Subsequently, secondary histological analysis of brain tissue can be performed in order to verify the neuroprotective function of the gene by scoring neurodegeneration phenotypes. Gene mapping strategies include meiotic and deficiency mapping that rely on these same assays can be followed by DNA sequencing to identify possible nucleotide changes in the gene of interest.

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

We present a protocol using a forward genetic approach to screen for mutants exhibiting neurodegeneration in Drosophila melanogaster. It incorporates a climbing assay, histology analysis, gene mapping and DNA sequencing to ultimately identify novel genes related to the process of neuroprotection.

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic ...

Expression, Purification, and Liposome Binding of Budding Yeast SNX-BAR Heterodimers

SNX-BAR proteins are an evolutionarily conserved class of membrane remodeling proteins that play key roles in sorting and trafficking of protein and lipids during endocytosis, sorting within the endosomal system, and autophagy. Central to SNX-BAR protein function is the ability to form homodimers or heterodimers that bind membranes using highly conserved phox-homology (PX) and BAR (Bin/Amphiphysin/Rvs) domains. In addition, oligomerization of SNX-BAR dimers on membranes can elicit the formation of membrane tubules and vesicles and this activity is thought to reflect their functions as coat proteins for endosome-derived transport carriers. Researchers have long utilized in vitro binding studies using recombinant SNX-BAR proteins on synthetic liposomes or giant unilamellar vesicles (GUVs) to reveal the precise makeup of lipids needed to drive membrane remodeling, thus revealing their mechanism of action. However, due to technical challenges with dual expression systems, toxicity of SNX-BAR protein expression in bacteria, and poor solubility of individual SNX-BAR proteins, most studies to date have examined SNX-BAR homodimers, including non-physiological dimers that form during expression in bacteria. Recently, we have optimized a protocol to overcome the major shortcomings of a typical bacterial expression system. Using this workflow, we demonstrate how to successfully express and purify large amounts of SNX-BAR heterodimers and how to reconstitute them on synthetic liposomes for binding and tubulation assays.

Here, we present a workflow for the expression, purification and liposome binding of SNX-BAR heterodimers in yeast.

SNX-BAR proteins are an evolutionarily conserved class of membrane remodeling proteins that play key roles in sorting and trafficking of protein and ...

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

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Methyl-binding DNA capture Sequencing for Patient Tissues

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

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

Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells

Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

Expression, Purification, and Liposome Binding of Budding Yeast SNX-BAR Heterodimers