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 ...

<ol>
	<li>Vaquerizas, J. M., Kummerfeld, S. K., Teichmann, S. A., Luscombe, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+census+of+human+transcription+factors:+function,+expression+and+evolution.">A census of human transcription factors: function, expression and evolution.</a> Nature Reviews Genetics. 10 (4), 252-263 (2009).</li><li>Lambert, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Human+Transcription+Factors.">The Human Transcription Factors.</a> Cell. 172 (4), 650-665 (2018).</li><li>Arda, H. E., Walhout, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene-centered+regulatory+networks.">Gene-centered regulatory networks.</a> Briefings in Functional Genomics. 9 (1), 4-12 (2010).</li><li>Jolma, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-binding+specificities+of+human+transcription+factors.">DNA-binding specificities of human transcription factors.</a> Cell. 152 (1-2), 327-339 (2013).</li><li>Bulyk, M. L., Gentalen, E., Lockhart, D. J., Church, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+DNA-protein+interactions+by+double-stranded+DNA+arrays.">Quantifying DNA-protein interactions by double-stranded DNA arrays.</a> Nature Biotechnology. 17 (6), 573-577 (1999).</li><li>Robertson, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+profiles+of+STAT1+DNA+association+using+chromatin+immunoprecipitation+and+massively+parallel+sequencing.">Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing.</a> Nature Methods. 4 (8), 651-657 (2007).</li><li>Li, J. J., Herskowitz, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+ORC6,+a+component+of+the+yeast+origin+recognition+complex+by+a+one-hybrid+system.">Isolation of ORC6, a component of the yeast origin recognition complex by a one-hybrid system.</a> Science. 262 (5141), 1870-1874 (1993).</li><li>Sewell, J. A., Fuxman Bass, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Options+and+Considerations+When+Using+a+Yeast+One-Hybrid+System.">Options and Considerations When Using a Yeast One-Hybrid System.</a> Methods in Molecular Biology. 1794, 119-130 (2018).</li><li>Fuxman Bass, J. I., Reece-Hoyes, J. S., Walhout, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene-Centered+Yeast+One-Hybrid+Assays.">Gene-Centered Yeast One-Hybrid Assays.</a> Cold Spring Harbor Protocols. (12), (2016).</li><li>Reece-Hoyes, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+yeast+one-hybrid+assays+for+high-throughput+gene-centered+regulatory+network+mapping.">Enhanced yeast one-hybrid assays for high-throughput gene-centered regulatory network mapping.</a> Nature Methods. 8 (12), 1059-1064 (2011).</li><li>Fuxman Bass, J. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gene-centered+C.+elegans+protein-DNA+interaction+network+provides+a+framework+for+functional+predictions.">A gene-centered C. elegans protein-DNA interaction network provides a framework for functional predictions.</a> Molecular Systems Biology. 12 (10), 884 (2016).</li><li>Fuxman Bass, J. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+gene-centered+transcription+factor+networks+for+enhancers+and+disease+variants.">Human gene-centered transcription factor networks for enhancers and disease variants.</a> Cell. 161 (3), 661-673 (2015).</li><li>Reece-Hoyes, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+one-hybrid+assays+for+gene-centered+human+gene+regulatory+network+mapping.">Yeast one-hybrid assays for gene-centered human gene regulatory network mapping.</a> Nature Methods. 8 (12), 1050-1052 (2011).</li><li>Sahni, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Widespread+macromolecular+interaction+perturbations+in+human+genetic+disorders.">Widespread macromolecular interaction perturbations in human genetic disorders.</a> Cell. 161 (3), 647-660 (2015).</li><li>Fuxman Bass, J. I., Reece-Hoyes, J. S., Walhout, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+Bait+Strains+for+Yeast+One-Hybrid+Assays.">Generating Bait Strains for Yeast One-Hybrid Assays.</a> Cold Spring Harbor Protocols. (12), (2016).</li><li>Fuxman Bass, J. I., Reece-Hoyes, J. S., Walhout, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colony+Lift+Colorimetric+Assay+for+beta-Galactosidase+Activity.">Colony Lift Colorimetric Assay for beta-Galactosidase Activity.</a> Cold Spring Harbor Protocols. (12), (2016).</li><li>Fuxman Bass, J. I., Reece-Hoyes, J. S., Walhout, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zymolyase-Treatment+and+Polymerase+Chain+Reaction+Amplification+from+Genomic+and+Plasmid+Templates+from+Yeast.">Zymolyase-Treatment and Polymerase Chain Reaction Amplification from Genomic and Plasmid Templates from Yeast.</a> Cold Spring Harbor Protocols. (12), (2016).</li><li>Deplancke, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gene-centered+C.+elegans+protein-DNA+interaction+network.">A gene-centered C. elegans protein-DNA interaction network.</a> Cell. 125 (6), 1193-1205 (2006).</li><li>Reece-Hoyes, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+rewiring+and+complex+evolutionary+dynamics+in+a+C.+elegans+multiparameter+transcription+factor+network.">Extensive rewiring and complex evolutionary dynamics in a C. elegans multiparameter transcription factor network.</a> Molecular Cell. 51 (1), 116-127 (2013).</li><li>Carrasco Pro, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+landscape+of+mouse+and+human+cytokine+transcriptional+regulation.">Global landscape of mouse and human cytokine transcriptional regulation.</a> Nucleic Acids Research. 46 (18), 9321-9337 (2018).</li><li>Whitfield, T. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+transcription+factor+binding+sites+in+human+promoters.">Functional analysis of transcription factor binding sites in human promoters.</a> Genome Biology. 13 (9), R50 (2012).</li><li>Fuxman Bass, J. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factor+binding+to+Caenorhabditis+elegans+first+introns+reveals+lack+of+redundancy+with+gene+promoters.">Transcription factor binding to Caenorhabditis elegans first introns reveals lack of redundancy with gene promoters.</a> Nucleic Acids Research. 42 (1), 153-162 (2014).</li><li>Hens, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+protein-DNA+interaction+screening+of+Drosophila+regulatory+elements.">Automated protein-DNA interaction screening of Drosophila regulatory elements.</a> Nature Methods. 8 (12), 1065-1070 (2011).</li><li>Gubelmann, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+yeast+one-hybrid+and+microfluidics-based+pipeline+to+map+mammalian+gene+regulatory+networks.">A yeast one-hybrid and microfluidics-based pipeline to map mammalian gene regulatory networks.</a> Molecular Systems Biology. 9, 682 (2013).</li><li>Brady, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stele-enriched+gene+regulatory+network+in+the+Arabidopsis+root.">A stele-enriched gene regulatory network in the Arabidopsis root.</a> Molecular Systems Biology. 7, 459 (2011).</li><li>Pruneda-Paz, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-scale+resource+for+the+functional+characterization+of+Arabidopsis+transcription+factors.">A genome-scale resource for the functional characterization of Arabidopsis transcription factors.</a> Cell Reports. 8 (2), 622-632 (2014).</li><li>Burdo, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Maize+TFome--development+of+a+transcription+factor+open+reading+frame+collection+for+functional+genomics.">The Maize TFome--development of a transcription factor open reading frame collection for functional genomics.</a> The Plant Journal. 80 (2), 356-366 (2014).</li></ol>

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 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, 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 ...

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

enhanced-yeast-one-hybrid-screens-to-identify-transcription-factor

Research

JoVE Journal

Genetics

7.8K vistas.  Boston University. El objetivo de los ensayos de un híbrido de levadura mejorada es identificar el conjunto de factores de transcripción que se unen a una secuencia de ADN de interés. La ventaja de esta técnica es que los ensayos de un solo híbrido de levadura mejorada pueden evaluar más de 1000 factores de transcripción en un solo experimento. Por el contrario, la inmunoprecipitación de cromatina evalúa solo un factor de transcripción a la vez.Este método constituye una herramienta clave para...