We thank S.S. Wang, M. Amar and A. Galla for critical reading of the manuscript. Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award numbers RO1GM067837 and RO1GM056006 (to S.A.K). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

1. Construct and Reporter Plasmid Preparation
<ol>
	<li>PCR amplify the promoter regions / &#34;fragments&#34; (preferably ~250 - 500 bp) of the target DNA to be analyzed for further cloning into the entry vector using E. coli (TOP10).</li>
	<li>Upon sequencing confirmation, subclone the positive clone in a compatible pGLacZi expression vector 7 as previously described 8,9,10,11.</li>
	<li>Transform the yeast strain for promoter integration (YM4271) by homologous recombination of pGLacZi to generate yeast reporter strain as described earlier 12,13. The transformation and confirmation steps of the yeast strain with the DNA region along with the recipe of the media is not described here, as the steps are similar to classical Y1H protocol 9,10,11,12,13. The pEXP-AD502 control plasmid can be used for normalization purposes while quantifiying &#946;-galactosidase activity as previously described 8.</li>
	<li>Clone the TF or protein of interest and subsequently clone the interacting protein partner into pDEST22 and pDEST32&#916;DBD (pDEST32 minus DNA Binding Domain) expression vectors. The transformed yeast promoter fragments and the TF clones are subsequently used further in the protocol.</li>
</ol>
2. Modified Y1.5H Protocol
<ol>
	<li>On day 1 (afternoon), start with 5 mL of culture in SD-Ura from a Petri dish or glycerol stock and incubate overnight in a 30 &#176;C shaker. 
	NOTE: This culture can be started from a freshly streaked plate or directly from a 25% or 30% glycerol stock of the yeast clone with the DNA fragment.</li>
	<li>On day 2 (afternoon), inoculate 10 mL of YPDA media with 500 &#956;L of the overnight culture and incubate overnight in a 30 &#176;C shaker.</li>
	<li>Day 3 (early in the morning and whole afternoon): Prepare competent cells (early in the morning).
	<ol>
		<li>Inoculate 100 mL of YPDA media with the 2 mL of the overnight culture and incubate in a 30 &#176;C shaker. Grow this fresh culture until the OD600 reaches 0.4-0.8 (3-5 h). 
		NOTE: Check the OD600 of the overnight culture and make sure that the start point OD600 for this step is 0.1 - 0.2. Use of over- or under-grown culture may prolong the experiment.</li>
		<li>Centrifuge for 5 min at 1000 x g and 21 &#176;C. Discard the supernatant.</li>
		<li>Reconstitute pellets in 10 mL of sterile water using a vortex or pipetting up and down.</li>
		<li>Centrifuge for 5 min at 1000 x g and 21 &#176;C. Discard the supernatant.</li>
		<li>Reconstitute pellets in 2 mL of Tris-Ethylenediaminetetraaceticacid (TE)/ Lithium acetate (LiAc) using a vortex or pipetting up and down. 
		NOTE: Please see the recipe for TE/LiAc in Table 1.</li>
		<li>Centrifuge for 5 min 1000 x g and 21&#176;C. Discard the supernatant.</li>
		<li>Re-suspend pellet in 4 mL of TE/LiAc + 400 &#956;L of salmon sperm DNA (10 mg/mL) pipetting up and down and proceed to the next step.</li>
	</ol>
	</li>
	<li>Co-transformation: Heat shock the cells (late in the afternoon)
	<ol>
		<li>Distribute 20 &#956;L of cells per well in a 96-well mixing plate (U-bottom).</li>
		<li>Add 150 ng (~1.5 &#181;L) each of the pDEST22: TF plasmid, pDEST32&#916;DBD: TF plasmid and pEXP-AD502 plus pDEST32&#916;DBD: TF (normalization control) to the wells. 
		NOTE: Optimum results are expected with ~150 ng each of pDEST22: TF and pDEST32&#916;DBD: TF plasmid for successful co-transformation. Can vary with construct and plasmid expressions, thus need to be optimized with every experiment. Another control pDEST22-TF plus pDEST32deltaDBD can also be included at user&#8217;s discretion.</li>
		<li>Add 100 &#181;L of TE/ LiAc / polyethylene glycol (PEG) per well (mix three times). 
		NOTE: Please see the recipe for TE/LiAc/PEG in Table 1.</li>
		<li>Cover the plate with a breathable seal and incubate for 20 - 30 min (can be longer) at 30 &#176;C (no agitation necessary).</li>
		<li>Heat shock for 20 min (precisely) at 42 &#176;C.</li>
	</ol>
	</li>
	<li>Plate the cells.
	<ol>
		<li>Centrifuge for 5 min at 1000 x g and 21 &#176;C. Remove supernatant using a multichannel pipette. Add 110 &#956;L of TE and mix.</li>
		<li>Centrifuge for 5 min at 1000 x g and 21 &#176;C. Remove 100 &#956;L of the supernatant using a multichannel pipette. Re-suspend the pellet with the puncher.</li>
		<li>Transfer cells on SD-Trp-Leu agar plates. Incubate plates at 30 &#176;C until next step.</li>
	</ol>
	</li>
	<li>Day 5 (afternoon): Transfer the cells on new plates using puncher/microplate replicator.
	<ol>
		<li>Fill a sterile 96-well mixing plate (U-bottom) with 50 &#956;L of TE using a multichannel pipette. Pre-wet the puncher in TE. 
		NOTE: The puncher or the microplate replicator should be sterilized before this step and subsequently later in the protocol. The common way of sterilizing puncher such as immersing it in ethanol (100%; not molecular biology grade) and then burning it to the flame can be applied. Wait for 1 min to cool-off the puncher pins. 
		Caution: If performing the sterilization on the bench; make sure bench is pre-cleaned with ethanol and free of any combustible material around. Wear proper personal protective equipment (PPE).</li>
		<li>Punch colonies and re-suspend in TE-filled plate. 
		NOTE: If there is a smooth growth on the plate, colonies can be punched directly to the TE-filled plate or alternatively, single colony (in case of multiple colonies) should be picked using a sterilized toothpick to the TE filled plate and subsequently puncher can be used for the next step.</li>
		<li>Transfer them on new SD-Trp-Leu agar plates for optimal surface coverage. Incubate plates at 30 &#176;C until the next step.</li>
	</ol>
	</li>
	<li>Day 7 (afternoon): Start culture to measure &#946;-galactosidase activity.
	<ol>
		<li>Fill a sterile 96-well mixing plate (U-bottom) with 50 &#956;L of SD-Trp-Leu media using a multichannel pipette.</li>
		<li>Fill a sterile 96-deep well block with 180 &#956;L of SD-Trp-Leu media using a multichannel pipette.</li>
		<li>Pre-wet the puncher in media and transfer colonies from plate to the 50 &#956;L of media.</li>
		<li>Transfer 20 &#956;L of yeast to the 180 &#956;L of SD-Trp-Leu media and seal the block with a breathable seal. Incubate for 36 h at 30 &#176;C shaker.</li>
	</ol>
	</li>
	<li>Day 9 (morning): Start the culture for enzymatic measurement.
	<ol>
		<li>Transfer 100 &#956;L of culture to 500 &#956;L of YPDA media in a sterilized 96 deep well block.</li>
		<li>Cover the sterilized deep well plate with a breathable seal and incubate for 3&#160;- 5 h at 30 &#176;C with agitation at 200 rpm (until OD600 0.3-0.6).</li>
		<li>Re-suspend culture and transfer 125 &#956;L using a multichannel pipette on a spectrophotometer plate (measure OD600). 
		NOTE: Caution: Do not dispense the last drop to avoid bubbles, if the OD600 is below 0.3 - 0.6, incubate the remaining cells for 1 - 2 h more based on the reading. The 125 &#956;L culture used at this step can be discarded or transferred back to the original deep well block at user&#39;s discretion.</li>
		<li>Centrifuge the remaining cells in the deep well block for 10 min at 3000 x g and 21 &#176;C. Remove supernatant by inverting.</li>
		<li>Add 200 &#956;L of Z-buffer and vortex. 
		NOTE: Please see the recipe for Z-buffer in Table 1.</li>
		<li>Centrifuge for 5 min at 3000 x g and 21 &#176;C. Remove supernatant by inverting.</li>
		<li>Add 20 &#956;L Z-buffer and vortex.</li>
		<li>Cover the plate with sealing foil that can resist freeze thaw cycles. Perform 4 cycles of freeze/thaw using liquid nitrogen in a fume hood and then 42 &#186;C in a water bath (2 min each).</li>
		<li>Add 200 &#956;L Z-buffer/beta-mercaptoethanol (&#946;-ME)/Ortho-nitrophenyl-&#946;-galactoside (ONPG) (12 mL, 21 &#956;L, 8.4 mg respectively will yield 20 mL solution; always prepare fresh). 
		NOTE: ONPG will take some time to dissolve properly so prepare the solution an hour before reaching this step. ONPG serves as the substrate for measurement of &#946;-galactosidase activity.</li>
		<li>Incubate up to 17 - 24 h at 30 &#176;C (check in between, if color develops earlier proceed to the next step).</li>
	</ol>
	</li>
	<li>Day 10 (morning): Stop the reaction and measure.
	<ol>
		<li>Add 110 &#956;L 1M Na2CO3 to stop the reaction and record time.</li>
		<li>Vortex and centrifuge for 10 min at 3000 x g and 21 &#176;C.</li>
		<li>Take 125 &#956;L of supernatant using multichannel and measure OD420. 
		NOTE: Caution, do not dispense the last drop to avoid bubbles.</li>
		<li>Calculate &#946;-gal activity: 1000 x OD420 / (time (min) x volume (mL) x OD600 in a spreadsheet.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Saghbini, M., Hoekstra, D., Gautsch, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Media+formulations+for+various+two-hybrid+systems.">Media formulations for various two-hybrid systems.</a> Methods Mol Biol. 177, 15-39 (2001).</li><li>Reece-Hoyes, J. S., Marian 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=Yeast+one-hybrid+assays:+A+historical+and+technical+perspective.">Yeast one-hybrid assays: A historical and technical perspective.</a> Methods. 57 (4), 441-447 (2012).</li><li>Rigaut, 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=A+generic+protein+purification+method+for+protein+complex+characterization+and+proteome+exploration.">A generic protein purification method for protein complex characterization and proteome exploration.</a> Nat Biotechnol. 17 (10), 1030-1032 (1999).</li><li>Puig, O., 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+Tandem+Affinity+Purification+(TAP)+Method:+A+General+Procedure+of+Protein+Complex+Purification.">The Tandem Affinity Purification (TAP) Method: A General Procedure of Protein Complex Purification.</a> Methods. 24 (3), 218-229 (2001).</li><li>Gavin, A. -. 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=Functional+organization+of+the+yeast+proteome+by+systematic+analysis+of+protein+complexes.">Functional organization of the yeast proteome by systematic analysis of protein complexes.</a> Nature. 415 (6868), 141-147 (2002).</li><li>Tripathi, P., Carvallo, M., Hamilton, E. E., Preuss, S., Kay, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+B-BOX32+interacts+with+CONSTANS-LIKE3+to+regulate+flowering.">Arabidopsis B-BOX32 interacts with CONSTANS-LIKE3 to regulate flowering.</a> Proc Natl Acad Sci USA. 114 (1), 172-177 (2017).</li><li>Helfer, 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=LUX+ARRHYTHMO+encodes+a+nighttime+repressor+of+circadian+gene+expression+in+the+Arabidopsis+core+clock.">LUX ARRHYTHMO encodes a nighttime repressor of circadian gene expression in the Arabidopsis core clock.</a> Curr Biol : CB. 21 (2), 126-133 (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 Rep. 8 (2), 622-632 (2014).</li><li>Wanke, D., Harter, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Plant+Regulatory+DNA+sequences+by+the+Yeast-One-Hybrid+Assay.">Analysis of Plant Regulatory DNA sequences by the Yeast-One-Hybrid Assay.</a> Methods Mol Biol. 479, 291-309 (2009).</li><li>Klein, P., Dietz, K. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+DNA-Binding+Proteins+and+Protein-Protein+Interactions+by+Yeast+One-Hybrid+and+Yeast+Two-Hybrid+Screen.">Identification of DNA-Binding Proteins and Protein-Protein Interactions by Yeast One-Hybrid and Yeast Two-Hybrid Screen.</a> Methods Mol Biol. 639, 171-192 (2010).</li><li>Breton, G., Kay, S. A., Pruneda-Paz, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Arabidopsis+Transcriptional+Regulators+by+Yeast+One-Hybrid+Screens+Using+a+Transcription+Factor+ORFeome.">Identification of Arabidopsis Transcriptional Regulators by Yeast One-Hybrid Screens Using a Transcription Factor ORFeome.</a> Methods Mol Biol. 1398, 107-118 (2016).</li><li>Deplancke, B., Vermeirssen, V., Arda, H. E., Martinez, N. J., Walhout, A. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gateway-Compatible+Yeast+One-Hybrid+Screens.">Gateway-Compatible Yeast One-Hybrid Screens.</a> CSH Protoc. 2006 (5), (2006).</li><li>Deplancke, B., Dupuy, D., Vidal, M., Walhout, A. J. 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+gateway-compatible+yeast+one-hybrid+system.">A gateway-compatible yeast one-hybrid system.</a> Genome Res. 14 (10B), 2093-2101 (2004).</li></ol>

Authors declare no conflict of interest.

The existing Y1H standard procedure is suitable for identifying a single protein prey binding to its DNA bait. With various technical modifications, the existing system has been harnessed for defining transcriptional regulatory networks. However, TFs are known to function as a part of complexes involving two or more TFs or proteins, with only some of the TF capable of binding to the DNA. Proteins or TFs which possess only the protein-binding domains and lack the ability to bind to DNA are more likely to work in a complex...

In general, to understand protein-DNA interactions, the Y1H assay is the preferred system successfully used in a laboratory setting 1. The basic Y1H assay involves two components: a) a reporter construct with DNA of interest successfully cloned upstream of a gene encoding a reporter protein; and b) an expression construct which will generate a fusion protein between the TF of interest and a yeast transcription activation domain (AD). The DNA of interest is commonly referred to as &#39;bait&#39; while the fusion protein is known as &#39;prey&#39;. In past years, multiple versions of the Y1H assay have been developed to suit specific needs with their own advantages 2. The existing Y1H assay can be successfully implemented to identify and validate interaction of one protein at a time with its DNA bait, but lacks the capability to identify heterodimeric or multimeric protein-DNA interactions.
The method described here is a modified version of the existing Y1H enabling researchers to simultaneously study multiple proteins binding to their target DNA sequence(s). The goal of this assay is to express the protein of interest with an activation domain (pDEST22: TF) and evaluate the activation of the candidate DNA regions fused to a reporter in the presence and/or absence of the interacting protein partner (pDEST32&#916;DBD-TF) in the yeast system. This assay will allow us to determine whether the interaction between these two proteins is required for the activation of the target. This is a GATEWAY cloning compatible system and therefore feasible to use in a regular laboratory setting. Thisprotocol is based on direct transformation, which provides the ease of co-transformation of different TFs in a yeast system. Thus, it is an advantageous strategy to validate the protein complexes binding to their possible DNA targets for in vitro molecular and functional validation for any system. Current techniques such as Tandem affinity purification (TAP) methods are costly and labor intensive but allow detection of protein hetrocomplexes on a large scale 3,4,5. This modified yeast one-hybrid can be successfully performed in a regular lab setting on a small scale (Figure 1) and is also cost effective. The Y1.5H assay can facilitate researchers across the community to test and validate their hypothesis towards defining the role of multimeric protein complex binding to a common DNA target using a heterologous system. Based on the findings, the hypothesis could be validated in vivo in the preferred system of study. Recently, we have used this approach to define the role of a TF and its mode of action to regulate flowering in Arabidopsis 6.

<table><tbody><tr><td>pDEST22 vector</td><td>Invitrogen</td><td>PQ1000101</td><td>Pro-Quest Two hybrid system kit</td></tr><tr><td>pDEST32delatDBD</td><td>Invitrogen</td><td>PQ1000102</td><td>Pro-Quest Two hybrid system kit; this vecor is modified. It is pDEST32 minus DNA binding Domain</td></tr><tr><td>pENTR/D-TOPO Cloning Kit</td><td>Invitrogen</td><td>K240020</td><td></td></tr><tr><td>YM4271 Strain</td><td>Clontech</td><td>K1603-1</td><td>MATCHMAKER One-Hybrid System</td></tr><tr><td>pEXP-AD502</td><td>Invitrogen</td><td>PQ1000101</td><td>Pro-Quest Two hybrid system kit</td></tr><tr><td>GATEWAY LR Clonase II enzyme mix</td><td>Invitrogen</td><td>11791020</td><td></td></tr><tr><td>YPDA media</td><td>Clontech</td><td>630410</td><td></td></tr><tr><td>SD-Agar</td><td>Clontech</td><td>630412</td><td></td></tr><tr><td>SD minimal media</td><td>Clontech</td><td>630411</td><td></td></tr><tr><td>Uracil DO Supplement</td><td>Clontech</td><td>630416</td><td></td></tr><tr><td>Tryptophan Do Supplement</td><td>Clontech</td><td>630413</td><td></td></tr><tr><td>Tris Base</td><td>Fisher Scientific</td><td>BP152-1</td><td></td></tr><tr><td>EDTA</td><td>Fisher Scientific</td><td>S311-500</td><td></td></tr><tr><td>LiAc</td><td>Sigma-Aldrich</td><td>L4158</td><td></td></tr><tr><td>Saplmon Sperm (10mg/ml)</td><td>Invitrogen</td><td>15632011</td><td></td></tr><tr><td>96 well round bottom Plate</td><td>Greiner bio-one</td><td>650101</td><td></td></tr><tr><td>PEG3350</td><td>Sigma-Aldrich</td><td>1546547</td><td></td></tr><tr><td>96-deep well block</td><td>USA Scientific</td><td>1896-2000</td><td></td></tr><tr><td>Sealable Foil</td><td>USA Scientific</td><td>2923-0110</td><td></td></tr><tr><td>Araseal</td><td>Excel Scientific</td><td>B-100</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Fisher Scientific</td><td>034461-100</td><td></td></tr><tr><td>ONPG</td><td>Sigma-Aldrich</td><td>73660</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>223484</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S3264</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S3139</td><td></td></tr><tr><td>KCL</td><td>Fisher Scientific</td><td>BP366-500</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>83266</td><td></td></tr><tr><td>HCL</td><td>Fisher Scientific</td><td>SA54-4</td><td></td></tr><tr><td>Drybath</td><td>Thermo Fischer</td><td></td><td></td></tr><tr><td>Voretx</td><td>Thermo Fischer</td><td></td><td></td></tr><tr><td>Centrifgue</td><td>Eppendorf</td><td>Centrifuge 5810R</td><td></td></tr><tr><td>Plate Reader</td><td>Molecular Device</td><td></td><td>SPECTRAMAX PLUS Microplate Spectrophotometer</td></tr><tr><td>Incubator</td><td>Thermo Fisher</td><td>Model No. 5250- 37 Degree, 6250-30 degrees</td><td></td></tr><tr><td>Shaker Incubator</td><td>New Brunswick</td><td></td><td></td></tr><tr><td>Water Bath</td><td>Thermo Fisher</td><td>IsoTemp 205</td><td></td></tr><tr><td>Puncher</td><td></td><td></td><td></td></tr><tr><td>50 ml Falocn</td><td>BD Falcon</td><td></td><td></td></tr><tr><td>Beaker</td><td>Nalgene</td><td></td><td></td></tr><tr><td>Flask</td><td>Nalgene</td><td></td><td></td></tr><tr><td>Petriplates (150mm)</td><td>Greiner bio-one</td><td></td><td></td></tr></tbody></table>

a modified yeast-one hybrid system for heteromeric protein complex-dna interaction studies

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

The general plate set-up procedure for the co-transformation of DNA regions in the yeast cells with protein of interest (Figure 2). The plate set-up can be modified according to the need of the experiment and number of DNA regions/fragments tested. After day-5 of the protocol a well-grown and positive plate should be visible as in Figure 3. In our study, the promoter region of 2600 bp upstream from the start of the transcription ...

Watch this Scientific Journal Video about A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies at JoVE.com

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

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

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

a-modified-yeast-one-hybrid-system-for-heteromeric-protein-complex

Research

JoVE Journal

Biochemistry

11.3K Views.  University of Southern California.  De gemodificeerde gist-een-hybride analyse die hier wordt beschreven is een uitbreiding van de klassieke gist-hybride (Y1H) -assay om de heteromeer eiwitcomplex-DNA-interactie te onderzoeken en valideren in een heterologe systeem voor elke functionele genomica-studie. 

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

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

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

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

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

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

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.

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 ...

Genetics

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Biology

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

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

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

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

Immunology and Infection

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...

Biomolecular Interaction Studies Using Yeast Two-Hybrid Technique 

Isolating Interaction-Null&#47;Impaired Mutants Using the Yeast Two-Hybrid Assay

Protein-protein interactions are one of the most basic processes that underlie biological phenomena. One of the simplest and best ways to understand the role(s) and function(s) of a specific protein-protein interaction is to compare the phenotype of the wild-type (with the relevant protein-protein interaction) and those of mutants that lack the relevant interaction. Therefore, if such mutants can be isolated, they will help to elucidate the related biological processes. The yeast two-hybrid (Y2H) procedure is a powerful approach not only to detect protein-protein interactions but also to isolate interaction-null/impaired mutants. In this article, a protocol is presented to isolate interaction-null/impaired mutants using Y2H technology. First, a mutation library is constructed by combining the polymerase chain reaction and efficient seamless cloning technology, which efficiently excludes the empty vector from the library. Second, interaction-null/impaired mutants are screened by the Y2H assay. Because of a trick in the Y2H vector, undesired mutants, such as those with frameshift and nonsense mutations, are efficiently eliminated from the screening process. This strategy is simple and can, therefore, be applied to any combination of proteins whose interaction can be detected by the two-hybrid system.

Author Spotlight: Enhanced Isolation of Interaction-Null Mutants in Yeast

The interactions of biomolecules, such as protein-protein interactions, are the molecular basis of biological functions. If interaction-null/impaired mutants that specifically lack the relevant interaction can be isolated, they will greatly help to understand the function(s) of this interaction. This article presents an efficient way to isolate interaction-null/impaired mutants.

Protein-protein interactions are one of the most basic processes that underlie biological phenomena. One of the simplest and best ways to understand the ...

Modified Yeast-One Hybrid Assay to Detect Heteromeric Protein Complex-DNA Interactions

Source: Tripathi, P. et al., <a href="https://app.jove.com/v/56080">A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies.</a> J. Vis. Exp. (2017)
In this video, we demonstrate the modified yeast one-hybrid assay, which detects multiple proteins interacting with target DNA. The transformed yeast cells contain the target DNA sequence upstream of the reporter gene. Additionally, they express transcription factor proteins. When the proteins form complexes and bind to the target DNA, the reporter gene is expressed and can be detected enzymatically.

Source: Tripathi, P. et al., A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies. J. Vis. Exp. (2017)
In this ...

JoVE Encyclopedia of Experiments

Biological Techniques

Biomolecular Interaction Detection Techniques

Protein-nucleic acid interactions

Yeast Two-Hybrid Assay to Determine Protein Self-Association in Yeast Cells

Source: Stanczyk, P. J., et al., <a href="https://app.jove.com/v/54271">Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study.</a> J. Vis. Exp. (2016)
This video demonstrates the yeast two-hybrid assay to detect specific protein self-interactions. In this assay, the protein of interest is tagged to two domains of a transcription factor that encodes the &#946;-galactosidase enzyme. When the two proteins interact, this causes transcription and translation to form the enzyme, which is then detected using a biochemical assay.

Source: Stanczyk, P. J., et al., Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor ...

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

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