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. 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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 border="0" cellpadding="0" cellspacing="0" width="846">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">pDEST22 vector</td>
			<td style="width:132px;">Invitrogen</td>
			<td style="width:157px;">PQ1000101</td>
			<td style="width:332px;">Pro-Quest Two hybrid system kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">pDEST32delatDBD</td>
			<td style="width:132px;">Invitrogen</td>
			<td style="width:157px;">PQ1000102</td>
			<td>Pro-Quest Two hybrid system kit; this vecor is modified. It is pDEST32 minus DNA binding Domain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">pENTR/D-TOPO Cloning Kit</td>
			<td style="width:132px;">Invitrogen</td>
			<td style="width:157px;">K240020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">YM4271 Strain</td>
			<td style="width:132px;">Clontech</td>
			<td style="width:157px;">K1603-1</td>
			<td>MATCHMAKER One-Hybrid System</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">pEXP-AD502</td>
			<td style="width:132px;">Invitrogen</td>
			<td style="width:157px;">PQ1000101</td>
			<td>Pro-Quest Two hybrid system kit</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">GATEWAY LR Clonase II enzyme mix</td>
			<td style="width:132px;">Invitrogen</td>
			<td style="width:157px;">11791020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">YPDA media</td>
			<td style="width:132px;">Clontech</td>
			<td style="width:157px;">630410</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">SD-Agar</td>
			<td style="width:132px;">Clontech</td>
			<td style="width:157px;">630412</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">SD minimal media</td>
			<td style="width:132px;">Clontech</td>
			<td style="width:157px;">630411</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Uracil DO Supplement</td>
			<td style="width:132px;">Clontech</td>
			<td style="width:157px;">630416</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Tryptophan Do Supplement</td>
			<td style="width:132px;">Clontech</td>
			<td style="width:157px;">630413</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Tris Base</td>
			<td style="width:132px;">Fisher Scientific</td>
			<td style="width:157px;">BP152-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">EDTA</td>
			<td style="width:132px;">Fisher Scientific</td>
			<td style="width:157px;">S311-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">LiAc</td>
			<td style="width:132px;">Sigma-Aldrich</td>
			<td style="width:157px;">L4158</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Saplmon Sperm (10mg/ml)</td>
			<td style="width:132px;">Invitrogen</td>
			<td style="width:157px;">15632011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">96 well round bottom Plate</td>
			<td>Greiner bio-one</td>
			<td align="right">650101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">PEG3350</td>
			<td style="width:132px;">Sigma-Aldrich</td>
			<td style="width:157px;">1546547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">96-deep well block</td>
			<td style="width:132px;">USA Scientific</td>
			<td style="width:157px;">1896-2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Sealable Foil</td>
			<td style="width:132px;">USA Scientific</td>
			<td style="width:157px;">2923-0110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Araseal</td>
			<td style="width:132px;">Excel Scientific</td>
			<td style="width:157px;">B-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">2-mercaptoethanol</td>
			<td style="width:132px;">Fisher Scientific</td>
			<td style="width:157px;">034461-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">ONPG</td>
			<td style="width:132px;">Sigma-Aldrich</td>
			<td style="width:157px;">73660</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2CO3</td>
			<td style="width:132px;">Sigma-Aldrich</td>
			<td style="width:157px;">223484</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2HPO4&#160;</td>
			<td style="width:132px;">Sigma-Aldrich</td>
			<td style="width:157px;">S3264</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NaH2PO4&#160;</td>
			<td style="width:132px;">Sigma-Aldrich</td>
			<td style="width:157px;">S3139</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">KCL</td>
			<td style="width:132px;">Fisher Scientific</td>
			<td style="width:157px;">BP366-500</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MgSO4&#160;</td>
			<td style="width:132px;">Sigma-Aldrich</td>
			<td style="width:157px;">83266</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">HCL</td>
			<td style="width:132px;">Fisher Scientific</td>
			<td style="width:157px;">SA54-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Drybath</td>
			<td style="width:132px;">Thermo Fischer</td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Voretx</td>
			<td style="width:132px;">Thermo Fischer</td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Centrifgue</td>
			<td style="width:132px;">Eppendorf</td>
			<td style="width:157px;">Centrifuge 5810R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Plate Reader</td>
			<td style="width:132px;">Molecular Device</td>
			<td style="width:157px;"></td>
			<td>SPECTRAMAX PLUS Microplate Spectrophotometer</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:225px;">Incubator</td>
			<td style="width:132px;">Thermo Fisher</td>
			<td style="width:157px;">Model No. 5250- 37 Degree, 6250-30 degrees</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Shaker Incubator</td>
			<td style="width:132px;">New Brunswick</td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Water Bath</td>
			<td style="width:132px;">Thermo Fisher</td>
			<td style="width:157px;">IsoTemp 205</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Puncher</td>
			<td style="width:132px;"></td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">50 ml Falocn</td>
			<td style="width:132px;">BD Falcon</td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Beaker</td>
			<td style="width:132px;">Nalgene</td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Flask</td>
			<td style="width:132px;">Nalgene</td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Petriplates (150mm)</td>
			<td style="width:132px;">Greiner bio-one</td>
			<td style="width:157px;"></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. 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.

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 ...

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG

To overcome safety restrictions and regulations when studying genes and proteins from true pathogens, their homologues can be studied. Bacillus anthracis is an obligate pathogen that causes fatal inhalational anthrax. Bacillus cereus is considered a useful model for studying B. anthracis due to its close evolutionary relationship. The gene cluster ba1554 - ba1558 of B. anthracis is highly conserved with the bc1531- bc1535 cluster in B. cereus, as well as with the bt1364-bt1368 cluster in Bacillus thuringiensis, indicating the critical role of the associated genes in the Bacillus genus. This manuscript describes methods to prepare and characterize a protein product of the first gene (ba1554) from the gene cluster in B. anthracis using a recombinant protein of its ortholog in B. cereus, bc1531.

Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG

Bacillus anthracis is the obligate pathogen of fatal inhalational anthrax, so studies of its genes and proteins are strictly regulated. An alternative approach is to study orthologous genes. We describe biophysicochemical studies of a B. anthracis ortholog in B. cereus, bc1531, requiring minimal experimental equipment and lacking serious safety concerns.

To overcome safety restrictions and regulations when studying genes and proteins from true pathogens, their homologues can be studied. Bacillus anthracis ...

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 ...

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

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

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

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

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two &#963; factors, namely &#963;66 and &#963;28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the dissection and manipulation of biological pathways. Currently, only a limited number of chemically induced dimerization (CID) systems exist and engineering new ones with desired sensitivity and selectivity for specific small-molecule ligands remains a challenge in the field of protein engineering. We here describe a high throughput screening method, combinatorial binders-enabled selection of CID (COMBINES-CID), for the de novo engineering of CID systems applicable to a large variety of ligands. This method uses the two-step selection of a phage-displayed combinatorial nanobody library to obtain 1) &#34;anchor binders&#34; that first bind to a ligand of interest and then 2) &#34;dimerization binders&#34; that only bind to anchor binder-ligand complexes. To select anchor binders, a combinatorial library of over 109 complementarity-determining region (CDR)-randomized nanobodies is screened with a biotinylated ligand and hits are validated with the unlabeled ligand by bio-layer interferometry (BLI). To obtain dimerization binders, the nanobody library is screened with anchor binder-ligand complexes as targets for positive screening and the unbound anchor binders for negative screening. COMBINES-CID is broadly applicable to select CID binders with other immunoglobulin, non-immunoglobulin, or computationally designed scaffolds to create biosensors for in vitro and in vivo detection of drugs, metabolites, signaling molecules, etc.

Creating chemically induced protein dimerization systems with desired affinity and specificity for any given small molecule ligand would have many biological sensing and actuation applications. Here, we describe an efficient, generalizable method for de novo engineering of chemically induced dimerization systems via the stepwise selection of a phage-displayed combinatorial single-domain antibody library.

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the ...

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, and protein-protein interactions. FPOP utilizes a KrF excimer laser at 248 nm for photolysis of hydrogen peroxide to generate hydroxyl radicals which in turn oxidatively modify solvent-accessible amino acid side chains. Recently, we expanded the use of FPOP of in vivo oxidative labeling in Caenorhabditis elegans (C. elegans), entitled IV-FPOP. The transparent nematodes have been used as model systems for many human diseases. Structural studies in C. elegans by IV-FPOP is feasible because of the animal&#8217;s ability to uptake hydrogen peroxide, their transparency to laser irradiation at 248 nm, and the irreversible nature of the modification. The assembly of a microfluidic flow system for IV-FPOP labeling, IV-FPOP parameters, protein extraction, and LC-MS/MS optimized parameters are described herein.

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

In vivo fast photochemical oxidation of proteins (IV-FPOP) is a hydroxyl radical protein footprinting technique that allows for mapping of protein structure in their native environment. This protocol describes the assembly and set-up of the IV-FPOP microfluidic flow system.

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, ...

Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using &#967;CRAC

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be bound by many different RBPs that will regulate its production, stability, activity, and degradation. As such, much has been done to understand the dynamics that exist between these two types of molecules. A particularly important breakthrough came with the emergence of &#8216;cross-linking and immunoprecipitation&#8217; (CLIP). This technique allowed stringent investigation into which RNAs are bound by a particular RBP. In short, the protein of interest is UV cross-linked to its RNA substrates in vivo, purified under highly stringent conditions, and then the RNAs covalently cross-linked to the protein are converted into cDNA libraries and sequenced. Since its conception, many derivative techniques have been developed in order to make CLIP amenable to particular fields of study. However, cross-linking using ultraviolet light is notoriously inefficient. This results in extended exposure times that make the temporal study of RBP-RNA interactions impossible. To overcome this issue, we recently designed and built much-improved UV irradiation and cell harvesting devices. Using these new tools, we developed a protocol for time-resolved analyses of RBP-RNA interactions in living cells at high temporal resolution: Kinetic CRoss-linking and Analysis of cDNAs (&#967;CRAC). We recently used this technique to study the role of yeast RBPs in nutrient stress adaptation. This manuscript provides a detailed overview of the &#967;CRAC method and presents recent results obtained with the Nrd1 RBP.

Kinetic cross-linking and analysis of cDNA is a method that allows investigation of the dynamics of protein-RNA interactions in living cells at high temporal resolution. Here the protocol is described in detail, including the growth of yeast cells, UV cross-linking, harvesting, protein purification, and next generation sequencing library preparation steps.

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be ...

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

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

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG

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

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

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using χCRAC