Name: a modified yeast-one hybrid system for heteromeric protein complex-dna interaction studies
Uploaded: 2017-07-24T15:00:00
Description: Watch this Scientific Journal Video about A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies at JoVE.com

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,<sup cl...

<ol>
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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.

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		<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>
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		<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>
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			<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>
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			<td height="21" style="height:21px;width:225px;">YPDA media</td>
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			<td style="width:132px;">Clontech</td>
			<td style="width:157px;">630412</td>
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		<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>
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			<td style="width:157px;">BP152-1</td>
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			<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>
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			<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>
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		<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>
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			<td height="21" style="height:21px;width:225px;">96 well round bottom Plate</td>
			<td>Greiner bio-one</td>
			<td align="right">650101</td>
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			<td style="width:132px;">Sigma-Aldrich</td>
			<td style="width:157px;">1546547</td>
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			<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>
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			<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>
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			<td height="21" style="height:21px;width:225px;">Araseal</td>
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			<td style="width:157px;">B-100</td>
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		<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>
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			<td style="width:157px;">S3264</td>
			<td></td>
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			<td height="25" style="height:25px;">NaH2PO4&#160;</td>
			<td style="width:132px;">Sigma-Aldrich</td>
			<td style="width:157px;">S3139</td>
			<td></td>
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			<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>
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			<td height="25" style="height:25px;">MgSO4&#160;</td>
			<td style="width:132px;">Sigma-Aldrich</td>
			<td style="width:157px;">83266</td>
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			<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>
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		<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>
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		<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>
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		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Centrifgue</td>
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			<td style="width:157px;">Centrifuge 5810R</td>
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			<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>
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			<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>
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			<td height="21" style="height:21px;width:225px;">Puncher</td>
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			<td height="21" style="height:21px;width:225px;">50 ml Falocn</td>
			<td style="width:132px;">BD Falcon</td>
			<td style="width:157px;"></td>
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			<td height="21" style="height:21px;width:225px;">Beaker</td>
			<td style="width:132px;">Nalgene</td>
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			<td></td>
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		<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>
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		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Petriplates (150mm)</td>
			<td style="width:132px;">Greiner bio-one</td>
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	</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 ...

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