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 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.
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 'bait' while the fusion protein is known as 'prey'. 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 (pDEST32ΔDBD-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.
1. Construct and Reporter Plasmid Preparation
2. Modified Y1.5H Protocol
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 start site was segmented into six regions or fragments (FR 1-6) 6. In the representative figure (Figure 3), DNA regions 1 and 4 show positive interaction with the protein A and B complex. Upon the measurement of β-galactosidase activity (Supplemental Table 1) only fragment-1 shows four fold induction of the reporter gene activity while the fragment-4 did not pass our internal threshold of ≥ two fold.
Figure 1: An overview of the modified yeast-one hybrid (Y1.5H) assay. The layout describes step-by-step procedure of the assay. Please click here to view a larger version of this figure.
Figure 2: A simplified representation of the plate set-up for the co-transformation of the yeast cells with protein (TF) of interest. In a general experimental set-up, the plate can be arranged as shown in the figure. The combination of constructs and controls can be modified as per the need and is completely at user's discretion.
Figure 3: A representative image of the positive plate (SD-Trp-Leu) for replicate 1 after successful co-transformation. Similar result should be observed with more replicates. Fragment 1-6 represents DNA region; No Fragment is a negative control.
TE buffer 10X (10ml) | |
Desirable | From the stock |
100mM Tris-Cl (pH8.0 | 1mL of Tris 1M |
10mM EDTA (pH8.0) | 200uL of EDTA 0.5M |
Make up volume with water | |
TE/LiAc (10ml) | |
Desirable | From the stock |
1X | 1mL TE 10X |
0.1M LiAc | 1mL LiAc 1M |
Make up volume with water | |
TE/LiAc/PEG (50 mL) | |
Desirable | From the stock |
1X | 5 mL TE 10X |
0.1M LiAc | 5 mL LiAc 1M |
40 mL PEG3350 50% | |
Z-buffer (1L) pH 7.0 | |
Na2HPO4 16.1g of heptahydrated or 8.52g of anhydrous | |
NaH2PO4 5.5g of hydrated or 4g of anhydrous | |
KCl 0.75g | |
MgSO4 0.246g of hydrated or 0.12g of anhydrous | |
Maintain pH with HCL | |
Note: | |
All soultions are sterlized before use. | |
Media and agar plates used in the protocol (YPDA, SD-Ura; SD-Trp-Ura and SD-Trp-Ura agar plates) follow same recipe as in Y1H |
Table 1: The recipe for solutions used in the protocol. The table provides the recipe for the stock and working solutions of the major buffers used in the assay.
Supplemental Table 1: β-galactosidase activity measurement. The spreadsheet sheet presented here can be used as a template for the measurement of β-galactosidase activity using the formula given in the protocol. The combination of constructs can be modified as per the user's discretion. Please click here to download this file.
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, preferably with a TF that is capable of binding to the DNA. High-throughput screens using the traditional Y1H occasionally miss those biologically important interactions. Thus, it is imperative to adapt the Y1H assay to detect heteromeric protein-DNA interactions.
The method presented here is one such adaptation with the capability to detect protein-DNA interactions involving more than one protein or TF. The unique feature of the described method is the transformation of the yeast via co-transformation, which allows integration of different TFs in a yeast system and further applied to test the binding of the complex to the target DNA regions. A significant merit of this method is its ability to test two proteins with their DNA bait. This method does not provide the information of specific target (promoter) sites but would provide information for the DNA regions. At the same time, use of this method is advised only after the confirmation of the proposed protein-protein interaction. Based on the findings, further experiments such as electrophoretic mobility shift assay (EMSA) or Chromatin-immunoprecipitation (ChIP) assay or other assays suitable for the system of study should be performed to identify the target sites, preferably in vivo if desirable. The application of ONPG based measurement of β-galactosidase activity provides better confirmation than the classical qualitative X-gal assay. However, the overall experimental requirements and the properties of the protein(s) to be tested should be taken into consideration as needed. The implementation of the described method for the large-scale screen, using more than two protein partners with different DNA targets needs to be tested. The Y1.5H assay is a cost-effective method, which can be performed on a small scale in a regular laboratory setting. Perhaps, this assay could be performed with use of other vector systems but the protocol needs to be optimized if not using the gateway-compatible cloning system.
The presented protocol for the Y1.5H is an advantageous strategy to validate protein complex(s) with their DNA bait for any system of study; plants or mammals. This method is very useful for studying plant genomics where in vivo validation could be challenging given the level of ploidy of the plant and time and labor intensive transformation procedures. Thus, the use of this method can accelerate the hypothesis testing at least in a heterologous system.
The authors have nothing to disclose.
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.
pDEST22 vector | Invitrogen | PQ1000101 | Pro-Quest Two hybrid system kit |
pDEST32delatDBD | Invitrogen | PQ1000102 | Pro-Quest Two hybrid system kit; this vecor is modified. It is pDEST32 minus DNA binding Domain |
pENTR/D-TOPO Cloning Kit | Invitrogen | K240020 | |
YM4271 Strain | Clontech | K1603-1 | MATCHMAKER One-Hybrid System |
pEXP-AD502 | Invitrogen | PQ1000101 | Pro-Quest Two hybrid system kit |
GATEWAY LR Clonase II enzyme mix | Invitrogen | 11791020 | |
YPDA media | Clontech | 630410 | |
SD-Agar | Clontech | 630412 | |
SD minimal media | Clontech | 630411 | |
Uracil DO Supplement | Clontech | 630416 | |
Tryptophan Do Supplement | Clontech | 630413 | |
Tris Base | Fisher Scientific | BP152-1 | |
EDTA | Fisher Scientific | S311-500 | |
LiAc | Sigma-Aldrich | L4158 | |
Saplmon Sperm (10mg/ml) | Invitrogen | 15632011 | |
96 well round bottom Plate | Greiner bio-one | 650101 | |
PEG3350 | Sigma-Aldrich | 1546547 | |
96-deep well block | USA Scientific | 1896-2000 | |
Sealable Foil | USA Scientific | 2923-0110 | |
Araseal | Excel Scientific | B-100 | |
2-mercaptoethanol | Fisher Scientific | 034461-100 | |
ONPG | Sigma-Aldrich | 73660 | |
Na2CO3 | Sigma-Aldrich | 223484 | |
Na2HPO4 | Sigma-Aldrich | S3264 | |
NaH2PO4 | Sigma-Aldrich | S3139 | |
KCL | Fisher Scientific | BP366-500 | |
MgSO4 | Sigma-Aldrich | 83266 | |
HCL | Fisher Scientific | SA54-4 | |
Drybath | Thermo Fischer | ||
Voretx | Thermo Fischer | ||
Centrifgue | Eppendorf | Centrifuge 5810R | |
Plate Reader | Molecular Device | SPECTRAMAX PLUS Microplate Spectrophotometer | |
Incubator | Thermo Fisher | Model No. 5250- 37 Degree, 6250-30 degrees | |
Shaker Incubator | New Brunswick | ||
Water Bath | Thermo Fisher | IsoTemp 205 | |
Puncher | |||
50 ml Falocn | BD Falcon | ||
Beaker | Nalgene | ||
Flask | Nalgene | ||
Petriplates (150mm) | Greiner bio-one |