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

Summary

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.

Abstract

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.

Introduction

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.

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Protocol

1. Construct and Reporter Plasmid Preparation

1. PCR amplify the promoter regions / "fragments" (preferably ~250 - 500 bp) of the target DNA to be analyzed for further cloning into the entry vector using E. coli (TOP10).
2. Upon sequencing confirmation, subclone the positive clone in a compatible pGLacZi expression vector ⁷ as previously described ^8,9,10,11.
3. 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 β-galactosidase activity as previously described ⁸.
4. Clone the TF or protein of interest and subsequently clone the interacting protein partner into pDEST22 and pDEST32ΔDBD (pDEST32 minus DNA Binding Domain) expression vectors. The transformed yeast promoter fragments and the TF clones are subsequently used further in the protocol.

2. Modified Y1.5H Protocol

1. 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 °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.
2. On day 2 (afternoon), inoculate 10 mL of YPDA media with 500 μL of the overnight culture and incubate overnight in a 30 °C shaker.
3. Day 3 (early in the morning and whole afternoon): Prepare competent cells (early in the morning).
   1. Inoculate 100 mL of YPDA media with the 2 mL of the overnight culture and incubate in a 30 °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.
   2. Centrifuge for 5 min at 1000 x g and 21 °C. Discard the supernatant.
   3. Reconstitute pellets in 10 mL of sterile water using a vortex or pipetting up and down.
   4. Centrifuge for 5 min at 1000 x g and 21 °C. Discard the supernatant.
   5. 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.
   6. Centrifuge for 5 min 1000 x g and 21°C. Discard the supernatant.
   7. Re-suspend pellet in 4 mL of TE/LiAc + 400 μL of salmon sperm DNA (10 mg/mL) pipetting up and down and proceed to the next step.
4. Co-transformation: Heat shock the cells (late in the afternoon)
   1. Distribute 20 μL of cells per well in a 96-well mixing plate (U-bottom).
   2. Add 150 ng (~1.5 µL) each of the pDEST22: TF plasmid, pDEST32ΔDBD: TF plasmid and pEXP-AD502 plus pDEST32ΔDBD: TF (normalization control) to the wells.
      NOTE: Optimum results are expected with ~150 ng each of pDEST22: TF and pDEST32ΔDBD: 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’s discretion.
   3. Add 100 µL of TE/ LiAc / polyethylene glycol (PEG) per well (mix three times).
      NOTE: Please see the recipe for TE/LiAc/PEG in Table 1.
   4. Cover the plate with a breathable seal and incubate for 20 - 30 min (can be longer) at 30 °C (no agitation necessary).
   5. Heat shock for 20 min (precisely) at 42 °C.
5. Plate the cells.
   1. Centrifuge for 5 min at 1000 x g and 21 °C. Remove supernatant using a multichannel pipette. Add 110 μL of TE and mix.
   2. Centrifuge for 5 min at 1000 x g and 21 °C. Remove 100 μL of the supernatant using a multichannel pipette. Re-suspend the pellet with the puncher.
   3. Transfer cells on SD-Trp-Leu agar plates. Incubate plates at 30 °C until next step.
6. Day 5 (afternoon): Transfer the cells on new plates using puncher/microplate replicator.
   1. Fill a sterile 96-well mixing plate (U-bottom) with 50 μL 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).
   2. 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.
   3. Transfer them on new SD-Trp-Leu agar plates for optimal surface coverage. Incubate plates at 30 °C until the next step.
7. Day 7 (afternoon): Start culture to measure β-galactosidase activity.
   1. Fill a sterile 96-well mixing plate (U-bottom) with 50 μL of SD-Trp-Leu media using a multichannel pipette.
   2. Fill a sterile 96-deep well block with 180 μL of SD-Trp-Leu media using a multichannel pipette.
   3. Pre-wet the puncher in media and transfer colonies from plate to the 50 μL of media.
   4. Transfer 20 μL of yeast to the 180 μL of SD-Trp-Leu media and seal the block with a breathable seal. Incubate for 36 h at 30 °C shaker.
8. Day 9 (morning): Start the culture for enzymatic measurement.
   1. Transfer 100 μL of culture to 500 μL of YPDA media in a sterilized 96 deep well block.
   2. Cover the sterilized deep well plate with a breathable seal and incubate for 3 - 5 h at 30 °C with agitation at 200 rpm (until OD₆₀₀ 0.3-0.6).
   3. Re-suspend culture and transfer 125 μL 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 μL culture used at this step can be discarded or transferred back to the original deep well block at user's discretion.
   4. Centrifuge the remaining cells in the deep well block for 10 min at 3000 x g and 21 °C. Remove supernatant by inverting.
   5. Add 200 μL of Z-buffer and vortex.
      NOTE: Please see the recipe for Z-buffer in Table 1.
   6. Centrifuge for 5 min at 3000 x g and 21 °C. Remove supernatant by inverting.
   7. Add 20 μL Z-buffer and vortex.
   8. 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 ºC in a water bath (2 min each).
   9. Add 200 μL Z-buffer/beta-mercaptoethanol (β-ME)/Ortho-nitrophenyl-β-galactoside (ONPG) (12 mL, 21 μL, 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 β-galactosidase activity.
   10. Incubate up to 17 - 24 h at 30 °C (check in between, if color develops earlier proceed to the next step).
9. Day 10 (morning): Stop the reaction and measure.
   1. Add 110 μL 1M Na₂CO₃ to stop the reaction and record time.
   2. Vortex and centrifuge for 10 min at 3000 x g and 21 °C.
   3. Take 125 μL of supernatant using multichannel and measure OD₄₂₀.
      NOTE: Caution, do not dispense the last drop to avoid bubbles.
   4. Calculate β-gal activity: 1000 x OD₄₂₀ / (time (min) x volume (mL) x OD₆₀₀ in a spreadsheet.

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Results

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 ...

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Discussion

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...

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Materials

Name	Company	Catalog Number	Comments
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