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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

To identify novel regulators of transcription factors, we developed an approach to screen arrayed lentiviral or retroviral RNAi libraries using a dual-luciferase-based transcriptional reporter assay. This approach offers a quick and relatively inexpensive way to screen hundreds of candidates in a single experiment.

Abstract

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for anti-cancer therapies. However, directly targeting transcription factors is often difficult and can cause adverse side effects if the transcription factor is necessary in one or more adult tissues. Identifying upstream regulators that aberrantly activate transcription factors in cancer cells offers a more feasible alternative, particularly if these proteins are easy to drug. Here, we describe a protocol that can be used to combine arrayed medium-scale lentiviral libraries and a dual-luciferase-based transcriptional reporter assay to identify novel regulators of transcription factors in cancer cells. Our approach offers a quick, easy, and inexpensive way to test hundreds of genes in a single experiment. To demonstrate the use of this approach, we performed a screen of an arrayed lentiviral RNAi library containing several regulators of Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), two transcriptional co-activators that are the downstream effectors of the Hippo pathway. However, this approach could be modified to screen for regulators of virtually any transcription factor or co-factor and could also be used to screen CRISPR/CAS9, cDNA, or ORF libraries.

Introduction

The purpose of this assay is to use viral libraries to identify regulators of transcription factors in a relatively quick and inexpensive manner. Aberrant transcriptional activity is associated with cancer and metastasis1,2,3,4,5,6, so targeting transcription factors in cancer cells is a promising therapeutic approach. However, transcription factors are often difficult to target pharmacologically7 and many are required for normal cellular function in adult tissues8,9,10. Targeting the cancer-associated pathways that aberrantly activate transcription factors to drive disease is a more feasible approach with the potential to have less severe side effects. The commercial availability of arrayed lentiviral and retroviral RNAi, CRISPR/CAS9, cDNA, or ORF libraries allows researchers to test the importance of numerous genes in a single experiment. However, a reliable readout for altered transcriptional activity is required.

Here, we describe the use of a dual-luciferase-based transcriptional reporter assay and arrayed lentiviral libraries to identify proteins that regulate transcription factors in cancer cells. In this assay, shRNAs that target cancer-associated genes are delivered to mammalian cancer cells via lentiviral transduction and cells are selected for stable integration using puromycin. The cells are next transfected with a reporter construct that expresses firefly luciferase driven by a promoter specific to the transcription factor that is being investigated and a control construct that expresses Renilla luciferase from a constitutively active promoter that is not responsive to the transcription factor being investigated. We demonstrate this approach with a proof-of-concept screen for regulators of YAP and TAZ, the critical downstream effectors of the Hippo pathway8,10,11. Abnormal activity of YAP and TAZ promotes several steps of the metastatic cascade11 and is observed in many cancers11,12,13. However, how YAP and TAZ become aberrantly activated in some cancer cells is not yet fully understood. YAP and TAZ do not bind DNA, but instead are recruited to promoters by other transcription factors. Members of the TEA domain (TEAD) family of transcription factors are the major binding partners for YAP and TAZ, and are critical for most YAP and TAZ-dependent functions. Our reporter construct expresses firefly luciferase from a YAP/TAZ-TEAD-responsive promoter and previous studies have demonstrated that it faithfully detects changes in YAP-TEAD and TAZ-TEAD transcriptional activity2,14,15.

Our approach is rapid, medium-throughput, and does not require screening facilities, automated robots, or deep sequencing of pooled libraries. The costs are relatively low and there are numerous commercially available libraries to choose from. The required equipment and reagents are also relatively standard in most laboratories. It can be used to screen for regulators of virtually any transcription factor if a luciferase-based reporter exists or is generated. We use this approach to screen shRNAs in cancer cells, but any cell line that can be transfected with reasonable efficiency could be used with any type of arrayed library.

Protocol

NOTE: A schematic summary of this protocol is shown in Figure 1.

1. Lentiviral vector library preparation

NOTE: The demonstrated screen used an arrayed shRNA library purchased as glycerol stocks in 96-well plates, but libraries can also be assembled manually based on a list of candidates. Appropriate controls should be considered and included in any library. This includes a non-targeting control shRNA (shNTC), a control shRNA targeting the transcription factor being investigated, and if possible, an shRNA targeting firefly luciferase.

  1. Add 1.3 mL of Luria Broth (LB) (1% bacto-trypton, 0.5% yeast extract, 1% NaCl, pH 7.5) containing 100 µg/mL of ampicillin to each well of a 96-well deep well plate. Inoculate each well with 2 µL of glycerol stock and grow at 37 °C overnight with constant agitation at 225 rpm.
  2. Transfer each bacterial culture into a 1.5 mL centrifuge tube and pellet the bacteria by centrifugation at 21,000 x g at 4 °C for 10 min.
  3. Purify each vector using a bacterial mini-prep kit by following the manufacturer's protocol.
  4. Determine the concentration of each vector using a spectrophotometer.
  5. Store the plasmids at -20 °C.
    NOTE: The protocol can be paused here.

2. Packaging of the arrayed lentiviral library

NOTE: All work involving lentivirus, including packaging, infection, and subsequent culturing of infected cells should strictly follow the institutional biosafety rules and regulations.

  1. Expand the 293FT cells using complete growth media (Dulbecco's modified Eagle medium (DMEM) containing 4 mM L-glutamine, 4,500 mg/L glucose and sodium pyruvate, supplemented with 10% fetal bovine serum (FBS), 100 units/mL of penicillin, 100 µg/mL streptomycin antibiotic and 2 mM L-glutamine).
  2. For each vector in the library from step 1.4, seed one 24-well with 1 x 105 293FT cells.
    NOTE: It is recommended that some extra wells of a control viral vector be packaged and used to test the titer of the virus prior to proceeding to step 3 (see below).
  3. Incubate the cells at 37 °C with 5% CO2 for 24 h.
    NOTE: A general protocol for packaging lentivirus that was described previously14 has been scaled down to 24 wells for this protocol. It uses psPAX2 for lentiviral packaging and VSVG as a coat protein. If ectropic virus is desired, a vector delivering Eco can be used instead of VSVG. It is highly recommended that this protocol is optimized to achieve a viral titer that gives between 30%-70% infection efficiency of the target cells (see Discussion). See Supplemental Table 1 for a list of all vectors used.
  4. Set up a transfection mixture for each viral vector from Step 1.4 as described below. Each transfection should contain 250 ng of the viral vector, 125 ng of psPAX2, 125 ng of VSVG, 1.25 µL of transfection reagent 1, and 23.75 µL of transfection buffer (see Table of Materials).
    1. Dilute each lentiviral vector to 50 ng/µL with nuclease-free water, and then transfer 5 µL (250 ng) into a well of a 96-well PCR plate.
    2. Make the transfection super mix by mixing 1.25 µL * X of transfection reagent 1 and 23.75 µL * X of pre-warmed transfection buffer where "X" is the total number of transfections plus several extra to account for volume loss during pipetting.
    3. Incubate the transfection super mix at room temperature for 5 min.
    4. Add 125 ng * X of psPAX2 and 125 ng * X of VSVG to the tube of transfection super mix from Step 2.4.3 and gently pipet up and down to mix. Rapidly proceed to Step 2.4.5.
    5. Immediately aliquot the mixture from Step 2.4.4 into each tube of a PCR strip, and then use multi-channel pipette to transfer 25 µL the mixture into each well containing viral vector from Step 2.4.1.
    6. Incubate at room temperature for 20 min.
    7. Transfer all 30 µLs from each 96-well from Step 2.4.6 into a well of the 24-well containing 293 FT cells from Step 2.3.
  5. Incubate the cells at 37 °C with 5% CO2 for 24 h and then replace the media in every well of the 24-well plate with 500 µL of fresh complete growth media. Incubate the cells at 37 °C with 5% CO2 for another 24 h.
  6. Using a multi-channel pipette, collect the viral supernatant from each well and aliquot 220 µL (enough for 1 infection in Step 3 plus some extra volume) into two 96-wells each. These are the arrayed viral supernatant plates.
  7. Store the arrayed viral supernatant plates at -80 °C.
    NOTE: The protocol can be paused here. It is also recommended to test some of the extra control virus that was packaged (see above) on the cells to be infected before proceeding to Step 3. This is to ensure that the titer is sufficient to achieve at least 30% infection efficiency.

3. Infection of the cells for the screen

NOTE: Human melanoma cells (A375) were used to demonstrate this approach, but this method can be applied to any adherent cells that infect with lentivirus. However, cell culture and plating conditions should be optimized for each cell line (see Discussion).

  1. Expand the cells to be infected in complete growth media.
  2. Seed 24-well plates with 1 x 105 cells in 0.5 ml of complete growth media per well. Seed one well for each viral vector to be tested (including controls) and include an extra well that will not be infected which will serve as a control for drug selection in Step 3.7.
  3. Incubate the cells at 37 °C with 5% CO2 for 24 h.
  4. Infect each well from Step 3.2 with a different viral supernatant from the frozen arrayed lentiviral supernatant as follows.
    1. Prepare complete growth media that contains 20 µg/mL polybrene.
    2. Thaw the arrayed lentiviral library supernatants from Step 2.7 to room temperature.
    3. Aspirate the growth media from the 24-well plates from Step 3.2 and immediately add 200 µL of polybrene-containing growth media to each well.
    4. Using a multi-channel pipette, transfer 200 µL of viral supernatant from each 96-well from Step 3.4.2 to the 24 wells from Step 3.4.3.
  5. Incubate the cells at 37 °C with 5% CO2 for 24 - 48 h.
    NOTE: Some cell lines may require longer than 24 h to express the shRNAs and become puromycin resistant. The viral vector used here delivers a Turbo-GFP-IRES-puroR with a miR30-based shRNA in the 3'UTR of puroR (Figure 1). Infection efficiency and expression of the puromycin resistance gene and the shRNA were monitored by green fluorescent protein.
  6. Prepare complete growth media that contains 2.5 µg/mL puromycin.
    NOTE: The selection concentration of puromycin varies between cell lines. It is recommended that an antibiotic kill curve be performed for each cell line to be assayed prior to the screen.
  7. Aspirate the media from each well and replace with 500 µL of puromycin-containing complete growth media.
    NOTE: Be sure to also add puromycin to a control non-infected well that can be used in subsequent steps to ensure the puromycin selection is complete.
  8. Incubate the cells at 37 °C with 5% CO2 for 48 h.
    NOTE: It is best to select for 48 h, so plating density and viral titer should be optimized so that the cells are not overconfluent prior to 48 h.
  9. Ensure that the infected cells are green under the fluorescent microscope, and that cells on a control non-infected well treated with puromycin are all dead before proceeding to Step 4.

4. Seeding cells for transfection of dual-luciferase reporter

NOTE: A test transfection should be done to determine the optimal seeding density for each new cell line.

  1. Trypsinize each well from Step 3.9 and transfer roughly 1 x 105 cells into wells at the corresponding position on the new 24-well plate as follows.
    NOTE: This protocol is designed for the screening of libraries with hundreds of shRNAs so it is not feasible to count every well of infected cells. Therefore, the steps below were used to estimate cell numbers in each well to help ensure roughly equal plating density.
    1. Group the wells from Step 3.9 into 3 - 4 groups such that all wells in a group have a similar cell density.
    2. Trypsinize 1 representative well from each group with 200 µL of trypsin-EDTA (1x PBS supplemented with 0.5 mM EDTA and 0.1% trypsin) for 5 min at 37 °C. Then neutralize the trypsin-EDTA by adding 400 µL of puromycin containing complete growth media.
    3. Count each representative well to determine the total cell number and dilute the cell suspension from each representative well to 2 x 105 cells/mL of using complete growth media.
    4. Seed 0.5 mL (1 x 105 cells) of each well from Step 4.1.3 into the corresponding position on a new 24-well plate and incubate this new 24-well plate at 37 °C with 5% CO2 for 24 h.
    5. For each group from Step 4.1.1, use the total cell number determined in Step 4.1.3 to calculate the volume of trypsin-EDTA to add to each well so that the resulting cell suspension will be 1 x 106 cells/mL.
    6. Add the appropriate volume of trypsin-EDTA to each well and incubate for 5 min at 37 °C.
      NOTE: For larger screen it is recommended that the 24-well plates be trypsinized and replated in groups rather than all at once to ensure the viability of the cells.
    7. During the above incubation, use a multi-channel pipette to add 400 µL of puromycin-containing complete growth media to the corresponding wells on a new 24-well plate.
    8. Transfer 100 µL of cell suspension (approximately 1 x 105 cells) from each well to the corresponding position on the new 24-well plates prepared in Step 4.1.7.
    9. Repeat Steps 4.1.6 through 4.1.8 for all plates of grouped wells from Step 4.1.1, one plate at a time.
  2. Incubate the cells at 37 °C with 5% CO2 for 24 h.

5. Transfection of dual-luciferase reporter

  1. Transfect each well from Step 4.2 with the dual-luciferase reporter constructs as follows.
    NOTE: The total amount of DNA and the optimal ratio of firefly luciferase reporter vector to control Renilla luciferase vector should be determined prior to starting this assay. Here, 400 ng of a DNA mixture that contains 20 parts firefly luciferase reporter and 1 part control Renilla luciferase was used.
    1. Make the transfection dilution mixture (Tube A) and the reporter dilution mixture (Tube B) by mixing the indicated volumes of each reagent (Table 1) multiplied by the total number of transfections (plus several extra).
      NOTE: This protocol is optimized for transfection reagent 2 (see Table of Materials). If a different transfection reagent is used, the transfection should be optimized prior to this step.
    2. Mix the transfection dilution mixture (Tube A) with reporter dilution mixture (Tube B) and incubate at room temperature for 15 min to produce transfection mixture.
    3. During the above incubation, rinse each 24-well from Step 4.2 with 0.25 mL of phosphate buffer saline (PBS), and add 447 µL of complete growth media to each well.
    4. After the 15 min incubation, use a multi-channel pipette to distribute 53 µL of transfection mix to each well of the 24-well plates.
  2. Incubate the cells at 37 °C with 5% CO2 for 24 h.

6. Quantification of dual-luciferase activity

  1. Measure luciferase activity using a plate reader and a dual-luciferase reporter assay kit as described below.
    NOTE: This protocol is optimized for the indicated reporter assay kit (see Table of Materials) and follows the manufacturer's recommended protocol.
    1. Prepare enough 1x passive lysis buffer for all wells plus several extra (75 µL is needed per well) by diluting 5x passive lysis buffer (provided in kit) 1 to 5 with deionized water. Also thaw reagent A and reagent B Buffer (provided in kit, 100 µL of each is needed for each well).
    2. Aspirate the media from each well of the 24-well plate from Step 5.2.
    3. Add 75 µL of 1x passive lysis buffer to each well and incubate at room temperature for 30 min with occasionally shaking.
    4. Prepare reagent B by diluting 50x reagent B substrate (provided in kit) 1:50 with thawed reagent B buffer.
    5. Add 30 µL of 1x passive lysis buffer to 4 wells for blanking (see Supplemental Table 2).
    6. Transfer 30 µL of lysate from Step 6.1.3 into duplicate wells of a 96-well flat bottom white assay plate.
    7. Use a multi-channel pipette to add 50 µL of reagent A to each well and read the firefly luciferase signal with a plate reader.
    8. Use a multi-channel pipette to add 50 µL of reagent B from Step 6.1.4 to each well and read the Renilla luciferase signal with a plate reader.
  2. Process the raw data as follows (for a detailed description, see Supplemental Table 2).
    1. Exclude samples with very low Renilla luciferase signal as low values indicate that the viral construct was toxic or that too few transfected cells were assayed.
      NOTE: As explained in the Discussion, significantly "low" Renilla luciferase signal can result in anomalous results. Here, wells in which the Renilla luciferase signal was more than 1 standard deviation below the mean were excluded (see Supplemental Table 2). This was based on previous studies performed using this reporter system in these cells14, but may differ in other cell lines.
    2. Normalize the raw firefly luciferase value of each well to the raw Renilla luciferase value of the same well to obtain the firefly/Renilla ratio.
    3. Average the firefly/Renilla ratios of all replicate control wells and then divide the firefly/Renilla ratio of every other well by that number to get a fold change.
    4. Assign the control sample and set its firefly/Renilla ratio value to 1.
    5. Average the firefly/Renilla ratios of the duplicate wells and plot with the standard deviation.
      NOTE: The standard deviation is not used for statistical analysis, but instead as a means to identify wells where the replicates differ significantly.

Results

Our YAP/TAZ-TEAD reporter construct (pGL3-5xMCAT (SV)-492,14,15) contains a minimal SV-49 promoter with 5 repeats of the canonical TEAD binding element (MCAT)15 driving the firefly luciferase gene (Figure 1). It is co-transfected into cells along with the PRL-TK control vector (Promega), which expresses Renilla luciferase from the con...

Discussion

In this study, we demonstrate an approach for medium-throughput screening of arrayed viral libraries in combination with a dual-luciferase-based transcriptional reporter assay that can be used to identify and test novel regulators of transcription factors. It is critical to characterize and optimize the reporter system for each cell line prior to any screen. Experiments should be done to confirm that the reporter is responsive to altered activity of the transcription factor being investigated and the magnitude of change ...

Disclosures

The authors have nothing to disclose.

Acknowledgements

We would like to thank Emily Norton and Mikaelan Cucciarre-Stuligross for assisting in the preparation of shRNA vectors. This work was supported in part by a Susan G. Komen Career Catalyst Grant that awarded to J.M.L. (#CCR17477184).

Materials

NameCompanyCatalog NumberComments
2.0 ml 96-well deep well polypropylene plateUSA Scientific1896-2000For bacterial mini-prep
Trypsin - 2.50%Gibco15090-046Component of trypsin-EDTA
96 well flat bottom white assay plateCorning3922For dual-luciferase assay
Ampicillin - 100 mg/mlSigma-Aldrich45-10835242001-EAFor bacterial mini-prep
Bacto-tryptone - powderSigma-Aldrich95039Component of LB broth
Dual-luciferase reporter assay system, which include LAR II reagent (reagent A), Stop & Glo substrate (reagent B substrate) and Stop & Glo buffer (reagent B buffer) - KitPromegaE1960For dual-luciferase assay
Dulbecco's phosphate buffered saline w/o calcium, magnesium and phenol red - 9.6 g/LHimediaTS1006For PBS
EDTA - 0.5 MVWR97061-406Component of trypsin-EDTA
Ethanol - 100%Pharmco-AAPER111000200For bacterial mini-prep
Foetal Bovine Serum - 100%VWR97068-085Component of complete growth media
Hexadimethrine bromide (Polybrene) - 8 mg/mlSigma-Aldrich45-H9268For virus infection
HyClone DMEM/High glucose - 4 mM L-Glutamine; 4500 mg/L glucose; sodium pyruvateGE Healthcare life sciencesSH30243.01Component of complete growth media
I3-P/i3 Multi-Mode Microplate/EAMolecular devicesFor dual-luciferase assay
L-Glutamine - 200 mMGibco25030-081Component of complete growth media
Lipofectamine 3000 (Transfection Reagent 2) - 100%Life technologiesL3000008For transfections
Molecular Biology Water - 100%VWR02-0201-0500For dilution of shRNA vector for virus packaging
NaCl - powderBDHBDH9286Component of LB broth
NanoDrop One Microvolume UV-Vis SpectrophotometerThermo scientificFor measuring vector DNA concentration
Opti-MEM (Transfection Buffer) - 100%Gibco31985-062For transfections
Penicillin Streptomycin - 10,000 Unit/ml (Penicillin); 10,000 µg/ml (Streptomycin)Gibco15140-122Component of complete growth media
PureLink Quick Plasmid Miniprep Kit - KitThermo Fisher ScientificK210010For bacterial mini-prep
Puromycin - 2.5 mg/mlSigma-Aldrich45-P7255For antibiotic selection after infection
TC20 automated cell counterBio-RadFor cell counting
X-tremeGENE 9 DNA transfection reagent (Transfection Reagent 1) - 100%Roche6365787001For virus packaging
Yeast extract - powderVWRJ850Component of LB broth
P3000 (Transfection Reagent 3) - 100%Life technologiesL3000008For transfections

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