Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

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 metastasis^1,2,3,4,5,6, 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 i....

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% ba....

Results

Our YAP/TAZ-TEAD reporter construct (pGL3-5xMCAT (SV)-49^2,14,15) 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 .......

Materials

Name	Company	Catalog Number	Comments
2.0 ml 96-well deep well polypropylene plate	USA Scientific	1896-2000	For bacterial mini-prep
Trypsin - 2.50%	Gibco	15090-046	Component of trypsin-EDTA
96 well flat bottom white assay plate	Corning	3922	For dual-luciferase assay
Ampicillin - 100 mg/ml	Sigma-Aldrich	45-10835242001-EA	For bacterial mini-prep
Bacto-tryptone - powder	Sigma-Aldrich	95039	Component 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) - Kit	Promega	E1960	For dual-luciferase assay
Dulbecco's phosphate buffered saline w/o calcium, magnesium and phenol red - 9.6 g/L	Himedia	TS1006	For PBS
EDTA - 0.5 M	VWR	97061-406	Component of trypsin-EDTA
Ethanol - 100%	Pharmco-AAPER	111000200	For bacterial mini-prep
Foetal Bovine Serum - 100%	VWR	97068-085	Component of complete growth media
Hexadimethrine bromide (Polybrene) - 8 mg/ml	Sigma-Aldrich	45-H9268	For virus infection
HyClone DMEM/High glucose - 4 mM L-Glutamine; 4500 mg/L glucose; sodium pyruvate	GE Healthcare life sciences	SH30243.01	Component of complete growth media
I3-P/i3 Multi-Mode Microplate/EA	Molecular devices		For dual-luciferase assay
L-Glutamine - 200 mM	Gibco	25030-081	Component of complete growth media
Lipofectamine 3000 (Transfection Reagent 2) - 100%	Life technologies	L3000008	For transfections
Molecular Biology Water - 100%	VWR	02-0201-0500	For dilution of shRNA vector for virus packaging
NaCl - powder	BDH	BDH9286	Component of LB broth
NanoDrop One Microvolume UV-Vis Spectrophotometer	Thermo scientific		For measuring vector DNA concentration
Opti-MEM (Transfection Buffer) - 100%	Gibco	31985-062	For transfections
Penicillin Streptomycin - 10,000 Unit/ml (Penicillin); 10,000 µg/ml (Streptomycin)	Gibco	15140-122	Component of complete growth media
PureLink Quick Plasmid Miniprep Kit - Kit	Thermo Fisher Scientific	K210010	For bacterial mini-prep
Puromycin - 2.5 mg/ml	Sigma-Aldrich	45-P7255	For antibiotic selection after infection
TC20 automated cell counter	Bio-Rad		For cell counting
X-tremeGENE 9 DNA transfection reagent (Transfection Reagent 1) - 100%	Roche	6365787001	For virus packaging
Yeast extract - powder	VWR	J850	Component of LB broth
P3000 (Transfection Reagent 3) - 100%	Life technologies	L3000008	For transfections