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).

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.
<ol>
	<li>Add 1.3 mL of Luria Broth (LB) (1% bacto-trypton, 0.5% yeast extract, 1% NaCl, pH 7.5) containing 100 &#181;g/mL of ampicillin to each well of a 96-well deep well plate. Inoculate each well with 2 &#181;L of glycerol stock and grow at 37 &#176;C overnight with constant agitation at 225 rpm.</li>
	<li>Transfer each bacterial culture into a 1.5 mL centrifuge tube and pellet the bacteria by centrifugation at 21,000 x g at 4 &#176;C for 10 min.</li>
	<li>Purify each vector using a bacterial mini-prep kit by following the manufacturer&#39;s protocol.</li>
	<li>Determine the concentration of each vector using a spectrophotometer.</li>
	<li>Store the plasmids at -20 &#176;C. 
	NOTE: The protocol can be paused here.</li>
</ol>
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.
<ol>
	<li>Expand the 293FT cells using complete growth media (Dulbecco&#39;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 &#181;g/mL streptomycin antibiotic and 2 mM L-glutamine).</li>
	<li>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).</li>
	<li>Incubate the cells at 37 &#176;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.</li>
	<li>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 &#181;L of transfection reagent 1, and 23.75 &#181;L of transfection buffer (see Table of Materials).
	<ol>
		<li>Dilute each lentiviral vector to 50 ng/&#181;L with nuclease-free water, and then transfer 5 &#181;L (250 ng) into a well of a 96-well PCR plate.</li>
		<li>Make the transfection super mix by mixing 1.25 &#181;L * X of transfection reagent 1 and 23.75 &#181;L * X of pre-warmed transfection buffer where &#34;X&#34; is the total number of transfections plus several extra to account for volume loss during pipetting.</li>
		<li>Incubate the transfection super mix at room temperature for 5 min.</li>
		<li>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.</li>
		<li>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 &#181;L the mixture into each well containing viral vector from Step 2.4.1.</li>
		<li>Incubate at room temperature for 20 min.</li>
		<li>Transfer all 30 &#181;Ls from each 96-well from Step 2.4.6 into a well of the 24-well containing 293 FT cells from Step 2.3.</li>
	</ol>
	</li>
	<li>Incubate the cells at 37 &#176;C with 5% CO2 for 24 h and then replace the media in every well of the 24-well plate with 500 &#181;L of fresh complete growth media. Incubate the cells at 37 &#176;C with 5% CO2 for another 24 h.</li>
	<li>Using a multi-channel pipette, collect the viral supernatant from each well and aliquot 220 &#181;L (enough for 1 infection in Step 3 plus some extra volume) into two 96-wells each. These are the arrayed viral supernatant plates.</li>
	<li>Store the arrayed viral supernatant plates at -80 &#176;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.</li>
</ol>
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).
<ol>
	<li>Expand the cells to be infected in complete growth media.</li>
	<li>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.</li>
	<li>Incubate the cells at 37 &#176;C with 5% CO2 for 24 h.</li>
	<li>Infect each well from Step 3.2 with a different viral supernatant from the frozen arrayed lentiviral supernatant as follows.
	<ol>
		<li>Prepare complete growth media that contains 20 &#181;g/mL polybrene.</li>
		<li>Thaw the arrayed lentiviral library supernatants from Step 2.7 to room temperature.</li>
		<li>Aspirate the growth media from the 24-well plates from Step 3.2 and immediately add 200 &#181;L of polybrene-containing growth media to each well.</li>
		<li>Using a multi-channel pipette, transfer 200 &#181;L of viral supernatant from each 96-well from Step 3.4.2 to the 24 wells from Step 3.4.3.</li>
	</ol>
	</li>
	<li>Incubate the cells at 37 &#176;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&#39;UTR of puroR (Figure 1). Infection efficiency and expression of the puromycin resistance gene and the shRNA were monitored by green fluorescent protein.</li>
	<li>Prepare complete growth media that contains 2.5 &#181;g/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.</li>
	<li>Aspirate the media from each well and replace with 500 &#181;L 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.</li>
	<li>Incubate the cells at 37 &#176;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.</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>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.
	<ol>
		<li>Group the wells from Step 3.9 into 3 - 4 groups such that all wells in a group have a similar cell density.</li>
		<li>Trypsinize 1 representative well from each group with 200 &#181;L of trypsin-EDTA (1x PBS supplemented with 0.5 mM EDTA and 0.1% trypsin) for 5 min at 37 &#176;C. Then neutralize the trypsin-EDTA by adding 400 &#181;L of puromycin containing complete growth media.</li>
		<li>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.</li>
		<li>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 &#176;C with 5% CO2 for 24 h.</li>
		<li>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.</li>
		<li>Add the appropriate volume of trypsin-EDTA to each well and incubate for 5 min at 37 &#176;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.</li>
		<li>During the above incubation, use a multi-channel pipette to add 400 &#181;L of puromycin-containing complete growth media to the corresponding wells on a new 24-well plate.</li>
		<li>Transfer 100 &#181;L 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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Incubate the cells at 37 &#176;C with 5% CO2 for 24 h.</li>
</ol>
5. Transfection of dual-luciferase reporter
<ol>
	<li>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.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>During the above incubation, rinse each 24-well from Step 4.2 with 0.25 mL of phosphate buffer saline (PBS), and add 447 &#181;L of complete growth media to each well.</li>
		<li>After the 15 min incubation, use a multi-channel pipette to distribute 53 &#181;L of transfection mix to each well of the 24-well plates.</li>
	</ol>
	</li>
	<li>Incubate the cells at 37 &#176;C with 5% CO2 for 24 h.</li>
</ol>
6. Quantification of dual-luciferase activity
<ol>
	<li>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&#39;s recommended protocol.
	<ol>
		<li>Prepare enough 1x passive lysis buffer for all wells plus several extra (75 &#181;L 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 &#181;L of each is needed for each well).</li>
		<li>Aspirate the media from each well of the 24-well plate from Step 5.2.</li>
		<li>Add 75 &#181;L of 1x passive lysis buffer to each well and incubate at room temperature for 30 min with occasionally shaking.</li>
		<li>Prepare reagent B by diluting 50x reagent B substrate (provided in kit) 1:50 with thawed reagent B buffer.</li>
		<li>Add 30 &#181;L of 1x passive lysis buffer to 4 wells for blanking (see Supplemental Table 2).</li>
		<li>Transfer 30 &#181;L of lysate from Step 6.1.3 into duplicate wells of a 96-well flat bottom white assay plate.</li>
		<li>Use a multi-channel pipette to add 50 &#181;L of reagent A to each well and read the firefly luciferase signal with a plate reader.</li>
		<li>Use a multi-channel pipette to add 50 &#181;L of reagent B from Step 6.1.4 to each well and read the Renilla luciferase signal with a plate reader.</li>
	</ol>
	</li>
	<li>Process the raw data as follows (for a detailed description, see Supplemental Table 2).
	<ol>
		<li>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 &#34;low&#34; 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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Assign the control sample and set its firefly/Renilla ratio value to 1.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

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 ...

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.

<table><tbody><tr><td>2.0 ml 96-well deep well polypropylene plate</td><td>USA Scientific</td><td>1896-2000</td><td>For bacterial mini-prep</td></tr><tr><td>Trypsin - 2.50%</td><td>Gibco</td><td>15090-046</td><td>Component of trypsin-EDTA</td></tr><tr><td>96 well flat bottom white assay plate</td><td>Corning</td><td>3922</td><td>For dual-luciferase assay</td></tr><tr><td>Ampicillin - 100 mg/ml</td><td>Sigma-Aldrich</td><td>45-10835242001-EA</td><td>For bacterial mini-prep</td></tr><tr><td>Bacto-tryptone - powder</td><td>Sigma-Aldrich</td><td>95039</td><td>Component of LB broth</td></tr><tr><td>Dual-luciferase reporter assay system, which include LAR II reagent (reagent A), Stop &amp;amp; Glo substrate (reagent B substrate) and Stop &amp;amp; Glo buffer (reagent B buffer) - Kit</td><td>Promega</td><td>E1960</td><td>For dual-luciferase assay</td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline w/o calcium, magnesium and phenol red - 9.6 g/L</td><td>Himedia</td><td>TS1006</td><td>For PBS</td></tr><tr><td>EDTA - 0.5 M</td><td>VWR</td><td>97061-406</td><td>Component of trypsin-EDTA</td></tr><tr><td>Ethanol - 100%</td><td>Pharmco-AAPER</td><td>111000200</td><td>For bacterial mini-prep</td></tr><tr><td>Foetal Bovine Serum - 100%</td><td>VWR</td><td>97068-085</td><td>Component of complete growth media</td></tr><tr><td>Hexadimethrine bromide (Polybrene) - 8 mg/ml</td><td>Sigma-Aldrich</td><td>45-H9268</td><td>For virus infection</td></tr><tr><td>HyClone DMEM/High glucose - 4 mM L-Glutamine; 4500 mg/L glucose; sodium pyruvate</td><td>GE Healthcare life sciences</td><td>SH30243.01</td><td>Component of complete growth media</td></tr><tr><td>I3-P/i3 Multi-Mode Microplate/EA</td><td>Molecular devices</td><td></td><td>For dual-luciferase assay</td></tr><tr><td>L-Glutamine - 200 mM</td><td>Gibco</td><td>25030-081</td><td>Component of complete growth media</td></tr><tr><td>Lipofectamine 3000 (Transfection Reagent 2) - 100%</td><td>Life technologies</td><td>L3000008</td><td>For transfections</td></tr><tr><td>Molecular Biology Water - 100%</td><td>VWR</td><td>02-0201-0500</td><td>For dilution of shRNA vector for virus packaging</td></tr><tr><td>NaCl - powder</td><td>BDH</td><td>BDH9286</td><td>Component of LB broth</td></tr><tr><td>NanoDrop One Microvolume UV-Vis Spectrophotometer</td><td>Thermo scientific</td><td></td><td>For measuring vector DNA concentration</td></tr><tr><td>Opti-MEM (Transfection Buffer) - 100%</td><td>Gibco</td><td>31985-062</td><td>For transfections</td></tr><tr><td>Penicillin Streptomycin - 10,000 Unit/ml (Penicillin); 10,000 &amp;micro;g/ml (Streptomycin)</td><td>Gibco</td><td>15140-122</td><td>Component of complete growth media</td></tr><tr><td>PureLink Quick Plasmid Miniprep Kit - Kit</td><td>Thermo Fisher Scientific</td><td>K210010</td><td>For bacterial mini-prep</td></tr><tr><td>Puromycin - 2.5 mg/ml</td><td>Sigma-Aldrich</td><td>45-P7255</td><td>For antibiotic selection after infection</td></tr><tr><td>TC20 automated cell counter</td><td>Bio-Rad</td><td></td><td>For cell counting</td></tr><tr><td>X-tremeGENE 9 DNA transfection reagent (Transfection Reagent 1) - 100%</td><td>Roche</td><td>6365787001</td><td>For virus packaging</td></tr><tr><td>Yeast extract - powder</td><td>VWR</td><td>J850</td><td>Component of LB broth</td></tr><tr><td>P3000 (Transfection Reagent 3) - 100%</td><td>Life technologies</td><td>L3000008</td><td>For transfections</td></tr></tbody></table>

identification of transcription factor regulators using medium-throughput screening of arrayed libraries and a dual-luciferase-based reporter

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.

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...

Watch this Scientific Journal Video about Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter at JoVE.com

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

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.

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for ...

identification-transcription-factor-regulators-using-medium

Research

JoVE Journal

Cancer Research

6.7K Views.  Albany Medical College. Dit protocol kan worden gebruikt om arrayed lentiviral of retroviral test bibliotheken te screenen om nieuwe regelgevers van transcriptiefactoren in kankercellen te identificeren. De belangrijkste voordelen van deze techniek zijn dat het snel, medium doorvoer, en goedkoop, en dat het gebruik maakt van apparatuur en reagentia algemeen toegankelijk voor de meeste onderzoekers. Om elke lentivirale vector in de bibliotheek uit te breiden en te zuiveren, voeg u eerst 1,3 milliliter Luria-bouillon ...

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

Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to tissue and cell-type specific regulation of gene expression during the cellular differentiation. Thus, their identification and further investigation is important in order to understand how cell fate is determined. Integration of gene expression data (e.g., microarray or RNA-seq) and results of chromatin immunoprecipitation (ChIP)-based genome-wide studies (ChIP-seq) allows large-scale identification of these regulatory regions. However, functional validation of cell-type specific enhancers requires further in vitro and in vivo experimental procedures. Here we describe how active enhancers can be identified and validated experimentally. This protocol provides a step-by-step workflow that includes: 1) identification of regulatory regions by ChIP-seq data analysis, 2) cloning and experimental validation of putative regulatory potential of the identified genomic sequences in a reporter assay, and 3) determination of enhancer activity in vivo by measuring enhancer RNA transcript level. The presented protocol is detailed enough to help anyone to set up this workflow in the lab. Importantly, the protocol can be easily adapted to and used in any cellular model system.

In this work we provide an experimental workflow of how active enhancers can be identified and experimentally validated.

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to ...

Developmental Biology

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Medicine

Describing a Transcription Factor Dependent Regulation of the MicroRNA Transcriptome

While the transcription regulation of protein coding genes was extensively studied, little is known on how transcription factors are involved in transcription of non-coding RNAs, specifically of microRNAs. Here, we propose a strategy to study the potential role of transcription factor in regulating transcription of microRNAs using publically available data, computational resources and high throughput data. We use the H3K4me3 epigenetic signature to identify microRNA promoters and chromatin immunoprecipitation (ChIP)-sequencing data from the ENCODE project to identify microRNA promoters that are enriched with transcription factor binding sites. By transfecting cells of interest with shRNA targeting a transcription factor of interest and subjecting the cells to microRNA array, we study the effect of this transcription factor on the microRNA transcriptome. As an illustrative example we use our study on the effect of STAT3 on the microRNA transcriptome of chronic lymphocytic leukemia (CLL) cells.

Herein we propose a strategy to study the effect of a transcription factor of interest on the microRNA transcriptome using publically available data, computational resources and high throughput data from microRNA arrays after transfecting cells with small hairpin (sh)RNA targeting a transcription factor of interest.

While the transcription regulation of protein coding genes was extensively studied, little is known on how transcription factors are involved in ...

Bioengineering

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors to recognize thousands of odorants. Odorant ligands have been published for fewer than 6% of human receptors1-11. This lack of data is due in part to difficulties functionally expressing these receptors in heterologous systems. Here, we describe a method for expressing the majority of the olfactory receptor family in Hana3A cells, followed by high-throughput assessment of olfactory receptor activation using a luciferase reporter assay. This assay can be used to (1) screen panels of odorants against panels of olfactory receptors; (2) confirm odorant/receptor interaction via dose response curves; and (3) compare receptor activation levels among receptor variants. In our sample data, 328 olfactory receptors were screened against 26 odorants. Odorant/receptor pairs with varying response scores were selected and tested in dose response. These data indicate that a screen is an effective method to enrich for odorant/receptor pairs that will pass a dose response experiment, i.e. receptors that have a bona fide response to an odorant. Therefore, this high-throughput luciferase assay is an effective method to characterize olfactory receptors&#8212;an essential step toward a model of odor coding in the mammalian olfactory system.

Olfactory receptor activation patterns encode odor identity, but the lack of published data identifying odorant ligands for mammalian olfactory receptors hinders the development of a comprehensive model of odor coding. This protocol describes a method to facilitate high-throughput identification of olfactory receptor ligands and quantification of receptor activation.

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors ...

Neuroscience

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

Biology

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the structure of native control circuits. In both cases, the ability to program gene expression in a rapid and tunable fashion, with no off-target effects, can be useful. We have constructed yeast strains containing the ACT1 promoter upstream of a URA3 cassette followed by the ligand-binding domain of the human estrogen receptor and VP16. By transforming this strain with a linear PCR product containing a DNA binding domain and selecting against the presence of URA3, a constitutively expressed artificial transcription factor (ATF) can be generated by homologous recombination. ATFs engineered in this fashion can activate a unique target gene in the presence of inducer, thereby eliminating both the off-target activation and nonphysiological growth conditions found with commonly used conditional gene expression systems. A simple method for the rapid construction of GFP reporter plasmids that respond specifically to a native or artificial transcription factor of interest is also provided.

This protocol describes an experimental procedure for the rapid construction of artificial transcription factors (ATFs) with cognate GFP reporters&#160;and quantification of the ATFs ability to stimulate GFP expression via flow cytometry.

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

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

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