The authors thank Drs. Sueyoshi and Wang at the National Institute of Environmental Health Sciences (NIEHS) for their critical reading of the manuscript, and&#160;Tanner Jefferson for performing procedures on the video.&#160;This work was supported by the National Institutes of Health Grant 1ZIAES070065 (to K.S.K.) from the Division of Intramural Research of the NIEHS.

1. Preparation of Plasmids for the Mammalian Two-hybrid Assay
<ol>
	<li>Use the following plasmids: pG5-Luc, pBIND-ER&#945;EF, and pACT-ER&#945;EF. 
	NOTE: The plasmids are available from the authors upon request and are sent on filter paper. pG5-Luc is the reporter plasmid that contains five repeats of GAL4 responsive elements fused with the luciferase expression unit (Figure 1A). pBIND-ER&#945;EF is the protein expression plasmid for GAL4DBD-fused ER&#945; LBD proteins. pBIND-ER&#945;EF contains a Renilla luciferase expression unit for normalizing the transfection efficiency (Figure 1B). pACT-ER&#945;EF is the protein expression plasmid for the VP16AD-fused ER&#945; LBD proteins (Figure 1C). The diagram of the expressed protein structure from the plasmids is shown in Figure 2.</li>
	<li>Prepare plasmid DNAs.
	<ol>
		<li>Cut out the plasmid-loaded area from the paper using clean scissors and put it into a 1.5 mL tube. Wear gloves and use clean tweezers to pick up the plasmid-loaded paper. Add 100 &#181;L of Tris-EDTA (TE) buffer (pH 8.0) and vortex well.</li>
		<li>Add 1 &#181;L of the plasmid DNA solution from step 1.2.1 to competent Escherichia coli cells (see Table of Materials) and keep the tube on ice for 15 min.</li>
		<li>Heat-shock the plasmid-added competent cells; incubate the tube(s) in 42 &#176;C water bath for 30 s and, then, keep them on ice for 2 min.</li>
		<li>Add 250 &#181;L of super optimal broth with catabolite repression (SOC) medium (room temperature) to the tube(s) and culture with shaking at 37 &#176;C for 30 min.</li>
		<li>Plate 100 &#181;L of the cultured E. coli onto a prewarmed Luria broth (LB) plate with 100 &#181;g/mL ampicillin (10 cm-diameter dish) and culture at 37 &#176;C overnight.</li>
		<li>Pick one colony from the plate and add it into a 14 mL tube containing 2 mL of terrific broth (TB) with 100 &#181;g/mL ampicillin. Culture with shaking at 37 &#176;C until the medium is faintly clouded.</li>
		<li>Add 1 mL of the cultured medium from step 1.2.6 to an autoclaved 250 mL glass flask containing 50 mL of TB with 100 &#181;g/mL ampicillin. Culture with shaking at 37 &#176;C for 20 - 24 h.</li>
		<li>Transfer the cultured E. coli to a 50 mL conical tube and centrifuge at 3,450 x g at room temperature for 30 min using the swing-bucket rotor. Discard the medium. Save the E. coli pellet at -80 &#176;C.</li>
		<li>Add 3 mL of cell resuspension solution (see Table of Materials) to the E. coli pellet and suspend it completely. Add 3 mL of cell lysis solution (see Table of Materials), mix the tube gently by inverting it 10x, and keep it on ice for 5 min (keep the time punctually). Add 6 mL of neutralization solution (see Table of Materials), mix the tube steadily with tapping, and then, keep it on ice for 10 min.</li>
		<li>Centrifuge the tube at 3,450 x g at 4 &#730;C for 30 min using the swing-bucket rotor. Transfer the DNA-containing solution to a new 50 mL conical tube and add 10 mL of DNA purification resin (see Table of Materials). Suspend the resin gently.</li>
		<li>Centrifuge the tube at 625 x g for 1 min using the swing-bucket rotor. Discard the solution and keep the resin at the bottom.</li>
		<li>Add 15 mL of column wash solution (80 mM potassium acetate, 8.5 mM Tris-HCl [pH 7.5], 40 &#181;M EDTA, and 55% ethanol) to the 50 mL conical tube and shake it gently to suspend the resin. Repeat step 1.2.11. 
		NOTE: The column wash solution must be prepared using diethyl pyrocarbonate (DEPC)-treated water or nuclease-free water.</li>
		<li>Add 5 mL of column wash solution to the 50 mL conical tube and suspend the resin with a 5 mL pipet. Apply the resin to the column (see Table of Materials) that is set on the vacuum manifold. Vacuum the column until the resin dry. Add 6 mL of column wash solution to the column and vacuum.</li>
		<li>Once the resin dries visually, detach the reservoir from the column. Transfer the bottom section of the column (resin) to a 1.5 mL tube. Centrifuge the column at maximum speed for 2 min using a microcentrifuge to dry the resin completely.</li>
		<li>Transfer the bottom section of the column (resin) to a new 1.5 mL tube. Add 300 &#181;L of nuclease-free water to the column and wait until the whole resin is soaked.</li>
		<li>Centrifuge the column at maximum speed for 1 min to collect the plasmid DNA-containing solution (200 - 250 &#181;L).</li>
		<li>Treat the DNA-containing solution with phenol-chloroform (1:1), then with chloroform as follows.
		<ol>
			<li>Add the same volume of phenol-chloroform (1:1) to the DNA-containing solution (200 - 250 &#181;L), shake well, and centrifuge at maximum speed for 2 min using a microcentrifuge. Collect the upper layer (DNA-containing solution) and add it to a new 1.5 mL tube. Repeat this step 1x.</li>
			<li>Add the same volume of chloroform to the DNA-containing solution (200 - 250 &#181;L), shake well, and centrifuge at maximum speed for 1 min. Collect the upper layer (DNA-containing solution) and add it to a 1.5 mL tube. 
			NOTE: Endotoxin (lipopolysaccharide) induces cellular signaling in some cells. To avoid such an unpredicted effect, step 1.2.17 is recommended, which can reduce endotoxin contamination.</li>
		</ol>
		</li>
		<li>Precipitate the plasmid DNA using the ethanol precipitation technique as follows.
		<ol>
			<li>Add 1/9 of DNA mixture volume&#160;of 3 M sodium acetate (pH 5.5) and 20x of 3M sodium acetate volume&#160;of 100% ethanol to the DNA-containing solution. For example, add 27.8 &#181;L of 3 M sodium acetate and 278 &#181;L of 100% ethanol to 250 &#181;L of the DNA solution (the final volume is 555.8 &#181;L).</li>
			<li>Keep the solution at -80 &#176;C for 20 min at least. Centrifuge at maximum speed for 20 min at 4 &#730;C. Discard the solution and keep the pellet. Add 200 &#181;L of 75% ethanol and rinse the inside of the tube. Centrifuge for 20 min at 4 &#176;C, discard the solution, and keep the pellet. Dry the DNA pellet using a vacuum concentrator.</li>
			<li>Add 100 - 250 &#181;L of TE buffer (pH 8.0) to the tube. Dissolve the DNA pellet and check the DNA concentration using a spectrophotometer at a wavelength of 260 nm. Keep the DNA concentration around 0.5 mg/mL.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Mammalian Two-hybrid Assay
<ol>
	<li>Maintain the cells.
	<ol>
		<li>Culture human hepatocellular carcinoma HepG2 cells in a 750 mL, 175 cm2 tissue culture flask with the phenol red-free minimum essential media (MEM) supplemented with 10% fetal bovine serum (FBS; heat-inactivated), 2 mM L-glutamine, and 1% penicillin-streptomycin solution (antibiotics) for maintaining the cells.</li>
		<li>Culture the cells in a 5% CO2-supplemented, 37 &#176;C incubator until the cells are approximately 90% confluent. 
		NOTE: To reduce estrogenic background activity, phenol red-free medium should be used for all M2H experiments. This step should be performed inside a clean bench/biological safety cabinet. The cells should be maintained following the general mammalian cell culture protocol7.</li>
	</ol>
	</li>
	<li>Prepare the cells for transfection. 
	NOTE: The following procedural steps should be performed inside the clean bench/biological safety cabinet. The cells are cultured/incubated in a 5% CO2-supplemented, 37 &#176;C incubator in this step every time.
	<ol>
		<li>Replate the 90% confluent cells to the 24-well plates; rinse the cells 1x with phosphate-buffered saline (PBS).</li>
		<li>Add 7 mL of 0.05% trypsin-EDTA solution to the culture flask and soak the cells for 1 - 2 min while keeping the culture flask in a clean bench at room temperature. Remove a part of the trypsin-EDTA solution and leave 1 - 2 mL of the solution in the culture flask. Incubate the culture flask in the incubator for 1 - 2 min.</li>
		<li>Smack the bottom of the flask to detach the cells and check the cells under the microscope. 
		NOTE: If the cells are still attached to the flask, incubate for another 1 - 2 min and, then, repeat step 2.2.3.</li>
		<li>Suspend the cells in the phenol red-free MEM supplemented with 10% charcoal-stripped FBS (heat-inactivated), 2 mM L-glutamine, and 1% penicillin-streptomycin solution. 
		NOTE: Charcoal-stripped FBS must be used to reduce the effect of serum-derived endogenous estrogen.</li>
		<li>Count the cell number and seed cells in a 24-well plate at a density of 1.2 x 105 cells/well. Assign three wells for each data point (triplicate).</li>
		<li>Culture the cells overnight. Continue to step 2.4.1.</li>
	</ol>
	</li>
	<li>Prepare a DNA mixture for transfection. 
	NOTE: The following plasmid DNAs are transfected in one well: 100 ng of pG5-Luc reporter plasmid, 50 ng of expression plasmids for GAL4DBD fusion proteins (pBIND), and 50 ng of expression plasmids for VP16AD fusion proteins (pACT).
	<ol>
		<li>To analyze the ligand-dependent ER&#945; LBD dimerization activity, prepare the following combinations (Figure 3). Combination (i): pG5-Luc + pBIND-hER&#945;EF + pACT, and pG5-Luc + pBIND-mER&#945;EF + pACT. Combination (ii): pG5-Luc + pBIND-hER&#945;EF + pACT-hER&#945;EF, and pG5-Luc + pBIND-mER&#945;EF + pACT-mER&#945;EF. Combination (iii): pG5-Luc + pBIND + pACT-hER&#945;EF, and pG5-Luc + pBIND + pACT-mER&#945;EF. 
		NOTE: Combination (i) represents the basal experimental level of human ER&#945; LBD and mouse ER&#945; LBD, respectively. If the result shows a remarkably high level with ligand alone, this method is unfeasible for use with that ligand (e.g., diethylstilbestrol [DES]). Combination (ii) represents the levels of dimerized human ER&#945; LBD and dimerized mouse ER&#945; LBD, respectively. Combination (iii) represents negative controls; use this set only when the experiment is performed for the first time to evaluate the background level of the system.</li>
		<li>Before mixing, calculate the total DNA amount required based on the number of wells for transfection. For experiments conducted in triplicates and at five data points, mix the following plasmid DNAs in two 1.5 &#181;L tubes for combinations (i) and (ii): pG5-Luc, 5 (data points) x 3 (wells) x 100 ng = 1,500 ng (15 &#181;L); pBIND-mER&#945;EF, 5 (data points) x 3 (wells) x 50 ng = 750 ng (7.5 &#181;L); pACT (for combination i) or pACT-mER&#945;EF (for combination ii), 5 (data points) x 3 (wells) x 50 ng = 750 ng (7.5 &#181;L). 
		NOTE: The concentration of each plasmid DNA solution is 0.1 &#181;g/&#181;L.</li>
		<li>Precipitate the DNA mixture using the ethanol precipitation technique. Add 1/9 of DNA mixture volume of 3 M sodium acetate (pH 5.5) and 20x of 3M sodium acetate volume of 100% ethanol to the DNA mixture. Then, vortex the tube. 
		NOTE: For example, add 3.4 &#181;L of 3 M sodium acetate, and 34 &#181;L of 100% ethanol to the 30 &#181;L of plasmid DNA mixture exemplified in step 2.3.2. This step helps to keep an invariable transfection condition, and it is not necessary to perform this step in a biological safety cabinet.</li>
		<li>Keep the mixture overnight at -20 &#176;C. Centrifuge it at maximum speed for 20 min at 4 &#176;C. Discard the solution (the pellet is invisible). Add 120 &#181;L of 75% ethanol to the tube; then, rinse the inside of the tube. Centrifuge for 20 min at 4 &#176;C. Discard the solution and dry the DNA mixture using a vacuum concentrator for 30 min.</li>
	</ol>
	</li>
	<li>Perform the transfection. 
	NOTE: The following steps should be performed in a clean bench or a biological safety cabinet.
	<ol>
		<li>The following morning, rinse the cells seeded in a 24-well plate (from step 2.2.6) 2x with 0.5 mL of PBS (room temperature).</li>
		<li>Add warm (37 &#176;C) fresh phenol red-free MEM supplemented with 10% charcoal-stripped FBS (heat-inactivated) and 2 mM L-glutamine without antibiotics to each well (0.5 mL medium/well). Keep the cells in a 5% CO2-supplemented, 37 &#176;C incubator.</li>
		<li>Suspend the dried plasmid DNA mixture (from step 2.3.4) in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) (no serum, phenol red-free, without glutamine; 13 &#181;L/well). Calculate the amounts of DMEM from the number of wells for transfection. 
		NOTE: 15 wells for combination (i) x 13 &#181;L = 195 &#181;L, and 15 wells for combination (ii) x 13 &#181;L = 195 &#181;L.</li>
		<li>Suspend the transfection reagent (see Table of Materials; 1.5 &#181;L/well) in DMEM (12.5 &#181;L/well) in another 1.5 mL tube. Calculate the amounts of transfection reagent and DMEM from the number of wells for transfection. 
		NOTE: 30 [15 wells for combination (i) and 15 wells for combination (ii)] x 1.5 &#181;L of transfection reagent + 30 x 12.5 &#181;L of DMEM = 420 &#181;L.</li>
		<li>Add an equal amount of the transfection reagent medium (from step 2.4.4) to each tube (from step 2.4.3). Incubate the tubes for 5 - 10 min at room temperature. 
		NOTE: 200 &#181;L of transfection reagent medium + 195 &#181;L of combination (i) DNA mixture = 395 &#181;L, and 200 &#181;L of transfection reagent medium + 195 &#181;L of combination (ii) DNA mixture = 395 &#181;L. It is important to add an equal volume of transfection reagent medium to the tubes of combination (i) and (ii). It is not necessary to use up the medium in this step.</li>
		<li>Add the combined mixture (from step 2.4.5) to each well (25 &#181;L/well) of the 24-well plate (from step 2.4.2) and incubate the cells for 6 h in the 5% CO2-supplemented, 37 &#176;C incubator.</li>
		<li>Remove the transfection medium from the 24-well plate. Add warm (37 &#176;C) fresh phenol red-free MEM supplemented with 10% charcoal-stripped FBS (heat-inactivated), 2 mM L-glutamine, 1% penicillin-streptomycin solution, and ligand (suspended in ethanol) (0.5 mL medium/well). Culture the cells for 16 - 18 h in the 5% CO2-supplemented, 37 &#176;C incubator.</li>
	</ol>
	</li>
	<li>Harvest the samples.
	<ol>
		<li>Rinse the cells with 1 mL of PBS and, then, add 100 &#181;L of 1x passive lysis buffer (see Table of Materials).</li>
		<li>Vigorously shake the 24-well plates with a vortex mixer to detach the cells.</li>
		<li>Freeze the plates (cell lysates) 1x by liquid nitrogen or save them at -80 &#176;C before analysis. Immediately before the assay, vigorously shake the plates on a shaker until they are at room temperature. 
		NOTE: This process prevents irregularly appearing anomalous values of luciferase activity.</li>
	</ol>
	</li>
	<li>Perform a dual-luciferase assay.
	<ol>
		<li>Prepare the reagents for the dual-luciferase assay system (see Table of Materials).
		<ol>
			<li>For making the luciferase assay substrate (LAS) buffer, add all luciferase assay buffer into the luciferase assay substrate which is in a glass brown bottle. Save the LAS buffer at -20 &#176;C. 
			NOTE: The LAS buffer should be warmed to room temperature before use.</li>
			<li>For making the Renilla luciferase substrate (RLS) buffer, mix Renilla luciferase substrate and Renilla luciferase buffer in a ratio of 1:50. Make fresh RLS buffer for every assay and use a black tube or a tube shaded by tinfoil. Calculate the amount of RLS buffer making a fresh buffer. 
			NOTE: The RLS buffer should be warmed to room temperature before use.</li>
		</ol>
		</li>
		<li>Set the reagents in the microplate reader.
		<ol>
			<li>Wash the injection lines 2x with 70% ethanol; then, wash the lines 2x with water. 
			NOTE: This is important to prevent disturbing the assay results.</li>
			<li>Set the LAS buffer to the P-injector line (first injection) and set the RLS buffer to the M-injector line (second injection). Do not mix these buffers. RLS buffer inhibits luciferase activity.</li>
		</ol>
		</li>
		<li>Apply 10 &#181;L of harvested samples (from step 2.5.3) to the 96-well white plastic plate.</li>
		<li>Apply the following settings to the microplate reader. For the P-injector, use a volume of 50 &#181;L, a delay of 2 s, and a speed of 50 &#181;L/s. For the M-injector, use a volume of 50 &#181;L, a delay of 2 s, and a speed of 50 &#181;L/s. Set an integration time of 15 s. Set the temperature to 25 &#176;C.</li>
		<li>Run the microplate reader.</li>
		<li>Record the values of the luciferase activity (IPValue) and the values of the Renilla luciferase activity (IMValue) using the data acquisition program (see Table of Materials).</li>
		<li>Wash the injection lines after the data collection (see step 2.6.2.1).</li>
	</ol>
	</li>
	<li>Perform data analysis.
	<ol>
		<li>Use the normalized values (IPValue/IMValue) for data analysis.</li>
		<li>Set the normalized value of combination (i) [pG5-Luc + pBIND-ER&#945;EF + pACT] with vehicle (0 nM ligand) as 1 for the calculation of the basal level and the homodimerized ER&#945; LBD level. The levels are represented as &#34;Relative activity&#34;.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Arao, Y., Korach, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+F+domain+of+estrogen+receptor+&#945;+is+involved+in+species-specific,+tamoxifen-mediated+transactivation.">The F domain of estrogen receptor &#945; is involved in species-specific, tamoxifen-mediated transactivation.</a> Journal of Biological Chemistry. 293 (22), 8495-8507 (2018).</li><li>Brzozowski, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+agonism+and+antagonism+in+the+oestrogen+receptor.">Molecular basis of agonism and antagonism in the oestrogen receptor.</a> Nature. 389 (6652), 753-758 (1997).</li><li>Shiau, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structural+basis+of+estrogen+receptor/coactivator+recognition+and+the+antagonism+of+this+interaction+by+tamoxifen.">The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen.</a> Cell. 95 (7), 927-937 (1998).</li><li>Yang, J., Singleton, D. W., Shaughnessy, E. A., Khan, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+F-domain+of+estrogen+receptor-alpha+inhibits+ligand+induced+receptor+dimerization.">The F-domain of estrogen receptor-alpha inhibits ligand induced receptor dimerization.</a> Molecular and Cellular Endocrinology. 295 (1-2), 94-100 (2008).</li><li>Montano, M. M., M&#252;ller, V., Trobaugh, A., Katzenellenbogen, B. 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R., Hughes, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Human Cell Culture Protocols. , (2011).</li><li>Arao, Y., Coons, L. A., Zuercher, W. J., Korach, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transactivation+Function-2+of+Estrogen+Receptor+&#945;+Contains+Transactivation+Function-1-regulating+Element.">Transactivation Function-2 of Estrogen Receptor &#945; Contains Transactivation Function-1-regulating Element.</a> Journal of Biological Chemistry. 290 (28), 17611-17627 (2015).</li><li>Huang, H. -. J., Norris, J. D., McDonnell, D. 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The authors have nothing to declare.

Herein, we described the protocol for the M2H assay, focusing on the assay conditions for detecting the homodimerization activity of ER&#945; LBD as an example. In general, the M2H assay is not popular for the assessment of ligand-dependent ER&#945; LBD dimerization activity. This is due to the ER&#945; LBD possessing a transcriptional activation function; the activity of which disturbs, in some cases, the results of the M2H assay. However, as we demonstrate here, the M2H assay can be used for analyzing the LBD dimerizat...

Estrogen receptor alpha (ER&#945;) is an estrogenic ligand-dependent transcription regulator. The amino acid sequenceof ER&#945; is highly conserved among species. Because of the higher homology between human and mouse ER&#945;, the function of these receptors is thought of as identical, and the differential activity of estrogenic substances (e.g., tamoxifen) in those species is caused by the species&#39; differences in chemical metabolisms rather than by the structural differences of ER&#945;. ER&#945; has highly conserved domain structures that are common among the nuclear receptor (NR) superfamily, designated A to F domains. The E domain or ligand-binding domain (LBD) includes the ligand-binding pocket and the transcriptional activation function 2, named AF-2. The F domain is localized immediately adjacent to the E domain and is the most variable domain among the NRs. Even between human and mouse ER&#945;, the homology of the F domain is significantly lower than that of the other domains1. The ligand-bound LBD of ER&#945; enhances the homodimerization of the ER&#945; protein to bind the specific estrogen-responsive DNA element directly for regulating the ligand-dependent gene transcription (classical action of ER&#945;). Crystallographic studies have revealed the differential positioning of helix 12 (AF-2 core domain) with estradiol (E2)- or 4-hydroxy-tamoxifen (4OHT)-bound LBD dimers2,3. The ER&#945; F domain (45 amino acids) connect helix 12 directly. However, there is no information regarding the effect of this extension of 45 amino acids (F domain) from the helix 12 on the ER&#945; LBD dimerization. In this study, we demonstrate the contribution of the F domain to the species-specific 4OHT-dependent ER&#945; LBD homodimerization using a mammalian two-hybrid (M2H) assay.
The M2H assay is a method to demonstrate protein-protein interactions in mammalian cells introducing the three different plasmid DNAs: two protein expression plasmids, which express GAL4 DNA-binding domain (DBD) fusion ER&#945; LBD and VP16AD fusion ER&#945; LBD, and a GAL4RE-fused luciferase expression reporter plasmid. When the GAL4DBD fusion ER&#945; LBD and the VP16AD fusion ER&#945; LBD interact (make a dimer) in the cells, this protein complex binds to the GAL4RE and, then, activates luciferase gene expression through the VP16AD-dependent transactivation function. The level of LBD homodimerization can be evaluated by the luciferase activity.
The yeast two-hybrid (Y2H) assay is an alternative method based on the same principle that uses yeast as the host environment. Previous reports using the Y2H system demonstrated that F-domain-truncated human ER&#945; LBD increases E2-dependent coactivator recruitment, concluding that the F domain prevents E2-mediated transcription4. This result is inconsistent with other reports which have demonstrated the attenuated transcriptional activity of F-domain-truncated human ER&#945; in the mammalian cells5,6. Recently, our study, using the M2H system, demonstrated that the E2-dependent coactivator recruitment activity of human ER&#945; LBD is decreased by F domain truncation in mammalian cells and is consistent with transcription activity1. These observations suggest that the physiological role of protein-protein interaction differs in a cell type-specific manner and context. The M2H assay can demonstrate protein-protein interaction activity in the same cellular context that is used for determining the transcriptional activity. This provides an advantage of the M2H assay compared to the Y2H or other in vitro protein-protein interaction analyses.
There remain questions regarding the molecular mechanisms of the partial agonist activity of selective estrogen receptor modulators (SERMs) (e.g., 4OHT) to regulate ER&#945;-mediated transcription. ER&#945; LBD-based M2H assays may be applied to study the mechanism of the partial agonist activity of SERMs in various mammalian cell types.

<table><tbody><tr><td>pG5-Luc</td><td>Promega</td><td>E249A</td><td>Component of CheckMate Mammalian Two-Hybrid System</td></tr><tr><td>pBIND</td><td>Promega</td><td>E245A</td><td>Component of CheckMate Mammalian Two-Hybrid System</td></tr><tr><td>pACT</td><td>Promega</td><td>E246A</td><td>Component of CheckMate Mammalian Two-Hybrid System</td></tr><tr><td>One Shot TOP10 Chemically Competent E. coli</td><td>Invitrogen</td><td>C404010</td><td></td></tr><tr><td>Centrifuge Rotor</td><td>SORVALL</td><td>75006445</td><td></td></tr><tr><td>Swing Buckets</td><td>SORVALL</td><td>75006441</td><td></td></tr><tr><td>Cell Resuspension Solution (CRA)</td><td>Promega</td><td>A7112</td><td></td></tr><tr><td>Cell Lysis Solution (CLA)</td><td>Promega</td><td>A7122</td><td></td></tr><tr><td>Neutralization Solution (NSA)</td><td>Promega</td><td>A7131</td><td></td></tr><tr><td>Column Wash Solution (CWB)</td><td>Promega</td><td>A8102</td><td></td></tr><tr><td>Wizard Midipreps DNA Purification Resin</td><td>Promega</td><td>A7701</td><td></td></tr><tr><td>Wizard Midicolumns</td><td>Promega</td><td>A7651</td><td></td></tr><tr><td>MEM, no glutamine, no phenol red</td><td>Gibco</td><td>51200038</td><td></td></tr><tr><td>L-glutamine (200 mM)</td><td>Gibco</td><td>A2916801</td><td></td></tr><tr><td>0.5% Trypsin-EDTA (10x)</td><td>Gibco</td><td>15400054</td><td></td></tr><tr><td>Penicillin-Streptomycin (100x)</td><td>Sigma-Aldrich</td><td>P0781</td><td></td></tr><tr><td>BenchMark fetal bovine serum (FBS)</td><td>Gemini-Bio</td><td>100-106</td><td>Heat inactivated</td></tr><tr><td>Charcoal:dextran stripping fetal bovine serum</td><td>Gemini-Bio</td><td>100-119</td><td>Heat inactivated</td></tr><tr><td>DMEM, high glucose, no glutamine, no phenol red</td><td>Gibco</td><td>31053028</td><td>for transfection</td></tr><tr><td>Lipofectamine 2000</td><td>Invitrogen</td><td>11668027</td><td></td></tr><tr><td>Passive Lysis 5X Buffer</td><td>Promega</td><td>E1941</td><td></td></tr><tr><td>Dual-Luciferase Reporter Assay System</td><td>Promega</td><td>E1980</td><td></td></tr><tr><td>SpectraMax L microplate reader</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>SoftMax Pro Software</td><td>Molecular Devices</td><td></td><td></td></tr></tbody></table>

detecting the ligand-binding domain dimerization activity of estrogen receptor alpha using the mammalian two-hybrid assay

Estrogen receptor alpha (ER&#945;) is an estrogenic ligand-dependent transcription regulator. The sequence of ER&#945; protein is highly conserved among species. It has been thought that the function of human and mouse ER&#945;s is identical. We demonstrate the differential 4-hydroxy-tamoxifen (4OHT) effect on mouse and human ER&#945; ligand-binding domain (LBD) dimerization activity using the mammalian two-hybrid (M2H) assay. The M2H assay can demonstrate the efficiency of LBD homodimerization activity in mammalian cells, utilizing the transfection of two protein expression plasmids (GAL4 DNA-binding domain [DBD] fusion LBD and VP16 transactivation domain [VP16AD] fusion LBD) and a GAL4-responsive element (GAL4RE) fused luciferase reporter plasmid. When the GAL4DBD fusion LBD and the VP16AD fusion LBD make a dimer in the cells, this protein complex binds to the GAL4RE and, then, activates a luciferase gene expression through the VP16AD dependent transcription activity. The 4OHT-mediated luciferase activation is higher in the HepG2 cells that were transfected with the mouse ER&#945; LBD fusion protein expression plasmids than in the human ER&#945; LBD fusion protein expression plasmid transfected cells. This result suggests that the efficacy of the 4OHT-dependent mouse ER&#945; LBD homodimerization activity is higher than human ER&#945; LBD. In general, the utilization of the M2H assay is not ideal for the evaluation of nuclear receptor LBD dimerization activity, because agonistic ligands enhance the basal level of the LBD activity and that impedes the detection of LBD dimerization activity. We found that 4OHT does not enhance ER&#945; LBD basal activity. That is a key factor for being able to determine and detect the 4OHT-dependent LBD dimerization activity for successfully using the M2H assay. ER&#945; LBD-based M2H assays may be applied to study the partial agonist activity of selective estrogen receptor modulators (e.g., 4OHT) in various mammalian cell types.

Figure 3&#160;displays the scheme of possible responses in the combination (i) and combination (ii) plasmid-transfected cells. The experimental results are shown in Figure 4. The activity of combination (i) (pG5-Luc + pBIND-mER&#945;EF + pACT) shows stimulation by 10 nM E2 (Figure 4A), because the ER&#945; LBD contains the ligand-dependent transactivation functional domain, AF-2. Ago...

Watch this Scientific Journal Video about Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay at JoVE.com

Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay

We present a method for analyzing the 4-hydroxy-tamoxifen-dependent estrogen receptor alpha ligand-binding domain dimerization activity using the mammalian two-hybrid assay.

Estrogen receptor alpha (ER&#945;) is an estrogenic ligand-dependent transcription regulator. The sequence of ER&#945; protein is highly conserved among ...

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Research

JoVE Journal

Cancer Research

6.3K Views.  National Institutes of Health (NIH). We present a method for analyzing the 4-hydroxy-tamoxifen-dependent estrogen receptor alpha ligand-binding domain dimerization activity using the mammalian two-hybrid assay.

Video: Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in understanding how such pathways are organized and how they function. There are many methods to detect protein-protein interactions in cells, but relatively few can be used to detect interactions between endogenous proteins. One such method, the proximity ligation assay (PLA), has several advantages to recommend its use. Compared to other common methods of protein-protein interaction analysis, PLA has relatively high sensitivity and specificity, can be performed with minimal cell manipulation, and, in the protocol described herein, requires only two target-specific antibodies derived from different species (e.g., from mouse and rabbit) and one specialized reagent: a set of secondary antibodies that are covalently linked to specific oligonucleotides that, when brought in close proximity of one another, create an amplifiable platform for in situ PCR or rolling circle amplification. In this presentation, we show how to apply the PLA technique to visualize changes in MST1 and MST2 proximity in fixed cells. The technique described in this manuscript is particularly applicable for the analysis of cell signaling studies.

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Here, we show how to use a Proximity Ligation Assay (PLA) to visualize MST1/MST2 heterodimerization in fixed cells with high sensitivity.

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in ...

Biochemistry

Screening for Phytoestrogens using a Cell-based Estrogen Receptor  &#946; Reporter Assay

Plants are a source of food for many animals, and&#160;they can produce thousands of chemicals. Some of these compounds affect physiological processes in the vertebrates that consume them, such as endocrine function. Phytoestrogens, the most well studied endocrine-active phytochemicals, directly interact with the hypothalamo-pituitary gonadal axis of the vertebrate endocrine system. Here we present the novel use of a cell-based assay to screen plant extracts for the presence of compounds that have estrogenic biological activity. This assay uses mammalian cells engineered to highly express estrogen receptor beta (ER&#946;) and that have been transfected with a luciferase gene. Exposure to compounds with estrogenic activity results in the cells producing light. This assay is a reliable and simple way to test for biological estrogenic activity. It has several improvements over transient transfection assays, most notably, ease of use, the stability of the cells, and the sensitivity of the assay.

We have optimized a commercially available estrogen receptor &#946; reporter assay for screening human and nonhuman primate foods for estrogenic activity. We validated this assay by showing that the known estrogenic human food soy registers high, while other foods show no activity.

Plants are a source of food for many animals, and&#160;they can produce thousands of chemicals. Some of these compounds affect physiological processes in ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

Bioengineering

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Medicine

A High-Throughput Screening Approach for Evaluating the Binding Affinity of Environmental Pollutants to Nuclear Receptors

Increasing levels of compounds have been detected in the environment, causing widespread pollution and posing risks to human health. However, despite their high environmental occurrence, there is very limited information regarding their toxicological effects. It is urgent to develop high-throughput screening (HTS) methods to guide toxicological studies. In this study, a receptor-ligand binding assay using an HTS system was developed to determine the binding potency of environmental pollutants on nuclear receptors. The test is conducted using a microplate reader (i.e., a 96-well plate containing various chemicals) by measuring the fluorescence polarization (FP) of a specific fluorescent probe. This assay consists of four parts: the construction and transformation of recombinant vectors, the expression and purification of the receptor protein (ligand-binding domain), receptor-probe binding, and competitive binding of chemicals with the receptor. The binding potency of two environmental pollutants, perfluorooctanesulfonic acid (PFOS) and triphenyl phosphate (TPHP), with peroxisome proliferator-activated receptor gamma (PPAR&#947;) was determined to illustrate the assay procedure. Finally, the advantages and disadvantages of this method and its potential applications were also discussed.

This protocol describes a high-throughput screening system that uses fluorescence polarization of a specific fluorescent probe binding to a nuclear receptor as a readout for screening environmental pollutants.

Increasing levels of compounds have been detected in the environment, causing widespread pollution and posing risks to human health. However, despite ...

Environment

Systems Biology of Metabolic Regulation by Estrogen Receptor Signaling in Breast Cancer

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective mining of the information in the large-scale datasets that are obtained from gene expression microarrays, deep sequencing experiments or metabolic profiling is essential to uncover and then effectively target the critical regulators of diseased cell phenotypes. Estrogen Receptor &#945; (ER&#945;) is one of the master transcription factors regulating the gene programs that are important for estrogen responsive breast cancers. In order to understand to role of ER&#945; signaling in breast cancer metabolism we utilized transcriptomic, cistromic and metabolomic data from MCF-7 cells treated with estradiol. In this report we described generation of samples for RNA-Seq, ChIP-Seq and metabolomics experiments and the integrative computational analysis of the obtained data. This approach is useful in delineating novel molecular mechanisms and gene regulatory circuits that are regulated by a particular transcription factor which impacts metabolism of normal or diseased cells.

Integration of data from genome-wide sequencing experiments and metabolomics experiments is a challenge. In this paper we report, for the first time, generation, analysis and integration of transcriptome, cistrome and metabolome data from breast cancer cells treated with estradiol.

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Biology

Detecting Estrogenic Ligands in Personal Care Products using a Yeast Estrogen Screen Optimized for the Undergraduate Teaching Laboratory

The Yeast Estrogen Screen (YES) is used to detect estrogenic ligands in environmental samples and has been broadly applied in studies of endocrine disruption. Estrogenic ligands include both natural and manmade &#34;Environmental Estrogens&#34; (EEs) found in many consumer goods including Personal Care Products (PCPs), plastics, pesticides, and foods. EEs disrupt hormone signaling in humans and other animals, potentially reducing fertility and increasing disease risk. Despite the importance of EEs and other Endocrine Disrupting Chemicals (EDCs) to public health, endocrine disruption is not typically included in undergraduate curricula. This shortcoming is partly due to a lack of relevant laboratory activities that illustrate the principles involved while also being accessible to undergraduate students. This article presents an optimized YES for quantifying ligands in personal care products that bind estrogen receptors alpha (ER&#945;) and/or beta (ER&#946;). The method incorporates one of the two colorimetric substrates (ortho-nitrophenyl-&#946;-D-galactopyranoside (ONPG) or chlorophenol red-&#946;-D-galactopyranoside (CPRG)) that are cleaved by &#946;-galactosidase, a 6-day refrigerated incubation step to facilitate use in undergraduate laboratory courses, an automated application for LacZ calculations, and R code for the associated 4-parameter logistic regression analysis. The protocol has been designed to allow undergraduate students to develop and conduct experiments in which they screen products of their choosing for estrogen mimics. In the process, they learn about endocrine disruption, cell culture, receptor binding, enzyme activity, genetic engineering, statistics, and experimental design. Simultaneously, they also practice fundamental and broadly applicable laboratory skills, such as: calculating concentrations; making solutions; demonstrating sterile technique; serially diluting standards; constructing and interpolating standard curves; identifying variables and controls; collecting, organizing, and analyzing data; constructing and interpreting graphs; and using common laboratory equipment such as micropipettors and spectrophotometers. Thus, implementing this assay encourages students to engage in inquiry-based learning while exploring emerging issues in environmental science and health.

This article presents an optimized yeast estrogen screen for quantifying ligands in Personal Care Products (PCPs) that bind estrogen receptors alpha (ER&#945;) and/or beta (ER&#946;). The method incorporates two colorimetric substrate options, a six-day refrigerated incubation for use in undergraduate courses, and statistical tools for data analysis.

The Yeast Estrogen Screen (YES) is used to detect estrogenic ligands in environmental samples and has been broadly applied in studies of endocrine ...

Cell Aggregation Assay to Detect Trans Interactions between Cell Adhesion Molecules

Source: Restrepo, S., et al., <a href="https://app.jove.com/v/61237">Measuring Transcellular Interactions through Protein Aggregation in a Heterologous Cell System.</a> J. Vis. Exp. (2020)
In this video, we demonstrate the technique to measure transcellular interactions between synaptic cell adhesion molecules through the HEK cell aggregation assay. In this assay, an equal number of HEK cells expressing a desired presynaptic cell adhesion molecule and a fluorescent protein are mixed with HEK cells expressing a post-synaptic cell adhesion molecule and another fluorescent protein. The interaction between the cell adhesion molecules forces the cells to aggregate, which can be measured by fluorescence microscopy.

Test agregacji komórek w celu wykrycia interakcji trans między cząsteczkami adhezyjnymi komórek

Source: Restrepo, S., et al., Measuring Transcellular Interactions through Protein Aggregation in a Heterologous Cell System. J. Vis. Exp. (2020)
In this ...

Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay

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Rozdziały w tym wideo

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Screening for Phytoestrogens using a Cell-based Estrogen Receptor β Reporter Assay

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

A High-Throughput Screening Approach for Evaluating the Binding Affinity of Environmental Pollutants to Nuclear Receptors

Systems Biology of Metabolic Regulation by Estrogen Receptor Signaling in Breast Cancer

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

Detecting Estrogenic Ligands in Personal Care Products using a Yeast Estrogen Screen Optimized for the Undergraduate Teaching Laboratory

Test agregacji komórek w celu wykrycia interakcji trans między cząsteczkami adhezyjnymi komórek