Part:BBa_K2100001
pENTR pEREx5
pEREx5 is a synthetic mammalian promoter that responds to estrogen receptor activated by estradiol (E2). pEREx5 consists of five repeats of the estrogen response element (ERE) consensus sequence upstream of a minimal promoter (minCMV).
In endometrial cells, estrogen receptors facilitate the cell's response to estrogen, initiating the proliferative phase of the menstrual cycle. Mechanistically, estrogen diffuses through the cell membrane and binds to the ligand binding domain of the ER. The DNA-binding domain of the estrogen receptor becomes exposed. The receptor then relocalizes to the nucleus from the cytoplasm, recruits co-activators, and acts as a transcription factor. There are two isoforms of the estrogen receptor, ER alpha and ER beta. The two differ in their ligand selectivity. In the context of our project, we are working with ER alpha.
Our team tested different possible variations of an estrogen-responsive promoter by varying the amount of estrogen response elements (EREs) present for the activated ER complex to bind to. This particular construct contains five estrogen responsive elements with the sequence 5′-GGTCAnnnTGACC-3′. We interspaced these elements with 22 randomly selected bases in between, the same spacing between TREs in the TRE promoter. As noted from the results of the paper Klinge et. al. 1997, increasing the number of EREs does not correlate with an increase in transcriptional activity, possibly due to helical turns in the DNA that change the orientation of an ERE depending on the number and spacing of them. When an ERE is not properly oriented, it has trouble interacting with ERα and polymerases. Since we wanted to decrease the probability of helical turns inhibiting the transcriptional activity of promoters, we modeled the spacing after the TRE promoter.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 6
Illegal EcoRI site found at 271
Illegal EcoRI site found at 281 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 6
Illegal EcoRI site found at 271
Illegal EcoRI site found at 281 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 6
Illegal EcoRI site found at 271
Illegal EcoRI site found at 281 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 6
Illegal EcoRI site found at 271
Illegal EcoRI site found at 281 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 6
Illegal EcoRI site found at 271
Illegal EcoRI site found at 281 - 1000COMPATIBLE WITH RFC[1000]
We used our synthetic promoter to build multiple constructs including:
- pERE5:eYFP to characterize its base level functionality across the cell lines
- pERE5:TALER14 and pERE5:BM3R1 to characterize the cascade of our promoter with different repressors
- pERE5:TP901 to characterize the cascade of our promoter with a serine recombinase
First, we characterized the synthetic ERE5 promoter in three cell lines: MCF-7, ISH, and tHESC. All cell lines have endogeneous Estrogen Receptor alpha. We analyzed data from cells induced with estradiol (E2) and uninduced as a control. The estradiol is diluted and mixed with ethanol at small percents, so we also tested an ethanol vehicle to account for the proliferation the cells undergo after being induced.
Experiment in MCF-7:
We transfected MCF-7 cells with 250ng of hEF1a:mKate as a transfection marker and 250 ng pERE5:eYFP to examine the promoter's transcriptional activity by observing increases in yellow fluorescence upon cells being induced with 5 nM E2. This ratio was chosen to be 1:1 based on the small amount of plasmids being transfected.
The y-axis represents the measured yellow fluorescence intensity from the eYFP on our reporter plasmid, whereas the x-axis represents the measured red fluorescence intensity from the mKate on our constitutively active transfection marker. Since transient transfection results in an uneven distribution of plasmids, it is important to bin our data by transfection marker so that cells which received roughly the same number of plasmids can be compared against one another.
The results show an 11 fold difference in yellow fluorescent output between the induced MCF-7 cells and the uninduced cells, which improves on the results seen in Klinge et al. [1] for three estrogen responsive elements.
Additionally, in MCF7 we attempted to stratify the amount of activation of our promoter based on the amount of estrogen used to induce the cells. We ran an experiment where we kept the previously mentioned 1:1 ratio of transfection marker to pERE5:eYFP, but induced with varying levels of estrogen at .25 nM, .5 nM, 1 nM, 2.5 nM, 5 nM, 10 nM. We had hypothesized that our promoters would demonstrate a graded response in eYFP production to this graded induction of E2 levels.
For the plot above, the y axis measures yellow fluorescent output and the x axis measure red fluorescent output from the transfection marker. The colored contours represent different levels of E2 induction ranging from 0.25 nM to 10 nM. The pink contour in each graph represents the uninduced population. We did not observe a graded response in eYFP production in response to the sweep of E2 induction, instead observing saturation at 0.25 nM E2. We hypothesize that, because MCF7 overexpresses the estrogen receptor, relatively small E2 signals can still be transduced to large responses.
Our promoters were able to successfully sense changes in estrogen signaling in the MCF7 cell line. All three promoters demonstrate a fold increase of different magnitude upon exposure to estrogen. We have not yet been able to demonstrate a graded response of our promoters to changing E2 levels in MCF7. Instead we observed saturation at our lowest concentration tested, .25 nM.
Experiment in tHESC:
We transfected tHESC cells with 250ng of hEF1a:mKate as a transfection marker and 250 ng pERE5:eYFP (the same 1:1 ratio as the experiments ran in MCF-7) to examine the promoter's transcriptional activity by observing increases in yellow fluorescence upon cells being induced with 50 nM E2. This was to test the on-off functionality of our promoter pERE5 in the tHESC cell line.
The y-axis represents the measured yellow fluorescence intensity from the eYFP on our reporter plasmid, whereas the x-axis represents the measured red fluorescence intensity from the mKate on our constitutively active transfection marker.
Unfortunately, we did not observe a clear fold difference for pERE5 upon induction with 10 nM E2. We suspected that perhaps we had not induced the system with enough E2 and included 50 nM in our finer sweep of E2 levels.
We also attempted to demonstrate a graded response of our promoters to changing E2 levels in tHESC.
We induced cells transfected with pERE5 to either 2 nM, 10 nM, or 50 nM E2(represented by the color contours in the above plot) in an attempt to obtain graded eYFP production. However, we still did not observe a clear fold difference for pERE5:eYFP.
Experiment in ISH:
We additionally transfected ISH cells with 250ng of hEF1a:mKate as a transfection marker and 250 ng pERE5:eYFP (the same 1:1 ratio as the experiments ran in MCF-7 and tHESC) to examine the promoter's transcriptional activity increase when induced with estrogen.
The y-axis represents the measured yellow fluorescence intensity from the eYFP on our reporter plasmid, whereas the x-axis represents the measured red fluorescence intensity from the mKate on our constitutively active transfection marker.
Our promoter pERE5 demonstrated a 3 fold increase in activity between the induced and uninduced populations. We suspect the differences in promoter activity between this transfection and those done in MCF7 are due to different basal levels of ER in the two different cell lines.
We also attempted to stratify the response of our promoter to varying levels of estrogen in ISH. We sought to obtain a finer characterization through exposing transfected cells to a sweep of E2 concentrations including 0.25 nM, 0.5 nM, 1 nM, 2.5 nM, 5 nM, 10 nM. Just like before, we had hypothesized that our promoters would demonstrate a graded response to estrogen induction.
Colored contours represent different levels of E2 induction ranging from 0.25 nM to 10 nM. The pink contour in each graph represents the uninduced population.
We did not observe a graded response in eYFP production in response to the sweep of E2 induction, instead observing saturation at 0.25 nM E2 just as seen with MCF7.
Cascade of pERE5:BM3R1 and pERE5:TALER14
Next we experimented with our promoter pENTR pERE5 cloned with repressors (pENTR TALER14 and pENTER BM3R1) to test the functionality of the promoters in a cascade. These experiments entailed multiple plasmids to be activated: pERE5:TALER14 or BM3R1 (500ng), pTALER14 or pBM3R1:EYFP (170ng), hEF1a:mKate (170ng), hEF1a:Gal4VP16 (170ng), which is approximately a 3:1:1:1 ratio of plasmids.
(1) Estrogen diffuses into the cell and binds with the estrogen receptor. (2) Estrogen receptors will homodimerize with one another forming an activation complex. (3) Estrogen receptor will bind to our synthetic promoter (4) Production of repressor protein (5) Repressor binds to binding sites upstream of an eYFP reporter (6) Transactivator Gal4-VP16 is constitutively produced (7) Gal4-VP16 binds to sites on pRep (8) eYFP is produced as readout depending upon how active repressors are (9) Constituively active transfection marker hEF1a:mKate allows us to bin and analyze the data.
Our estrogen sensitive promoters respond to increases in E2 levels by producing more of the repressor. The repressors then bind to binding sites in a promoter upstream of fluorescent reporter eYFP. The constitutively active trans-activator Gal4-VP16 sets a large basal eYFP expression when there is no repressor, so that a measurable drop in signal can be observed when repressors are active. Constituvely active hEF1a mKate serves as a transfection marker by which we bin our data.
The first cell line in which we deployed our genetic circuit was ISH, the endometrial epithelial cell line. We had expected eYFP expression to decrease after induction of our promoter - repressor cascades with E2.
Blue contours represent the cell population that was left uninduced, green contours represent the cell population that was induced with 5 nM E2.
However, we were unable to resolve a clear fold difference between the uninduced and induced population in any of the pERE5 and TAL14, BM3R1 cascades. This is probably an artifact of poor transfection in the ISH cell line for this experiment (less than 2 percent transfected after cationic lipid transfection), which leads to erratic jumps in the data after binning by constitutive marker. In the future, we may want to try other modes of transfection for ISH to improve the transfection efficiency.
We next proceeded to deploy this experiment in MCF7. We hypothesized that we were unable to resolve a clear fold difference in our pERE5 - repressor cascades transfected into ISH because of the limited functionality of our promoters in the ISH cell line. So, we proceeded to transfect our cells into the MCF7 cell line where we had observed up to a 11 fold difference in the activity of some of our promoters.
Blue contours represent the cell population that was left uninduced, green contours represent the cell population that was induced with 5 nM E2.
Similarly, we had expected eYFP expression to decrease after induction of our promoter - repressor cascades with E2. However, we were still unable to resolve a clear fold difference between the uninduced and induced population in any of the pERE5 and TAL14, BM3R1 cascades. Given more time, we would like to explore whether transfecting our entire circuit on one plasmid instead of four separate plasmids would lead to better results.
Cascade of pERE5:TP901
Also, we experimented with our promoter pENTR pERE5 cloned with a serine recombinase to test the functionality of the promoters in a cascade. These experiments entailed multiple plasmids to be activated: pERE5:TP901, hEF1a:Flipped_eYFP-recombinase sites, and a transfection marker, hEF1a:BFP. Upon activation of TP901, the inverted eYFP gene flanked by recombinase recognition sites is flipped to the correct orientation and expresses fluorescence. We tested these constructs in the cell line MCF7.
We have previously demonstrated activation of TP901 under the inducible promoter EGSH, which is activated by transactivator VgEcR along with estrogen analog PonA. Despite high levels of TP901 basal expression, we observed a clear difference in activation between the induced and uninduced wells. We expected to see similar results in this experiment with TP901 under estrogen inducible promoters.
For our transfection experiment, we induced half the wells with 5.0 nM E2 in order to compare on vs. off states of the promoter.
Unfortunately, we had poor transfection efficiency in this experiment, and thus the results are inconclusive. The data showed no clear fold difference between the induced and uninduced populations. We would like to try this experiment again in the future to get better results.
Overall, our promoter pERE5 demonstrates extremely successful fold differences when induced with estrogen in multiple cell lines. With more time, we believe that our promoter will be able to have a stratified induction series of estrogen concentrations and will also be successfully cascaded with various genes leading to activation differences in the genes since the promoter has a significant fold difference between induced and uninduced.
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