Team:MIT/Experiments/Promoters/Experiment-Details
Promoter Testing Workflow
Our synthetic mammalian promoters were designed and constructed by us, the students, using standard Golden Gate Assembly and Gateway Cloning starting from DNA ordered from IDT. After construction was complete, our promoters were tested in a variety of cell lines under different hormone conditions. Cells were first seeded into a 24 well plate and allowed to grow for one day in order to reach confluency. The next day cells were transiently transfected with a promoter readout plasmid that contained our promoter upstream of eYFP, as well as with a constitutively active transfection marker hEF1a mKate. Depending on the cell line and the ease with which it could be transfected, either cationic lipid transfection or electroporation were used to deliver plasmid DNA to cells.
Twelve to twenty four hours after transfection, cells were induced with either estradiol (E2), an estrogen hormone produce by the ovaries, or medroxyprogesterone actetate (MPA) , a progesterone analog. Twelve to twenty four hours after hormone induction, cells were prepared for flow cytometry in order to probe for the response to hormone on a population scale. Flow cytometry data was then analyzed using the open source Python toolbox, Cytoflow, which enabled us to bin our data by the transfection marker hEF1a mKate. This kind of analysis proves critical for comparing populations of cells that have been transiently transfected, because transient transfection procedures result in an uneven distribution of plasmids across the cell population. Binning the data allows us to compare cells which received roughly the same copy numbers of our two plasmids across different populations. Else, comparing cells that received a greater copy number of the plasmid to those which received less could result in skewing of the data towards a positive result.
Since we plan on deploying our diagnostic in an endometrial biopsy which contains a heterogeneous population of epithelial and stromal cells, it was critical to test the functionality of our promoters in a variety of cell lines. The cell lines our promoters were deployed in include the following:
- MCF7. A standard steroid receptor positive cell line derived from breast adenocarcinoma that was used to prototype our estrogen and progesterone signaling sensors.
- Ishikawa. An epithelial cell line derived from endometrial adenocarcinoma. These cells were used to test how our promoters would function in epithelial tissue from an endometrial biopsy.
- tHESC. A cell line of TERT-immortalized Human Endometrial Stromal Cells. These cells were used to test how our promoters would function in stromal tissue from an endometrial biopsy.
The MCF7, Ishikawa, and tHESC cell lines were provided through a collaboration that we initiated with the Griffith Lab at MIT affiliated with the Center for Gynepathology Research at MIT. Graduate students there advised us on the best practices for culturing cells and what concentrations of E2 and MPA would best simulate the proliferative and luteal phase of the menstrual cycle.
Promoter Architectures
We tested several promoters that contained binding sites (ERE) for the human estrogen receptor and binding sites (PRE) for the human progesterone receptor upstream of a minimal CMV promoter:
- pERE-#n. A promoter that contains n binding sites for ER in front of a minimal CMV promoter. n = 3, 5, 6.
- pPRE-#n. A promoter that contains n binding sites for ER in front of a minimal CMV promoter. n = 3, 4.
- pHybrid. A promoter that contains 5 binding sites for ER and 5 binding sites for ER alternatively spaced. This design roughly preserves distance from the TATA box between binding sites in the hybrid promoter and those tested in other constructs.
Promoter architectures with different copy numbers of binding sites for ER and PR.
Sensing Estrogen Signaling in MCF-7
We first deployed our sensors for estrogen signaling in the standard, easy-to-transfect cell line MCF7. Given that this cell line is known to over-express estrogen receptor, MCF7 provided a convenient platform for demonstrating the functionality of our promoters where we expected to see a large response.
Testing On - Off Functionality of Estrogen Sensitive Promoters
In our first set of experiments in this cell line, we sought to demonstrate our promoters basic on - off functionality by comparing uninduced cells to cells induced with 5 nM of E2. This concentration of E2 was selected as an appropriate "on" or saturating concentration with consideration of the Kd of the estrogen receptor [1], and previous work that had demonstrated functional estrogen inducible promoters with ERE1 - SV40, ERE2 - SV40, and ERE3 - SV40 architectures in MCF7 [2].
Of the constructs we tested, pERE3 demonstrated a 8 fold increase in activity between the induced and uninduced populations; pERE5 demonstrated a 11 fold increase in activity ; pERE6 demonstrated a 12 fold increase in activity . These results support the functionality of our promoters in the MCF7 cell line.
Each of the three panels represent results for promoters pERE3:eYFP, pERE5:eYFP, pERE6:eYFP. 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.
Finer Characterization Studies
In our next set of experiments, we sought to obtain a finer characterization of our promoters 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. We had hypothesized that our promoters would demonstrate a graded response in eYFP production to this graded induction of E2 levels.
Each of the three panels represent the results of fine induction experiments for promoters pERE3:eYFP, pERE5:eYFP, pERE6:eYFP. 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.
Conclusions: 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.
Sensing Estrogen Signaling in ISH
Testing On - Off Functionality of Estrogen Sensitive Promoters
In our next set of experiments, we sought to transfect our promoters into the ISH cell line. pERE5 demonstrated a 3 fold increase in activity between the induced and uninduced populations, and pERE6 demonstrated a 1.7 fold increase in activity. 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.
Each of the two panels represent results for promoters pERE5:eYFP and pERE6:eYFP when tested in ISH. 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.
Finer Characterization Studies
In our next set of experiments, we sought to obtain a finer characterization of the most promising promoter tested in ISH (pERE5) 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.
Each of the three panels represent the results of fine induction experiments for promoters pERE5:eYFP. 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.
Conclusions: One of our promoters was able to successfully sense changes in estrogen signaling in the ISH cell line. The pERE5 promoter demonstrated a 3 fold change upon hormone induction. We have not yet been able to demonstrate a graded response of our promoters to changing E2 levels in ISH.
Sensing Estrogen Signaling in tHESC
Transfecting our promoters into tHESC represented a major milestone in our project, because a sizable fraction of the cells in endometrial biopsies where we would ultimately deploy are diagnostic are derived from the same kind of endometrial stromal tissue that tHESC was derived from.
Testing On - Off Functionality of pERE5 and pERE6
We next sought to transfect our promoters into the tHESC cell line through electroporation. We began by testing our promoters with the greatest copy numbers of binding sites for the ER, pERE5, and pERE6. Unfortunately, we did not observe a clear fold difference for either pERE5 or pERE6 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.
Each of the two panels represent results for promoters pERE5:eYFP, pERE6:eYFP when tested in tHESC. 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.
Finer Characterization Studies for pERE5 and pERE6
We next induced cells transfected with pERE5 and pERE6 to either 2 nM, 10 nM, or 50 nM E2 in an attempt to obtain graded eYFP production. However, we still did not observe a clear fold difference for either pERE5 or pERE6.
The two panels represent the results of fine induction experiments for promoters pERE5:eYFP and pERE6:eYFP . Colored contours represent different levels of E2 induction ranging from 2 nM, 10 nM, to 5 nM.
Demonstrating ERE3's Functionality
Repeating the electroporation experiment done for pERE5 and pERE6 with pERE3 we were able to demonstrate a 9 fold difference between the 50 nM E2 induced population and an EtOH Vehicle Control which the E2 was dissolved in. Given more time, we would like to compare the induced populations in pERE5 and pERE6 to associated vehicle controls.
Results for our induction of pERE3:eYFP in tHESC as compared to an EtOH Vehicle Control. 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.
Conclusions: One of our promoters was able to successfully sense changes in estrogen signaling in the tHESC cell line. The pERE3 promoter demonstrated a 9 fold change upon hormone induction. We have not yet been able to demonstrate a graded response of our promoters to changing E2 levels in tHESC.
Sensing Progesterone Signaling in MCF7
We began by transfecting our sensors for progesterone signaling in the standard, easy-to-transfect cell line MCF7.
Testing On - Off Functionality of Progesterone Sensitive Promoters
In our experiment, we sought to demonstrate our promoters basic functionality by comparing uninduced cells to those induced with 1 uM of MPA. This concentration of MPA was recommended to us as an appropriate "on" or saturating concentration by the Griffith Lab which provided us with these cells. Unfortunately, due to poor transfection, we were unable to get results of the on-off functionality of pPRE3 in MCF7. However, while the results are not conclusive due to the poor transfection efficiency, pPRE4 has approximately a five fold increase in activity when induced with MPA for cells with lower amounts of plasmid as seen by the large fold difference at lower levels of red florescence -- our transfection marker. We can believe that with a better transfection efficiency, the cells that do receive a large amount of plasmids will demonstrate a similar fold difference between on and off when induced with MPA.
The panel above represents results for pPRE4:eYFP transfected into the MCF7 cell line, comparing the uninduced population to a population induced with 1 uM MPA. 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.
Sensing Progesterone Signaling in tHESC
Testing On - Off Functionality of Progesterone Sensitive Promoters
We also sought to demonstrate our progesterone sensitive promoters basic on - off functionality in tHESC by comparing uninduced cells to cells induced with 1 uM of MPA. Unfortunately, neither of our constructs demonstrated a noticeable fold difference upon MPA induction
The two panels represent the results of induction experiments for promoters pPRE3:eYFP, pPRE4:eYFP comparing the uninduced cell population to a population induced with 1 uM MPA.
Dual Sensing of Estrogen and Progesterone Signaling
Testing On - Off Functionality of Hybrid Promoters
We were unable to demonstrate dual sensing of both estrogen and progesterone in one cell line by our hybrid promoter (pHybrid) due to limited experimental time after completing its construction and cloning. However, our hybrid promoter was able to sucessfully sense progesterone signaling in tHESC with up to a 100 fold difference in activity upon MPA induction. As well, our hybrid promoter was able to sucessfully sense estrogen signaling in MCF7 with a 12 fold difference in activity upon induction.
pHybrid promoter transfected into MCF7, comparing the uninduced population to a population induced with 10 nM E2.
pHybrid promoter transfected into tHESC, comparing the uninduced population to a population induced with 1 uM MPA.
Overall Summary of Successful Results
Several of our promoters functioned as expected:
- pERE3, pERE5, pERE6 demonstrated successful estrogen signaling sensing in MCF7.
- pERE5 demonstrated successful estrogen signaling sensing in ISH.
- pERE3 demonstrated successful estrogen signaling sensing in tHESC
- pHybrid demonstrated successful estrogen signaling sensing in MCF7.
- pHybrid demonstrated successful progesterone signaling sensing in tHESC.
- pPRE4 demonstrated limited success of progesterone signaling sensing in MCF7.
Having demonstrated the functionality of our promoters for estrogen and progesterone signaling sensing, we then proceeded to create larger genetic circuits that incorporated our parts.
References
[1] The Binding Database https://www.bindingdb.org
[2] Sathya G, Li W, Klinge CM, Anolik JH, Hilf R, Bambara RA. Effects of multiple estrogen responsive elements, their spacing, and location on estrogen response of reporter genes. Mol Endocrinol. 1997;11(13):1994–2003.
