Team:MIT/Experiments/Promoters

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How does endometriosis respond to hormones?

Typical, healthy cells of the endometrium will respond in a coordinated fashion to the ovarian hormones, estrogen and progesterone, in order to create the cycles of cell turnover and growth characteristic of the menstrual cycle. Endometriosis, however, is characterized by aberrant cellular responses to estrogen and progesterone that ultimately lead to the disease phenotype.

Estrogen Signaling Dysregulation

Endometrial cells express endogenous estrogen receptors in two forms: ER-alpha and ER-beta. When a healthy cell senses estrogen, these two estrogen receptors will be activated and trigger downstream responses by binding to sequences in the genome known as estrogen responsive elements (EREs). In diseased or endometriotic cells, estrogen signaling is pathologically upregulated leading to proliferation and migration of cells outside the uterus. [1]

Progesterone Resistance

Endometrial cells express endogenous progesterone receptors in two forms: PR-A and PR-B. When a healthy cell senses progesterone, its PR receptors are activated and trigger downstream responses by binding to different sites in the genome known as progesterone responsive elements (PREs). However, in a diseased cell, while progesterone is present, it does not co-activate the progesterone receptors, and in turn does not result in any downstream effects. This disruption in the cell's normal response to progesterone is known as progesterone resistance. Research has implicated perturbations in key progesterone signaling intermediates such as HOXA10, FOX01, NFkB in causing progesterone resistance [1].

Hence, in order to resolve the diseased state, it was important for us to create sensors for estrogen signaling and progesterone signaling. Functional estrogen and progesterone signaling sensors could label a cell having both pathologically upregulated estrogen signaling and pathologically downregulated progesterone signaling as diseased.

[1] http://www.nature.com/nrendo/journal/v10/n5/full/nrendo.2013.255.html

How can our circuit detect hormones?

To create sensors for estrogen and progesterone signaling, our team decided to create synthetic mammalian promoters that could interact with the estrogen and progesterone receptors. We based our design off of a standard inducible system in mammalian synthetic biology, the TRE_tight promoter. Where the TRE_tight promoter has tetO binding sites for the reverse tetracycline-controlled trans-activator (rtTA) to bind, we replaced them with binding sites for the estrogen receptor, EREs, and binding sites for the progesterone receptor, PREs. We preserved the same minimal promoter used in the TRE_tight promoter, minCMV, derived from the cytomegalovirus.

Do our synthetic promoters work?

We demonstrated the success of our progesterone and estrogen inducible mammalian promoters in a variety of cell lines under different conditions:

• 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.

Figure . MCF7 cells above are transfected with our construct pEREx3 - eYFP, which contains three binding sites for the estrogen receptor upstream of a minimal promoter and yellow fluorescent gene. Cytometry data demonstrated an 11 fold increase in the transcriptional activity of our promoters when transfected into the MCF7 cell line. Since our transfection efficiency was poor (< 10%) in several of the more difficult-to-transfect cell lines we worked with, we relied primarily on the cytometry data linked below to reach conclusions about the functionality of our promoter instead of the brightfield imaging shown here.

Figure . A sampling of some of the cytometry data used to confirm the function of our promoters in various cell lines. Shown here is cytometry data of promoter function in MCF7 - full extended results for all promoters linked below.

Read more about the extended series of experiments testing the functionality of our promoters.

How do our promoters behave in larger circuits?

The true test of a robust part is its consistent behavior in larger genetic circuits. So, after demonstrating the functionality of our estrogen and progesterone inducible promoters, we next sought to create larger genetic circuits with them. We built estrogen inducible promoter - repressor cascades, as well as estrogen inducible promoter - recombinase and only met with limited success when transfecting our genetic circuits into various cell lines.

Figure . One of the larger genetic circuits we created for testing the behavior of our promoters in promoter-repressor cascades.

Read more about our experiments testing the functionality of our promoters in larger genetic circuits.