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How does endometriosis respond to hormones?

Endometriosis is characterized by aberrant cellular responses to the ovarian hormones, estrogen and progesterone.

Estrogen Signaling Dysregulation

There are endogenous estrogen receptors in two forms: ER-alpha and ER-beta. When a healthy cell senses estrogen, the these two estrogen receptor 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

There are also endoegenous 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. To do this, we employed bottom-up synthetic mammalian promoter engineering to create promoters that could interact with the estrogen and progesterone receptors.

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

How can our circuit detect hormones?

An important aspect of synthetic biology is having inducible systems so that the output is not produced constitutively. Since progesterone is a key biomarker of endometriosis and also one of the two components of the menstural cycle. We wanted to use the sensing of progesterone as a way to inhibit our system. In contrast we wanted to use the sensing of estrogen to activate our system. Currently, there had been some research on hormone inducible promoters, but this is largely lacking in the field of synthetic biology. We decided to tackle this problem by developing our own synthetic promoter, which was based off of the key components of the commmonly used synthetic promoter, Tetracyclin Response Element promoter (TRE). We kept the basic promoter elements, but rather than having tetO responsive sites, we used progesterone and estrogen responsive elements (PRE's and ERE's respectively.

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.

MCF7 Induction of pEREx3 - eYFP

How do our promoters behave in larger circuits?

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 met with limited success when transfecting our genetic circuits into various cell lines.