Using a Synthetic Gene Network to Model and Understand the Effects of Shuttling On Gene Expression Patterns

Using a Synthetic Gene Network to Model and Understand the Effects of Shuttling on Gene Expression Patterns

Gene Regulation Engineering

Session 15C23

Ashley A. Jermusyk and Gregory T. Reeves

In multicellular organisms, cellular signaling events are crucial for patterning tissues, as well as for maintaining healthy adult tissues, while improper signaling can lead to disease states, such as cancer.  Therefore, cellular signaling processes must be tightly regulated.  A complex system of gene regulatory circuits controls this signaling process and acts to buffer this system against noise, thereby minimizing mistakes in gene expression and preventing patterning defects or disease states.  Despite their importance to patterning and development, hypotheses regarding these gene regulatory circuits have been difficult to test experimentally due to their complexity and high connectivity.  Therefore, to better understand the fundamental processes involved, we created a synthetic gene network in the fruit fly Drosophila melanogaster.  This approach has the advantages that (1) the gene network is orthogonal to native Drosophila biology, and (2) the network is designed.  These two aspects imply the connectivity of the network is understood, and thus hypotheses regarding the designed network motif can be experimentally tested with this system.

As a multicellular organism, the fruit fly is an ideal system specifically for testing hypotheses regarding multicellular phenomena, such as tissue patterning.  The fundamental pattern in developing tissues is established by morphogens.  Morphogens determine the domains of gene expression in developing tissue by activating gene expression based on concentration thresholds.  Morphogen gradients are formed by localized protein production and subsequent diffusion and degradation.  A second protein can bind to the morphogen to facilitate transport and thus broaden the morphogen gradient.  It has been proposed that this “shuttling” mechanism can increase the robustness of gene expression (Eldar et al., 2003; Haskel-Ittah et al., 2012), but this hypothesis is difficult to test experimentally.  Therefore, we have created a synthetic network to test the effects of shuttling utilizing genes from yeast and E. coli, namely, gal4, gal80, and lacZ.  We expressed gal4 in a graded fashion along the anterior-posterior axis of the embryo, mimicking the intracellular diffusion of an endogenous transcription factor and morphogen, Bicoid.  As seen below in Figure 1, the Gal4 protein activates expression of UAS-linked gal80 and lacZ.  Gal80 binds to Gal4, preventing Gal4 from binding to UAS and activating expression of gal80 and lacZ, and this sequestration should create a negative feedback loop in our system.  However, due to the spatial dynamics of the system, Gal80 instead acts to facilitate the diffusion of Gal4, thereby creating a shuttling mechanism.  The effects of this shuttling mechanism on the network can be determined by looking at lacZ expression, specifically changes in expression due to the addition of Gal80 to the network.  These genes were chosen since they are endogenous to yeast (and therefore not to Drosophila), so all interactions in this network are fully understood.  Our goal is to measure the effect of Gal80-mediated Gal4 shuttling on the robustness of the location of the lacZ domain.  This system provides a direct experimental test of the effects of shuttling in cellular signaling events, specifically whether this shuttling can lead to increases in robustness of the system.  It is this robustness that is important for combating diseases and defects in development and maintenance of expression in the organism.

Figure 1. Network Diagram

Representation of the interactions in the studied network where arrows represent activation and flat arrowheads denote repression by binding of Gal80 to Gal4.

 

References

Eldar A, Rosin D, Shilo B, Barkai N. 2003. Self-enhanced ligand degradation underlies robustness of morphogen gradients. Dev Cell 5(4):635-46.

Haskel-Ittah M, Ben-Zvi D, Branski-Arieli M, Schejter ED, Shilo B, Barkai N. 2012. Self-organized shuttling: Generating sharp dorsoventral polarity in the early drosophila embryo. Cell 150(5):1016-28.