(12h) 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.