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Poster Full Abstracts - Regulation of Gene Expression
Poster board number is above title. The first author is the presenter
334
756C
Drosophila GAGA Factor is required for full activation of dE2F1 and Yki/Sd common targets in the wing.
Battuya Bayarmagnai
1
, Brandon Nicolay
1
,
Abul Islam
2
, Nuria Lopez-Bigas
2
, Maxim Frolov
1
. 1) Biochem & Mol Gen, Univ Illinois-Chicago, Chicago, IL; 2) Research Unit on Biomedical
Bioinformatics, Dept of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona 08003, Spain.
The Hippo signaling pathway regulates organ size: inactivation of the Hippo pathway or hyper-activation of its most downstream effector, transcriptional
co-activator Yorkie (Yki), leads to tissue overgrowth. Yki relies on its binding partners, including Scalloped (Sd), to recruit it to its target gene promoters.
However, this interaction only partially explains the mechanism of function of Yki. We recently showed that Yki/Sd cooperates with dE2F1 on a common
set of target genes to bypass cell cycle exit. Bioinformatics analysis suggested that GAGA Factor (GAF) may also play a role in regulating these genes.
Consistently, genome-wide location analysis of promoters bound by GAF (modENCODE) revealed significant enrichment for cell cycle targets common to
dE2f1 and Yki/Sd. Here we demonstrate that GAF, Yki/Sd and dE2f1, directly activate a common set of cell cycle genes. Reduction of GAF levels by RNAi
lead to a smaller wing, and lower expression of the identified subset of dE2f1, Yki/Sd cell cycle target genes. Furthermore, depletion of GAF rescued a lethal
phenotype induced by the overexpression of activated Yki in the wing. Consistently, Sd expression is essential for wing development. The interaction of
GAF with Yki/Sd was also observed in the eye, where expression of Yki induced ectopic proliferation, and co-depletion of GAF reduced the number of
interommatidial cells. GAF and RBF1, the negative regulator of dE2f1, co-localized on polytene chromosomes and bound common target gene promoters.
Furthermore, a direct physical interaction was revealed in co-immunoprecipitation experiments. Therefore, we propose that GAF is required for the full
activation of cell cycle targets common to dE2f1 and Yki/Sd, and suggest that GAF does so by physically interacting with RBF1, thereby limiting RBF1-
mediated repression of these genes.
757A
X-Signal amplification by Runt mediated antagonism of Groucho.
Sharvani Mahadevaraju, James W Erickson. Dept of Biol, Texas A&M Univ, College
Station, TX.
Runt is one of the X-linked signal elements (XSEs) that signals X chromosome dose to the establishment promoter (
Pe
) of the sex determination switch
gene
Sex-lethal
(
Sxl
). The mechanism of how a two-fold difference in X dose, and XSE protein concentrations, leads to the all-or-none response of
SxlPe
remains unknown. We are testing the idea that X-dose-sensitivity is achieved via a signal amplification mechanism that depends primarily on the maternal
co-repressor Groucho (Gro). In the absence of Gro,
Sxlpe
is expressed nearly in direct proportion to the X chromosome dose in both sexes. Several lines of
evidence led us to the hypothesis the Runt has a direct role in X signal amplification by interfering with Gro-dependent repression. Runt and other Runx
family members can act as both repressors and activators. Runt has conserved DNA binding domain and a C-terminal WRPY motif that interacts with Gro
co-repressor proteins. We tested the notion that Runt acts as an activator at
SxlPe
by antagonizing Gro through its WRPY motif by generating
runt
transgenes carrying runt with modified C-terminal Gro-interacting sequences. A
runt
transgene lacking the WRPY motif failed to activate
SxlPe
expression.
In contrast when the WRPY domain was replaced with the more potent Gro interaction domain, WRPW, Runt’s
Sxl
activation function was retained.
Because Runt’s XSE function at
SxlPe
may not require sequence-specific DNA binding we are testing the idea Runt can act independent of both of its
normal heterodimerization partners, the Bro, or Bgb, subunits. Neither maternal, nor zygotic,
Big-brother
(
Bgb
) or
brother
(
bro
) mutants have any effect at
Sxl
activation suggesting either that the genes are redundant or unnecessary for Runt function at
SxlPe
. We are attempting to create
Bgb bro
double mutants
using male recombination to determine whether either is required for
Sxl
activation.
758B
Nemo phosphorylates Eyes absent and enhances output from the Eya-Sine oculis transcriptional complex during Drosophila retinal determination.
Santiago A. Morillo
1
, Lorena Braid
2
, Esther M. Verheyen
2
, Ilaria Rebay
1,3
. 1) Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL; 2)
Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada; 3) Ben May Dept for Cancer Research, University of Chicago,
Chicago, IL.
The retinal determination gene network comprises a collection of transcription factors that respond to multiple signaling inputs to direct Drosophila eye
development. Previous genetic studies have shown that
nemo
, a gene encoding a proline-directed serine/threonine kinase, can promote retinal specification
through interactions with the retinal determination gene network, although the molecular point of cross-talk was not defined. We report that the Nemo kinase
positively and directly regulates Eyes absent (Eya). Genetic assays show that Nemo catalytic activity enhances Eya-mediated ectopic eye formation and
potentiates induction of the Eya-Sine oculis (So) transcriptional targets
dachshund
and
lozenge
. Biochemical analyses demonstrate that Nemo forms a
complex with and phosphorylates Eya at two consensus mitogen-activated protein kinase (MAPK) phosphorylation sites. These same sites appear crucial for
Nemo-mediated activation of Eya function in vivo. We propose that Nemo phosphorylation of Eya potentiates its transactivation function to enhance
transcription of Eya-So target genes during eye specification and development.
759C
Involvement of Polycomb/Trithorax group proteins in the regulation of the sex determination master switch,
Sex-lethal
.
Janel Rodriguez, Jamila
Horabin. Biomedical Sciences, Florida State University, Tallahassee, FL.
In
Drosophila
, one of the earliest developmental decisions made is that of determining sex. Key to the process of sex determination is the regulation of the
X chromosome sensing promoter of the master switch
Sex-lethal
(
Sxl
), at the establishment promoter
Sxl
Pe
.
Sxl
Pe
is only activated in female embryos
resulting in SXL protein. Splicing of the late
Sxl
transcripts from the maintenance promoter,
Sxl
Pm
, is then switched by SXL from the default male mode into
the female mode, to bypass inclusion of the male specific exon which prematurely truncates the translation open reading frame. Our lab has previously
shown that heterochromatin proteins are required for proper
Sxl
Pe
regulation. In analyzing the role of these proteins on the sex determination decision, we
have found that the Polycomb/Trithorax (Pc/Trx) group proteins also have an influence. Pc/Trx group proteins are chromatin-modifiers, known regulators of
gene expression. We have investigated the role of enhancer of zeste (E(Z)), absent, small or homeotic discs 1 (ASH1) and the heterogeneous nuclear
ribonucleoprotein
Drosophila
dodeca satellite protein 1 (DDP1) in regulating
Sxl
Pe
. DDP1 is known to regulate gene expression by interacting with
heterochromatin protein 1 (HP1) in an RNA dependent manner. We observe that E(Z), ASH1 and DDP1 are necessary for proper
Sxl
Pe
expression in female
embryos. Using chromatin immunoprecipitation (ChIP) assays we also observe that these proteins are necessary for proper histone 3 lysine 4 (H3K4) and