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Poster Full Abstracts - Regulation of Gene Expression
Poster board number is above title. The first author is the presenter
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there was extensive evolutionary shuffling of known TFBSs. We next built and trained a classifier to identify other functionally related enhancers based on
the presence or absence of known and putative TFBSs. Predicted FC enhancers were over-represented in proximity to known FC genes, and the presence of
a candidate enhancer was predictive of expression of the associated gene in FCs. In addition, we found that many of the TFBSs learned by the classifier are
critical for activity of FC enhancers, including those for POU homeodomain, Myb, Ets, Forkhead and T-box TFs. In particular, we used gene co-expression,
cis-trans tests of enhancer function and embryo RNAi to show that the T-box protein encoded by
optomotor-blind-related-gene-1
is a previously
uncharacterized regulator of muscle cell identity. In summary, machine learning combined with evolutionary sequence analysis can be used to recognize
novel TFBSs and to facilitate the identification of cognate
trans
-acting factors that coordinate cell type-specific developmental gene expression patterns.
772A
Regulatory DNA of the engrailed and invected genes.
Yuzhong Cheng, Judith Kassis. Program in Genomics of Differentiation, NICHD, Bethesda, MD.
engrailed (en) and invected (inv) form a gene complex that extends about 100kb. These two genes encode highly related homeodomain proteins that are
co-expressed in a complex manner throughout development and are co-regulated by enhancers that stretch over a 70 kb region. en/inv are co-regulated by the
Polycomb group genes (PcG), and the entire en/inv domain is covered with tri-methylated histone H3 (H3K27me3), the distinctive mark of PcG-regulated
genes. In addition, en and inv both have Polycomb-response elements (PREs), DNA elements that bind PcG-proteins. We would like to understand how
PREs act with en/inv enhancers to regulate en/inv expression. In order to do this, we need to identify the en/inv enhancers. We used two types of constructs
to identify en/inv enhancers: P-element based reporter constructs with small pieces of en/inv DNA fused to the en promoter driving the expression of lacZ
and large constructs using the phiC31 system and HA-tagged en and inv. We report two important findings. First, the sum of the parts is not equal to the
whole; that is, some of the enhancer activities we see in the small constructs are not recapitulated in the large constructs. Second, imaginal disk enhancer
activity was not found in any small reporter construct, but is present in a 45kb-en-HA transgene. We suggest that the activity of the imaginal disk enhancer
depends on two or more DNA regulatory elements and that chromatin structure may also play a role in its activity.
773B
A Common Sequence Motif Regulates
broad
and
pipe
Expression in Response to EGFR Signaling.
Lily S. Cheung
1,2
, Alisa Fuchs
3
, Enrica
Charbonnier
3,4
, Stanislav Y. Shvartsman
1,2
, George Pyrowolakis
3,4
. 1) Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ
08544; 2) Department of Chemical and Biological Engineering, Princeton University, Princeton NJ 08544; 3) Institute for Biology I, Faculty of Biology,
Albert-Ludwigs-University of Freiburg, Hauptstrasse 1, 79104 Freiburg, Germany; 4) BIOSS Centre for Biological Signalling Studies, Albert-Ludwigs-
University of Freiburg, 79104 Freiburg, Germany.
Activation of the
Drosophila
Epidermal Growth Factor Receptor (EGFR) homolog by Gurken, a ligand secreted from the oocyte, determines the dorso-
ventral (DV) axis of the follicular epithelium and the future embryo. Although multiple transcription factors involved in EGFR gene regulation in the follicle
cells have been characterized, the regulatory DNA sequences that interpret these factors remained unknown. In order to define
cis
-elements responsive to
EGFR signaling, we undertook an unbiased reporter analysis to identify regulatory sequences responsible for the regulation of
broad
, an EGFR target crucial
for the formation of dorsal respiratory structures in the eggshell. We found that the dynamic pattern of
broad
is regulated by two non-overlapping enhancers,
which are differentially regulated by the Iroquois transcription factor Mirror. We show that Mirror directly binds one of the enhancers through a non-
canonical binding site, and that this sequence is conserved in
pipe
, the first component in the cascade that will determine the DV axis of the embryo. We
further show that this sequence is sufficient to account for the cell autonomous repression of
pipe
by Mirror
in vivo
. Our work establishes Mirror as a key
effector of the EGFR pathway, and provides one of the first regulatory sequences controlling patterning during oogenesis.
774C
An In Vivo Titration of Transcription Factors in the Drosophila Embryo.
Matthew D. Davis
1
, Michael B. Eisen
2
. 1) Department of Molecular and Cell
Biology UC Berkeley, Berkeley, CA; 2) Howard Hughes Medical Institute Department of Molecular and Cell Biology UC Berkeley, Berkeley, CA.
The DNA binding domains of transcription factors bind their ligands with varying affinity depending on the sequence of the ligand. The thermodynamic
relationship between these molecules demonstrates classic cooperative Hill kinetics in vitro. In vivo, transcription factors bind their targets with
quantitatively varying specificity that is not well-explained by sequence affinities measured in vitro. Thus, the thermodynamic relationship established in
vitro is poorly understood in vivo, where other proteins and higher order interactions are presumably prevalent. We have titrated the in vivo concentration of
the transcription factors bicoid and Kruppel and measured their relative DNA binding with chromatin immunoprecipitation followed by short-read
sequencing. These experiments allow us to assess which sites in the genome are saturated by these transcription factors when they act in the early embryo,
and which sites are susceptible to differential binding as concentration varies. Similarly, it is unknown if increased activator or repressor bound to an
enhancer region will affect the output of the target promoter. To address this question, we have collected commensurate RNA sequencing and conofcal
microscopy data in these lines.
775A
The
Drosophila Niemann-Pick Type C-2 (NPC2c)
gene is a direct target of VP16-DHR96 protein.
Niloofar Farboodi. Biological Sciences, University of
Alberta, Edmonton, Alberta, Canada.
Excess cholesterol is associated with pathogenesis of cardiac and vascular diseases, diabetes and cancer. However, cholesterol is also essential for proper
functioning and structure of animal cells and therefore its concentrations have to be highly regulated. In
Drosophila
, this regulation occurs in part by a
nuclear receptor called
Drosophila hormone receptor 96
(
DHR96
).
DHR96
binds cholesterol
in vivo
and acts as a cellular cholesterol sensor. We recently
showed that
DHR96
1
mutants fail to survive on a low cholesterol diet, while control flies develop normally. Thus, it is presumed that DHR96 functions by
protecting cellular cholesterol levels from dropping below a critical threshold. Consistently, genes with roles in cholesterol metabolism and trafficking are
not properly regulated in
DHR96
1
mutants. It is unclear; however, which of these genes are direct targets of DHR96 and what DNA sequences are
recognition sites of this nuclear receptor. To approach this question, transgenic lines expressing
DHR96
fused to the activation domain of
VP16
were
generated. We predicted that direct target genes of DHR96 would be significantly induced as a result of VP16-mediated activation.
ACAT
and
NPC2c
were
the two genes with strong upregulation in their transcript levels.
NPC2c
, whose mammalian ortholog has known roles in intracellular cholesterol trafficking,