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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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expression in various degrees. These data directly prove that this part of
Lim3A
regulatory region is significant for
Lim3A
transcription control.
768C
Sequential activation of Pointed isoforms during eye development amplifies EGFR signaling.
Arkadi Shwartz, Eyal D Schejter, Ben-Zion Shilo.
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.
EGFR signaling in flies and in vertebrates involves activation of MAP kinase (MAPK), and culminates in triggering of ETS-domain transcriptional
activators, which in
Drosophila
is executed by Pointed (Pnt). The
pnt
gene harbors two promoters, and generates two alternative transcripts,
pntP1
and
pntP2
, which are under distinct modes of regulation. Transcription of
pntP1
is induced by an unknown MAPK-dependent activator, and PntP1 protein
activates transcription constitutively. PntP2 protein, on the other hand, is directly activated by MAPK phosphorylation, but its transcription is EGFR
independent. We have undertaken a detailed dissection of this process during
Drosophila
imaginal eye disc development. Full disruption of
pnt
function
revealed a photoreceptor recruitment phenotype, in which only R8 cells are present in the ommatidia of newly developed eye discs. Generation of flip-out
mutant clones for each isoform alone showed the same phenotype as complete
pnt
disruption, thus demonstrating that each isoform is essential for
photoreceptor recruitment. Further analysis showed that the Pnt isoforms are activated in a sequential manner: MAPK activates PntP2, which is only capable
of inducing
pntP1
transcription. Once expressed, PntP1 is sufficient to induce target genes essential for photoreceptor development. The induction of EGFR-
target genes may thus be sustained by PntP1 activity, beyond the time window where local MAPK activity is triggered by EGFR.
769A
Identification of a
wingless
pair rule response element.
Kimberly Bell
1,2
, Kevin Chen
1
, J. Peter Gergen
1
. 1) Department of Biochemistry and Cell Biology
and the Center for Developmental Genetics, Stony Brook University, Stony Brook, NY; 2) Graduate Program in Genetics, Stony Brook University, Stony
Brook, NY.
Prior studies on early transcriptional regulation in the Drosophila embryo identified two distinct pair rule response elements for the segment polarity gene
sloppy-paired-1
that interact in a non-additive manner to establish the initial striped expression pattern. These two elements are separated by more than 4 kb
of intervening DNA, suggesting that long distance non-additive enhancer interactions may be a common phenomenon in the pair rule to segment polarity
transition, and transcription regulation in general.
It was previously demonstrated that a 4.5kb region upstream of transcription unit of the segment polarity gene
wingless (wg)
drives expression in odd
numbered stripes with very weak expression of the even number stripes. The ChIP-on-Chip data from the University of California Berkeley Drosophila
Transcription Network Project identifies two regions within the
wg
locus that show association with four different pair rule transcription factors. One of
these regions spans from 3.9 kb to 1.1 kb upstream of the transcription start site and the other is located within an intron. Here we demonstrate that reporter
gene constructs containing the upstream 2.8kb region, in either orientation show robust expression that mimics the early striped pattern of
wg
. Both loss and
gain of function experiments demonstrate that this region is a bona fide pair-rule response element. Reporter expression is observed to come on earlier than
endogenous
wg
, and in some mutant backgrounds there are clear differences in the expression of the
lacZ
and endogenous
wg
mRNAs. We propose that non-
additive interactions between this upstream enhancer and other cis-regulatory sequences from the
wg
locus, potentially the putative pair-rule response
element within the intron, are responsible for generating the full
wg
pattern.
770B
EvoPrinter
and
cis
-Decoder Facilitate Analysis of Enhancer Structure.
Thomas Brody, Alexander Kuzin, Mukta Kundu, Jermaine Ross, Ward F.
Odenwald. Neural Cell-Fate Determinants, NINDS/NIH, Bethesda, MD.
We have developed two computer algorithms to discover and analyze cis-regulatory sequences. The phylogenetic footprinting program
EvoPrinter
identifies conserved sequence clusters (CSCs); functional analysis reveals that many of these serve as cis-regulatory modules.
cis
-Decoder identifies both
unique and repeat sequence elements that are shared among CSCs and are essential for enhancer function. We have identified several enhancers that consist
of multiple sub-modules that function semi-autonomously to drive expression in a sub-pattern of the total expression profile of the CSC. Analysis of a
castor
NB enhancer,
cas-6
, reveals that it consists of two sub-clusters separated by 250 bp of less conserved DNA.
cis
-Decoder reveals several conserved octamer
sequences in one of the sub-regions. One sub-region drives expression in a single pair of NBs per segment and a second, containing the octamer repeats and
a Single-minded site, drives expression in a single ventral midline NB. The entire enhancer expresses both in the midline and in a larger set of NBs,
indicating that the two halves can function semi-autonomously, but that both are necessary for the full biological function of the enhancer. Similarly, we
have found that another
cas
temporal network NB enhancer consists of three semi-autonomous modules that drive expression in a sub-pattern of the total
expression profile of the intact enhancer. Finally, one of the
D. melanogaster
NB enhancers for late temporal network determinant
grainyhead
appears in
several other
Drosophila
species as two separate CSCs. We tested these two regions, and found that one, containing three well-conserved POU transcription
factor binding sites, expresses in embryonic and larval brain precursors, while the second, containing two identical conserved novel 12mer sequences,
expresses in ventral cord NBs but does not express in larval precursors. We conclude that combined
EvoPrinter
and
cis
-Decoder analysis can reveal sub-
modules within CSCs and therefore prove of use in investigating the integrity of CSCs and the evolution of cis-regulatory DNA.
771C
A machine learning approach for identifying novel cell type-specific transcriptional regulators of myogenesis.
Brian Busser
1
, Leila Taher
2
, Yongsok
Kim
1
, Terese Tansey
1
, Ivan Ovcharenko
2
, Alan Michelson
1
. 1) National Heart Lung and Blood Institute, Bethesda, MD; 2) National Library of Medicine,
Bethesda, MD.
A complete understanding of the structure and function of transcriptional enhancers that are active in related cell types requires identification of the
sequence features to which co-regulatory transcription factors (TFs) bind. To address this problem, we developed a computational approach that profiles the
TF binding sites (TFBSs) governing the transcription of a set of co-expressed genes and applied it to discover novel components of the transcriptional
regulatory network controlling myoblast differentiation. Our approach involved assembling a small number of enhancers with activity in somatic muscle
founder cells (FCs). We then used evolutionary profiling to increase the size of this enhancer set by incorporating orthologous but diverged sequences from
other
Drosophila
species. Putative enhancer orthologs were found to be active in largely similar patterns as their
D. melanogaster
counterparts, even though