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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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was further examined to identify functional elements required for transcriptional activation. In a transgenic approach, a 1.7 kbp fragment upstream of
Npc2c
resulted in a 10-fold induction in a
lacZ
reporter gene, suggesting a regulatory role for this region. Chromatin immunoprecipitation was performed to identify
VP16-DHR96 DNA binding sites upstream of
NPC2c
. The highest enrichment level was found at about 2.6 kbp upstream of
Npc2c
transcription start site;
however, regions closer to this transcription start site were also enriched in the chromatin immunoprecipitate. These data demonstrate that VP16-DHR96
fusion protein binds
NPC2c
upstream sequences and consequently nominate this gene as a direct target of DHR96
in vivo
.
776B
REDfly:
The Regulatory Element Database for
Drosophila
.
Marc S. Halfon
1,2,3,4
, Steven M. Gallo
2,5
, Michael Simich
1,2
, Benjamin Des Soye
1,2
, Casey M.
Bergman
6
. 1) Department of Biochenistry, SUNY at Buffalo, Buffalo, NY; 2) NYS Center of Excellence in Bioinformatics & Life Sciences, Buffalo, NY; 3)
Department of Biological Sciences, SUNY at Buffalo, Buffalo, NY; 4) Molecular, Cellular, & Developmental Biology Department, Roswell Park Cancer
Institute, Buffalo, NY; 5) Center for Computational Research, SUNY at Buffalo, Buffalo, NY; 6) Faculty of Life Sciences, University of Manchester,
Manchester, UK.
The REDfly database is a highly-curated portal for
Drosophila cis
-regulatory data containing records for empirically validated
cis
-regulatory modules
(CRMs, “enhancers”) and transcription factor binding sites (TFBSs) curated from the published literature. REDfly includes any sequence reported as
functionally tested in a transgenic reporter gene assay regardless of whether it showed regulatory activity or has activity redundant with other, shorter
regulatory sequences. Graphical views show the position of each CRM within its genomic locus, the location of each CRM with respect to its associated
gene is provided, and conservation of local synteny between CRMs and their target genes across nine species of
Drosophila
is assessed. Curation of TFBSs
includes sites identified by electrophoretic mobility shift assay (EMSA, “gel shift”) and DNAase I footprinting. Extensive abilities exist for database
searching and results filtering. We have undertaken a major increase in curation activity over the past year: over 40% of records have been added within the
past six months, including over 1400 new reporter construct entries and over 400 new TFBS entries. In all, REDfly contains more than 4250 records of
reporter constructs and TFBSs drawn from over 560 publications. REDfly provides a comprehensive source of
Drosophila cis
-regulatory data and is a
powerful platform to facilitate high-throughput experimental and computational studies of gene regulation. REDfly is freely accessible at
http://redfly.ccr.buffalo.edu.
777C
Cis-regulatory contributions to the regulation of sloppy-paired-1 transcription initiation and elongation.
Saiyu Hang, J. Peter Gergen. Biochemistry
and Cell Biology and the Center for Developmental Genetics, Stony Brook University, Stony Brook, NY.
The Drosophila segmentation pathway provides a valuable system to study the in vivo mechanisms of transcription regulation. The expression of sloppy-
paired-1 (slp1) in the gastrula stage embryo is controlled by the interaction of 4 transcription factors Runt, Eve, Ftz, and Opa with 2 cis-regulatory elements
DESE and PESE. Both of the enhancers can be repressed by Runt, whereas only DESE mediates Runt-dependent activation. Runt and Opa activate DESE in
cells in the posterior half of odd-numbered parasegments, while Runt and Ftz repress DESE in cells in the anterior half of the even-numbered parasegments.
Using site-specific transgenesis and a number of reporter constructs, I found DESE facilitates pre-initiation complex formation at the promoter and that
DESE-dependent activation is influenced by the extent of promoter proximal DNA upstream of the transcription start site. Chromatin IP experiments that
compare activated versus repressed states of slp1 as well as a DESE-lacZ reporter gene indicate that Runt and Ftz repress expression by blocking the
elongation step of the transcription cycle which involves the regulated association of the elongation factor P-TEFb and phosphorylation of Ser2 in the C-
terminal domain of RNA polymerase II.
778A
Overlapping but distinct roles for Odd-paired and Unpaired in transcription activation in the Drosophila blastoderm embryo.
Michael L. Higgins
1,2
,
Liujing Xing
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 Biochemistry and Structural Biology, Stony Brook University, Stony Brook, NY.
The Drosophila sloppy-paired-1 (slp1) gene provides an attractive model for investigating the mechanisms of regulation by the primary pair-rule
transcription factor Runt. The slp1 expression pattern consists of 14 two-cell wide stripes in the posterior half of each parasegment in the early Drosophila
embryo. Runt works with the Zn-finger transcription factor Odd-paired (Opa), to activate the odd-numbered stripes, but the factor responsible for activation
of the even-numbered stripes is not yet established and has been referred to as Factor X. We present genetic experiments indicating that both Opa and D-
Stat, a transcription activator in the Drosophila JAK-STAT pathway, contribute to Factor X activity. The changes in expression of slp1 and different slp1-
lacZ reporter genes in embryos that are mutant for opa alone, unpaired (unpaired encodes a ligand that activates the JAK-STAT pathway) alone, and
embryos doubly mutant for these two factors reveal that Opa plays a major role in activating both even-numbered and odd-numbered stripes whereas JAK-
STAT signaling results in activation of the even-numbered stripes when opa is not present. We have identified DESE and PESE as two distinct pair-rule
response elements of slp1, both of which are capable of generating the even-numbered stripes. Interestingly, only DESE mediates activation in response to
JAK-STAT signaling. We will present results suggesting that JAK-STAT dependent activation of DESE is Runt-independent, and that the Runt-dependent
activation of DESE occurs via a distinct and mutually exclusive pathway. We hope that defining the specific roles of Runt, Opa and D-Stat in slp1 activation
will provide a foundation for future studies that are relevant to understanding the roles of homologs of these transcription factors in human development and
disease.
779B
A systems-level analysis of
giant
regulation in
Drosophila melanogaster
.
Astrid Hoermann, Damjan Cicin-Sain, Hilde Janssens, Johannes Jaeger.
EMBL/CRG Research Unit in Systems Biology, Centre de Regulació Genòmica, 08003 Barcelona, Spain.
Eukaryotic transcription is very complex, and we are far from a satisfactory biochemical understanding of it. We aim to use data-driven mathematical
modeling to investigate how different binding sites form a regulatory element, and how these elements then together establish the expression pattern of the
endogenous gene. These questions are addressed by quantitative analysis and mathematical modeling of the expression pattern of the gap gene
giant
(
gt
) in
the
Drosophila melanogaster
blastoderm. It is expressed in a posterior and an anterior domain, which refines into two stripes over time, and finally also