Basic HTML Version
Poster Full Abstracts - Regulation of Gene Expression
Poster board number is above title. The first author is the presenter
336
York Univ, New York, NY.
In the Drosophila eye, each ommatidium contains eight photoreceptors (PRs) arranged in a trapezoid shape. PR cell fates are specified at late larval stages.
After their recruitment, they are defined as outer PRs (R1-R6) vs. inner PRs (R7 and R8), which are located at the center of the trapezoid formed by the outer
PRs. Then, during late pupal stages, all PRs undergo terminal differentiation and express specific Rhodopsins (Rh). Even though outer PRs all express Rh1,
each of them can be distinguished by a unique transcriptional profile. Although some of the genetic programs controlling outer/inner fate determination are
well characterized, many regulatory steps remain unclear. To address the underlying mechanisms, we performed an RNAi screen knocking down all known
transcription factors (~950 TFs) and using rh1-GFP to visualize patterning phenotypes of PRs in the adult eye. The expression of UAS-RNAi is controlled
spatially and temporally by a combination of two eye-specific GAL4 drivers, ey-Gal4 and lGMR-Gal4.By using rh1>GFP as a readout, couple of genes has
been found with a mutant cell fate phenotype showing extra rh1-expressing PRs. Inner rhodopsins are normally expressed in the ectopic rh1-GFP expression
PR suggests those candidates function in repression of rh1 in inner PRs rather than in the determination of inner/outer fate. Further study shows that only de-
repression of rh1-GFP has been detected in mutant candidates rather than Rh1 protein. The result leads to the explanation that rh1 might be post-
transcriptionally regulated.
765C
Characterizing the Transcriptional and Metabolic Response to Hypoxia in
Drosophila Melanogaster
.
Yan Li
1
, Catherine Dumur
2
, Keith Baker
1
. 1)
Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA, 23298; 2) Department of Pathology, Virginia
Commonwealth University, Richmond, VA, 23298.
Low oxygen tension plays important roles in normal and pathological conditions, including tumorigenesis. The cellular response to hypoxia shifts
metabolic homeostasis at the transcriptional and post-transcriptional level. Hypoxic adaptation is thought primarily to proceed through the conserved
hypoxia-inducible factor 1(HIF-1), which is a member of the PAS domain family. Although HIF-1 biology has been examined extensively, HIF-mediated
metabolic transitions have never been characterized. In this study, we examine the response to low oxygen conditions in
Drosophila
. We measure the global
temporal-dependent changes in transcripts and metabolites in 4% hypoxia in wild type or
hif-1
mutants using microarray analysis and GC-MS and/or
LC/MS/MS. Unexpectedly, we find that in hypoxia the dHIF-independent pathway is responsible for the transcriptional induction of glycolytic enzymes.
Furthermore, the majority of hypoxia induced changes of intermediary metabolites still occurred in the absence of dHIF. We find that dHIF has a greater
impact on metabolism in normoxia than in hypoxia. Our data suggest that low oxygen induces both dHIF dependent and dHIF independent actions, which
elicit compensatory adaptation responses that impact transcription and metabolism. We conclude that dHIF-independent actions drive hypoxic transitions.
766A
Examining the role of EcR binding sites on ecdysone inducible polytene chromsome puffs.
Alexander D. Ostapenko, Rebecca F Spokony, Dmitri
Novikov, Kevin P. White. Institute for Genomics & Systems Biology, University of Chicago, Chicago, IL.
The steroid hormone ecdysone plays a crucial role in the development of insects. In
Drosophila melanogaster
, ecdysone pulses regulate the developmental
transition of both molting and metamorphosis by inducing genome-wide changes in gene expression. The ecdysone response can be visualized by puff
formation on polytene chromosomes from late third instar salivary glands. The
Ecdysone Receptor
(
EcR
) has been shown to be required for this response,
but it is not yet known which EcR binding sites are necessary and sufficient for puff formation and gene regulation. We hypothesized that EcR binding sites
contained within ecdysone responsive puff regions are necessary and sufficient for the puff response. We studied the ecdysone response by visual
examination of the puffing on polytene chromosomes. Our results indicate that EcR and its binding sites are required for puff formation. Using
immunohistochemistry on polytene squashes we confirmed that EcR binds to previously described ecdysone inducible puff regions. By inactivating EcR
with a heat-shock inducible RNAi, we produced alterations within the polytene structure as and confirmed a reduction of puff sizes at the primary ecdysone
response loci
74EF
,
75B
, and
Broad-Complex
. Using BAC recombineering, we generated transgenic fly lines with insertions of eGFP-tagged ecdysone-
inducible genes containing several EcR binding sites in large BACS at attP sites on chromosomes complementary to their endogenous locations. We found
that
74EF
and
EcR
genomic regions are sufficient to induce ectopic puffs. We confirmed EcR binding to both ectopic and endogenous puffs using
immunohistochemistry. We used Chromatin Immunopreciptiation and Sequencing to finely map EcR binding in these regions. We are currently testing
whether these binding sites are necessary and sufficient for puffing by using BAC recombineering techniques to delete EcR binding sites from the ectopic
puff-inducing loci, as well as inserting EcR binding sites (without the coding regions) into the attP sites and looking for a reduction or increase in puffing,
respectively.
767B
Molecular analysis of 5’ regulatory region of
Lim3
locus associated with
D. melanogaster
lifespan control.
Olga Y. Rybina, Elena G. Pasyukova. Inst
Molec Gen RAS, Moscow, Russian Federation.
Lim3
encodes an RNA polymerase II transcription factor with a key role in neuron development and specification. It was also identified as a candidate
gene that affects lifespan. These pleiotropic effects indicate the fundamental significance of the potential interplay between neural development/functioning
and lifespan control. The goal of this study was to analyze the causal relationships between
Lim3
structural variations, and gene expression and lifespan
changes.
Lim3A
, a transcript of
Lim3
locus, was shown to be functional during
Drosophila
neuron development. We sequenced a 2092 bp DNA fragment
including 5’ regulatory region of
Lim3A
in fifty
Drosophila
lines containing second chromosomes from Raleigh natural population. Five polymorphic
markers located within 380 to 680 bp upstream of the
Lim3A
transcription start sites (TSS) were significantly associated with the amount of
Lim3A
transcript, as evaluated by real time RT-PCR. Two of these five markers formed a haplotype which was also significantly associated with lifespan.
Haplotype variations could cause a six-fold change in gene transcription and a 25% change in lifespan. Several significant markers were located in binding
motifs of Polycomb/Trithorax group proteins (Rybina, Pasyukova, 2010). The DNA region located within 380 to 680 bp of the
Lim3A
TSS appeared to be
very conservative throughout
D. melanogaster
group, in accordance with low sequence variation of this region observed in Raleigh population. To evaluate
the role of different parts of
Lim3A
regulatory region in
Lim3A
transcription we used reporter constructions containing the firefly luciferase gene under the
control of fragments of
Lim3A
regulatory region of different length. The deletion of the whole region located within 380 to 680 bp of the
Lim3A
TSS
provided two fold decrease of expression of the reporter construct in S2
Drosophila
cell culture. Partial deletions of this region affected the reporter gene