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Full Abstracts – TECHNIQUES AND FUNCTIONAL GENOMICS
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136
Tissue-Specific Translation State Array Analysis in Drosophila melanogaster.
Patrick W-L Li, Artem Zycovich, Guiping Du, Marysia Kolipinski,
Pankaj Kapahi. Buck Institute for Research on Aging, Novato, CA.
Drosophila melanogaster is one of the most widely used model organisms in biology. The powerful genetic tools and the complex body plan have played a
key role in its popularity. Up till now, gene expression studies have been done in whole organism or dissected tissues, which either overlook any tissue-
specific changes or are constrained by the limitation of dissection methods. Furthermore, it has been shown that the correlation between mRNA and protein
levels is rather modest, likely due to differences in post-transcriptional events, a major one being translation. To overcome these shortcomings, we have
developed a method to capture mRNAs bound to tagged ribosomes in Drosophila melanogaster. Using the GAL4-UAS system, we can isolate ribosome
bound mRNAs from a variety of tissues or a subset of cells. This technique, combine with other transcriptomic tools such as microarray or RNASeq, can
generate insight into the translatome in a spatial (tissue-specific drivers) or temporal (inducible driver) manner. Here we demonstrated that overexpression of
this tagged ribosomal protein has no adverse effect on life history traits of the flies. In addition, data generated with our method is consistent with that
obtained from dissected tissues, and our method can even capture mRNA from specific cells that are difficult to dissect. In summary, we have established a
method to examine cell specific changes in mRNA translation which will be useful in examining the role of spatial and temporal influences on a given
phenotype in Drosophila melanogaster.
137
Accurate genome-wide identification of dynamic transcriptional enhancers during
Drosophila
development.
Daniel J. McKay
1
, Jason D. Lieb
1,2
. 1)
Dept of Biology, UNC Chapel Hill; 2) Carolina Center for Genome Sciences, UNC Chapel Hill, Chapel Hill, NC.
A longstanding goal in biology is to understand how a diversity of cell types is created from a single genome. Previous work has identified
cis
-regulatory
modules (CRMs) associated with nearly 5% of the genes in the
Drosophila
genome. However, the inability to accurately predict the location of regulatory
DNA within the genome leaves us with an incomplete understanding of the mechanisms controlling cell fate specification during development. To address
this gap, we have generated genome-wide open chromatin profiles at five developmental stages using a technique termed FAIRE-seq (Formaldehyde-
Assisted Isolation of Regulatory Elements, followed by high throughput sequencing). Although less than 4% of the genome is open at any particular stage,
we find that these regions behave dynamically over time, and cumulatively, over 12% of the genome is open across all samples. To test the ability of open
chromatin regions to function as CRMs, we performed transgenic reporter assays. Strikingly, all 26 of the cloned regions drive accurate reporter activity,
including regions from genes that have been heavily studied such as
hunchback
,
paired
, and
Distalless
. We conclude that open chromatin regions are highly
effective predictors of functional CRM activity. To examine the regulatory network underlying appendage development, we generated open chromatin
profiles of three different imaginal discs. While differences exist at key appendage regulators, the overall genome-wide profiles are nearly identical. To test
whether these similarities were a consequence of insufficient cellular determination, we generated open chromatin profiles from fully differentiated
appendages. To our surprise, we again found the profiles were nearly identical, while being distinct from imaginal discs. Taken together, these data suggest
that a core
cis
-regulatory network exists to control appendage development that is nevertheless capable of generating dramatic morphological differences.
138
High resolution association mapping in an outbred
Drosophila melanogaster
population using Pool-Sequencing (NGS speed mapping).
Christian W.
Schloetterer, Héloïse Bastide, Martina Visnovska, Raymond Tobler, Andrea Betancourt. Inst f Populationsgenetik, Vetmeduni Vienna, Wien, Austria.
Next Generation Sequencing (NGS) techniques provide powerful tools for the identification of the genetic basis responsible for variation in quantitative
phenotypes (QTLs). However, most of the methods to date only focus on inbred populations of laboratory organisms which poorly reflects natural variation.
Here, we test the performance of NGS for association mapping studies in an outbred
Drosophila melanogaster
population. Since,
D. melanogaster
has
extremely low levels of linkage disequilibrium, we reasoned that GWAS should be extremely powerful at an unprecedented level of resolution. To test our
approach, we focused on an extremely well-studied trait, abdominal pigmentation variation in
D. melanogaster
females. About 5,000 F1 females obtained
from naturally inseminated flies were scored for pigmentation and two replicates each of the 100 most extreme phenotypes were sequenced. Our results
confirm the efficiency of our approach. In addition to several genes with a proven role in pigmentation (e.g. bab), we identified additional candidates. Most
importantly, our analyses suggest that levels of linkage disequilibrium may be low enough to identify causative SNPs . Hence, we propose that our new
approach (NGS speed mapping) provides an excellent tool for GWAS studies, in particular for species with low levels of linkage disequilibrium.
139
Super-Resolution Imaging of Regulatory Chromatin Dynamics in Developing Embryos.
Alistair N. Boettiger, Xiaowei Zhuang. Chemistry and
Chemical Biology, Harvard University, Cambridge, Ma.
The differentiation of embryonic cells into their appropriate developmental fates is mediated in part by fine scale structural changes to chromatin.
Developmental specific transcription factors drive these changes through the repositioning of histones. This can facilitate or restrict access to transcriptional
machinery to the underlying genes or facilitate looping of distal regulatory sequences to target sites. These fine scale structural changes are mostly too small
(10s of nanometers) to be observed with conventional microscopy techniques (limited to several hundred nanometer resolution), and have so far evaded in
vivo observation in intact embryonic tissue.
We present super-resolution imaging techniques which allow for the detection of regulatory changes in chromatin on the scale of tens of nanometers in
developing embryos by imaging particular genomic regions of interest and chromatin associated proteins. These analyses provide a detailed view of
regulatory modifications at the single cell level. This allows for a direct causal relations between expression states and modification. It also allows for
variation between identical populations to be measured and the frequency of each state within the population to be determined. Additionally, because they
are applied within the intact embryo, cell identity and the spatial relation of the cell to its neighbors and embryonic signals are still maintained.