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Full Abstracts – REGULATION OF GENE EXPRESSION
129
28
Juxtaposed
cis
-regulatory elements contradict simple definitions of enhancer modularity.
Tara L. Martin, Meghan Bragdon, Kelly Eckenrode, Zeba
Wunderlich, Angela DePace. Department of Systems Biology, Harvard Medical School, Boston, MA.
Classically, enhancers are defined as modular
cis
-regulatory sequences which act independently of orientation or distance from the promoter to activate
gene expression. However, a growing body of evidence suggests that this definition does not fully capture the behavior of all
cis
-regulatory elements.
Multiple non-contiguous sequences can drive expression in the same domain (e.g., “redundant” enhancers), and
cis
-regulatory elements may interact with
each other to produce the endogenous pattern, (e.g.,
sloppy-paired-1
). To specifically test whether adjacent
cis
-regulatory elements would remain modular or
adopt a combined function, we juxtaposed elements that are regulated by the same factors but respond to them with different sensitivities. We created
transgenic lacZ reporter lines that combined the stripe 3+7 and stripe 4+6
cis
-regulatory elements from the
even-skipped
locus. Using a quantitative cellular
resolution imaging platform, we compared the average expression pattern from each line, and integrated it into an existing gene expression atlas for
D.melanogaster
. This method yields a dataset amenable to modeling where the expression patterns of all relevant regulators and their targets are present in
the same cellular resolution framework. Current thermodynamic models predict that our constructs will produce intermediate expression patterns because the
response to upstream regulators is an average of the sensitivities. However, in our experiments the expression domains did not blend. We find that the
spacing, order and orientation of the
cis
-regulatory elements influence expression in both subtle and dramatic ways. One
cis
-regulatory element sometimes
dominates, reducing the level of expression of it's neighbor without otherwise shifting the expression domain. We discuss models of gene regulation and
enhancer structure that are consistent with our results and suggest methods to incorporate higher order interactions between
cis
-regulatory elements into
expression pattern predictions.
29
Temporal Coordination of Gene Networks by Zelda in the Early
Drosophila
Embryo.
Hsiao-Lan Liang
1
, Chung-Yi Nien
1
, Stephen Butcher
2
, Yujia
Sun
1
, Shengbo Fu
1
, Tenzin Gocha
1
, Nikoai Kirov
1
, J. Robert Manak
2
, Christine Rushlow
1
. 1) Department of Biology, Center for Developmental Genetics,
New York University, New York, New York, USA; 2) Departments of Biology and Pediatrics, Roy J. Carver Center for Genomics, University of Iowa, Iowa
City, Iowa, USA.
Our recent discovery of the transcriptional activator Zelda (Zld), which binds to CAGGTAG and related sequences present in the enhancers of many early-
activated genes in
Drosophila
, suggested how a single molecule could collectively activate batteries of genes simultaneously. To explore further the function
of Zld and to unravel the gene circuitry regulated by Zld, we used genome-wide binding and expression assays to identify Zld target genes in the blastoderm
embryo. Our results showed that Zld binds to genes involved in early developmental processes such as cellularization, sex determination, neurogenesis,
pattern formation, as well as early-expressed microRNAs. In the absence of Zld, many target genes failed to be activated, while others, particularly the
patterning genes, exhibited delayed transcriptional activation, some of which also showed weak and/or sporadic expression. These effects disrupted the
normal sequence of patterning-gene interactions and resulted in highly altered spatial expression patterns, demonstrating the significance of a timing
mechanism in early development. In addition, we observed prevalent overlap between Zld-bound regions and genomic “hotspot” regions, which are bound
by many developmental transcription factors, especially the patterning factors. This, along with the finding that the most over-represented motif in hotspots,
CAGGTA, is the Zld binding site, implicates Zld in promoting hotspot formation. We propose that Zld promotes timely and robust transcriptional activation
of early-gene networks so that developmental events are coordinated and cell fates are established properly in the cellular blastoderm embryo.
30
The cis-regulatory code of Hox function in Drosophila.
Maria Polychronidou
1
, Sebastian Sorge
1
, Nati Ha
1
, Jana Friedrich
1
, Daniela Bezdan
2
, Petra
Kaspar
1
, Martin Schaefer
2,3
, Stephan Ossowski
2,4
, Stefan R. Henz
2
, Juliane Mundorf
1
, Jenny Raetzer
1
, Fani Papagiannouli
1
, Ingrid Lohmann
1
. 1) Centre for
Organismal Studies, Heidelberg, Heidelberg, Germany; 2) Max Planck Institute for Developmental Biology, Tübingen, Germany; 3) Max Delbrück Center
for Molecular Medicine, Berlin, Germany; 4) Center for Genomic Regulation, Barcelona, Spain.
Spatiotemporal control of gene expression is orchestrated by the combinatorial interplay of transcription factor (TF) complexes with cis-regulatory DNA
elements. However, it remains mostly unclear how TFs, many of which are active in several cell types, acquire cell-type specific functions. An ideal model
to study the mechanisms underlying TF tissue-specificity are the Homeobox (Hox) TFs, which despite their broad expression, activate or repress
transcriptional programs in a highly context dependent manner. To address how a widely expressed transcriptional regulator is able to modulate downstream
gene activity with high cellular specificity, we have quantitatively identified binding regions for the Hox TF Deformed (Dfd) in the Drosophila genome. By
analyzing Dfd bound cis-regulatory modules (CRMs), we show that architectural features like motif-pair associations and motif distance preferences are
essential for cell-type specific expression of associated target genes. CRM features indeed determine specificity, since they alone accurately predict target
gene function and expression patterns. We also find that Dfd and Ultrabithorax (Ubx), another Hox TF specifying different morphological traits, interact
exclusively with non-overlapping genomic regions in vivo, regardless of their similar DNA binding preferences. Despite their comparable basic design
principles, Dfd and Ubx CRMs show distinct motif compositions and motif-pair associations, explaining the high functional specificity of the two Hox
proteins. Our results uncover the regulatory code of Hox CRMs and elucidate the mechanisms underlying functional specificity of TFs in vivo.