Basic HTML Version
Full Abstracts – REGULATION OF GENE EXPRESSION II
136
46
Tissue specific analysis of chromatin marks identify temporal enhancer activity in development
.
Robert P. Zinzen
1
, Stefan Bonn
1
, Charles Girardot
1
, E. Hilary Gustafson
1
, Alexis Perez Gonzalez
2
, Nicolas Delhomme
1
, Yad Ghavi-Helm
1
, Bartek
Wilczynski
1
, Andy Riddell
2
, Eileen E.M. Furlong
1
. 1) Genome Biology Unit, EMBL, Heidelberg, Germany; 2) Flow Cytometry Core Facility, EMBL,
Heidelberg, Germany.
By using a new method to batch-isolate tissue-specific chromatin followed by immunoprecipitation (BiTS-ChIP), we uncovered mesoderm-specific
signatures for histone modifications and Pol II positioning from developing embryos. We find that enhancers exhibit heterogeneous chromatin/Pol II states,
and that specific states are highly correlated with spatio-temporal enhancer activity. While H3K4me1 enrichment provides no information on the activity
state of regulatory regions, other features such as H3K27ac, H3K79me3 and in particular Pol II enrichment mark active enhancers with temporal precision.
The uncovered enhancer signatures allowed for faithful
de novo
identification of new regulatory regions, which direct activity
in vivo
in the correct tissue at
the predicted time. This cell type-specific data therefore identifies enhancers in active use during development, which will be instrumental in deciphering
cis
-regulatory networks.
47
The transcriptional repressor Snail functions as activator during
Drosophila
mesoderm development.
Martina Rembold
1
, Lucia Ciglar
2
, Jorge Omar
Yáñez Cuna
3
, Charles Girardot
2
, Robert Zinzen
2
, Martina Braun
2
, Alexander Stark
3
, Eileen Furlong
2
, Maria Leptin
1
. 1) Institute of Genetics, University of
Cologne, Cologne, Germany; 2) European Molecular Biology Laboratory (EMBL), Heidelberg, Germany; 3) Research Institute of Molecular Pathology
(IMP), Vienna, Austria.
Gastrulation and the patterning of the mesoderm anlage in
Drosophila
are controlled by the activities of the transcription factors Twist and Snail. Twist
activates the expression of genes necessary for the morphogenetic changes leading to mesoderm internalisation and determination. Although Snail is known
as dedicated repressor delimiting the expression of ectodermal genes, increasing evidence indicates that it might also act as activator of transcription.
Whether Snail's activator function is direct or indirect is under debate.
We show that Snail directly activates the expression of mesodermal genes
in vitro
and
in vivo
. Chromatin immunoprecipitation and microarray (ChIP-on-
Chip) analysis for Twist and Snail combined with expression analysis in
twist
and
snail
mutant embryos allowed us to identify direct target genes on a
genome-wide basis. Snail shows extensive co-binding with Twist to active mesodermal enhancers for genes such as
Mef2
,
tinman
and
htl
, whose expression
is lost in
snail
mutant embryos. In cell culture Snail acts as co-activator of Twist by augmenting Twist induced expression of luciferase by up to two-fold. In
snail
mutant embryos
lacZ
expression driven by these enhancers is lost. This effect depends on Snail direct binding to the enhancer, as mutation of two snail
motifs in the
Mef2-I-D[L]
enhancer is sufficient to abrogate both the co-stimulatory effect in luciferase assays as well as
lacZ
expression
in vivo
. We are
currently investigating the defining features of enhancers that are activated versus those that are repressed by Snail and have uncovered a new factor
involved in this regulator switch.
Rembold, M and Ciglar, L contributed equally to this work.
48
Yan binding at the eve locus confers robustness.
Jemma L. Webber, Lauren Cote, Jie Zhang, Ilaria Rebay. Ben May Department for Cancer Research,
University of Chicago, Chicago, IL.
The Ets transcriptional repressor Yan functions as part of a conserved network downstream of receptor tyrosine kinase (RTK) signaling. This network
displays switch-like behavior, transitioning from a high-Yan to a low-Yan state following RTK activation. To ensure a reliable all-or-none response, such a
system must also include mechanisms to buffer gene expression against developmental noise that might induce inappropriate oscillations between states. In
our analysis of the genome-wide chromatin binding profile of Yan, we noticed that at developmentally important target genes, a significant fraction of Yan
occupancy occurs at regions of high peak density that span multiple kilobases. Focusing on the Yan target gene even-skipped (eve), to which we mapped
several high density Yan bound elements, including one that includes the previously identified muscle/heart enhancer (MHE), we used BAC recombineering
of a genomic Eve-YFP construct to examine the contribution of individual Yan-bound regulatory regions to robustness of mesodermal Eve expression. Our
results reveal a level of functional redundancy between the regions and suggest that robust regulation of mesodermal Eve expression requires Yan input at
multiple elements across the locus, not just at the MHE. Mechanistically, we observe changes in Yan occupancy that are consistent with long-range
interactions between elements spaced more than 10kb apart, suggesting that coordinated Yan occupancy at multiple sites across the eve locus may be critical
to ensure robust expression. We are currently investigating whether Yan SAM-domain mediated self-association can facilitate such long-range interactions
between enhancers. Extrapolating from our analysis at the eve locus, we speculate that the complex chromatin occupancy signatures observed with Yan may
provide a filter against noise to ensure robust regulation of gene expression across development.