Prsentation PowerPoint

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Methodology Chromatin ImmunoPrecipitation
(ChIP) Used to analyze how transcription factors
play their role. Engrailed is a homeodomain
transcription factor, acting either as a
repressor or an activator. This factor is highly
conserved during evolution, from fly to
human. Drosophila has been used to understand how
Engrailed is acting through the development.
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engrailed expression during Drosophila development
Larvae
Embryos
Adults
Central Nervous System
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• - Engrailed is important to define the Posterior
  cell identity in all the tissues from
• ectodermal cell layer and is necessary during
  the entire life of the animal.
• Engrailed is also important during neurogenesis.
  In humans it is implicated
• in Parkinsons disease and more recently in brain
  malformations, associated to autism,
• - Engrailed function during neurogenesis is
  probably highly conserved during evolution.
• One way to understand how Engrailed is playing
  these different
• roles, is to identify direct target genes that
  are regulated by Engrailed in vivo,
• during embryogenesis.

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I- Identification of direct Engrailed target
genes, at a genomic scale, using UV Chromatin
Immunopreciprecipitation approach
(UV-X-ChIP) II- How identification of targets
helps to understand Engrailed function Exemple
of frazzled and Engrailed function in axonal
guidance III- UV versus FA ChIP approaches
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I- UV Chromatin Immunopreciprecipitation approach
(UV-X-ChIP)
(Pascal Solano Bruno Mugat )
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Chromatin ImmunoPrecipitation (ChIP)
- Purification of nuclei from 0-16 hrs embryos -
UV cross-linking
- Sonication (0.1 - 3 Kb)
- in vivo IP with anti-EN Ab

• Addition of linkers
• PCR amplification

- in vitro IP with anti-EN Ab
PCR amplification. HindIII digestion and cloning
in Ks 603 individual clones isolated and
sequenced
315 independent clones
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Specificity and reproducibility of ChIP
Chromatin 1
sonication
Resin with no antibody
IP with anti-EN
Linkers / PCR
IP in vitro
Identification of 315 clones
Probe E1
Transfer of clones onto nitrocellulose
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Southern blot analysis of 315 clones
315 clones tested 85 fix Engrailed specifically
with the Probe E1 65 fix Engrailed specifically,
with both probes 203 new direct En targets
Chromatin 1
Chromatin 2
Probe E2
Probe E1
Background 2
Background 1
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Identification of EN consensus binding site
Motifs are present 5 fold more in EN
immunoprecipitated fragments, comparative to
random genomic fragments.
(David Martin, Bernard Jacq)
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Identification of potential En targets
203 positive
96 Intronic (47)
107 Intergenic (53)
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Potential Engrailed target genes
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81 targets are involved in different
developmental processes
slp2 fz2 nkd arm wnt2 scm corto, ash2
A/P patterning
Wing and tracheal development
vvl knrl sty vn bnl px
Muscle development and cytokinesis
Gsc twi mam hbsnrm aret Ca-alpha1D Pka-C2
CG1890 tensin GC15158 insc Grip84 Klp54D
CG12908 p120ctn
Neurogenesis
eg acj6 onecut Mio fracommbeatVa OR42b
OR22c OR83c ShaIPtp4E HD-14 Cad89D CG4509
fat2 stan 18W con Cha CG5559 unc-13-4A
ine SNF4A PK twins camKII CG9811 G-ia65A
robl62A huntingtin enb Fur1 ben tomosyn
Leucokinin trio
Eye development
ed Calx CdsA lama Pkg21D inaD CG12731 cno
drk
Others
p53 Ets98B dd4 Acp33A Ubp64E
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II- Engrailed function during neurogenesis
(Willy Joly Bruno Mugat)
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Engrailed and its targets during neurogenesis
Engrailed is expressed in a subset of neurons
Among the 203 potential Engrailed targets
identified by ChIP, 35 correspond to genes known
to be involved in neurogenesis
fra ena trio comm con OR42b OR22c OR83c
HD-14 Cad89D CG4509 fat2 beatVa stan 18W
eg acj6 ine onecut ben Mio ShaI Ptp4E
Cha CG5559 unc-13-4A SNF4A PK twins
camKII CG9811 G-ia65A robl62A Fur1 tomosyn
Leucokinin huntingtin
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Frazzled Netrin receptor (locus 49B3)
1Kb
GAAAGATCTCAATAAGCTGCTGCACATTGATTGAAATCGAAGACGGTAAC
GAGTAAATGTGGA ATGACACAACCTGAATTCCATGATGGCCATAATTA
CGATGAAGATGATGATGATGACGATGATA
TTTGGCTAGTTACACACAGTCGAGAACA
220bp 2C5 genomic fragment
2C5 binds Engrailed in vitro
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Study of 2C5 in vivo
Test of luciferase activity in Hela cells
EN?
Luciferase
2C5
Engrailed acts as an activator through 2C5 DNA
binding site
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Injected constructs
In the presence of different forms of EN proteins
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A
B
C
m-GFP
2C5
hsp 70
2C5 binds Engrailed in vivo
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Study of 2C5 in Drosophila
C
m-GFP
2C5
hsp 70
2C5 is a part of frazzled promoter
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Engrailed activity through 2C5
Engrailed acts as an activator through 2C5
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• - 2C5 genomic fragment isolated by ChIP,
  contains EN consensus binding sites, and
• is able to bind EN in vitro and in vivo.
• Engrailed is acting as an activator through 2C5
  sequences, both in Hela cells, and
• in Drosophila.

- Note that Engrailed consensus binding site
itself is driving repression by Engrailed,
which indicates that molecular mechanisms
responsible of activation are not known, but seem
more dependant of cofactors interacting with EN,
rather than depending on different EN binding
site.
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Engrailed and frazzled are involved in the
construction of the nerve cord
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Engrailed expressing cells during ventral nerve
cord development
- Formation of the VNC
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Engrailed-expressing cells send axons through
posterior PC commissures
- Wild-type stage 15 embryos
- en-Gal4 UAS-mCD8 GFP embryos
anti-HRP
anti-HRP anti-EN
anti-GFP anti-HRP
anti-GFP
enB86/ enB86
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Frazzled in involved in the formation of AC and
PC commissures
frazzled encodes the Netrin receptor that is
necessary for axons to cross the midline
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Genetic interactions between Engrailed and its
targets
S
PC
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• Frazzled, whose mammalian homolog is DCC
  (Deleted of Colorectal Cancer),
• is involved with Engrailed in the formation of
  the PC.
• Activation of frazzled by EN is necessary for PC
  axon growth
• The accessibility of EN targets allowed to better
  understand how EN is acting
• to construct the embryonic nerve cord, by acting
  in particular on axon growth,
• through the regulation of frazzled

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III- UV versus FA
(Nicolas Nègre and Grégory Chanas)
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UV versus FA
PcG
PcG
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En probe (UV)
En probe (FA)
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FA
UV
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• Ph is a Zn-finger protein, that might be able to
  bind DNA
• only when present in a complex
• UV vs FA might help to understand how proteins
  within the
• complexes can move according to different
  situations