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13/09/2014(
1(
The$ciliary$proteome$
Mar,jn(Huynen(
Centre(for(Molecular(and(Biomolecular(Informa,cs(Radboud(Ins,tute(of(Molecular(Life(Sciences(
(
Human ciliated tissues in movement, sensing and signalling
Inner ear: Stereocilia function in hearing and balance
Cerebral cavities, Bronchia & Fallopian tubes
Retina: Cones and Rods
Sperm cells
The$non0mo1le/$primary$cilium$as$a$signaling$hub$
• MechanoD,(ChemoD,(Thermosensa,on(• Sonic(Hedgehog(signaling(• Non(canonical(WNT(signaling(
• Effec,ve(as(cellular(antenna(• Large(specialized(membrane(surface(• Ac,ve(transport((IFT)(• Selec,ve(ga,ng(of(receptors(at(the(base((synapse)(
Fluid$flow$detec1on$by$cilia$regulates$proper$development$of$nephric$ducts$
Nephric(duct(Thome(et(al.(2007(Nat.(Cell.(Biol.(
Zang(et(al.(2004(Physiology(
13/09/2014(
2(
Cilia$in$renal$disease$
• Autosomal(polycys,c(kidney(disease((APKD)(• Nephronophthisis((medullary(cys,c(kidney(disease)(• Kidney(func,on(affected(in(mul,Dsyndromic(ciliopathies(
• BardetDBiedl,(Joubert,(Meckel,(Senoir(Loken,(…(
NPHP( APKD(
“A(systems(biology(approach(to(dissect(cilia(func,on((and(its(disrup,on(in(human(gene,c(disease”(
Systems$biology$of$the$cilium$ Large$scale$data$acquisi1on$for$the$predic1on$of$new$cilium$genes$
• ProteinDprotein(interac,ons(• Tandem(Affinity(Purifica,onDMS(• Stable(isotope(labeling(by(amino(acids(in(cell(culture(• YeastD2DHybrid(
• Bioinforma,cs(• CoDevolu,on(• Microarray(metaDanalysis((systems(coDexpression)(
• Func,onal(screens(• Genome(wide(siRNA(on(ciliogenesis(• Comprehensive(drug(panel(screen(on(ciliogenesis(
13/09/2014(
3(
Data$gathering$and$organiza1on$Predic1ng$new$cilium$proteins$by$comparing$genomes$
Gold$Standard$&$Nega1ve$set$
Extracellular$
Co0expression,$exploi1ng$>$1600$experiments,$35K$arrays$
13/09/2014(
4(
How$can$we$integrate$mul1ple$datasets?$
Proteomics(data(
Gene,c(data(Published(data(
Expression(data( Evolu,onary(data(
?(
A$Bayesian$classifier$to$iden1fy$novel$ciliary$genes$
Proteomics(data(
Gene,c(data(Published(datasets(
Expression(data( Evolu,onary(data(
Bayesian$integra1on$of$mul1ple$observa1ons$
• n(is(the(number(of(datasets(considered(• fi$=(dataset(i$• P(fi|T)(=(probability(that(a(gene(is(reported(by(dataset(i(given(it(is(a(known(ciliary(gene(
(
GeneScore = log2PCiliumP~Cilium
!
"#
$
%&
Pr ior! "## $##
+ log2i=1
n
∑P fi T( )P fi F( )!
"##
$
%&&
log(a ⋅b) = log(a)+ log(b)
13/09/2014(
5(
Bayesian$integra1on;$$
$dis1nguishing$between$ciliary$vs.$non0ciliary$genes$Ranking$poten1al$ciliary$genes$
18(
Ciliary(Predicted(NonDciliary(
FRD(cutoff(at(25%:(405(genes(of(which(283(unknown(to(be(ciliary(
Experimental$valida1on$
• 72(genes(of(the(283(will(be(tested(((25%(FDR)(• C.$elegans:(roaming(and(dyeDfilling(essays((phenotype)(• Zebrafish:(morpholino,(CRISPR((phenotype)(• Immunofluorescence(in(human(ciliated(cell(lines((localiza,on)(
• 35(tested(so(far(• 25(show(ciliary(phenotype(and/or(cilia(localiza,on((FDR(29%)(
N2 control
oscp-1
Roaming$assay$screen$in$C.#elegans#
Julie(Kennedy,(Blacque(lab(University(College(Dublin(
13/09/2014(
6(
C.#elegans#dye0filling$assay$N
2 co
ntro
l dy
s-1(
eg33
) /d
ystro
phi (
DM
D)
dys-
1(cx
18)
/dys
troph
in (D
MD
)
mag
i-1(g
k657
) /M
AG
I2
R10
F2.5
(gk7
48)
/OS
CP
1 R
10F2
.5(g
k699
) /O
SC
P1
Immunofluorescence$in$human$respiratory$cells$
Gerry(Dougherty,(Omran(lab((Wespälische(WilhelmsDUniversität(Münster(
Immunofluorescence$in$hTERT0RPE1$(re1na)$
Erik(de(Vrieze,(Kremer(lab(Radboudumc(
Analyzing$pa1ent$exome$sequence$data$
• Premise:(• Causal(genes(in(ciliopathy(pa,ents(perform(a(func,on(in(the(cilium(
• They(are(“ciliary(genes”(• And(“behave(like”(other(ciliary(genes(
• Our(Bayesian(classifier(is(a(probabilis,c(model(or(descrip,on(of(“ciliary(genes”(based(on(a(collec,on(of(experimental(and(bioinforma,cs(data(
• Genes(found(mutated(in(pa,ents(and(which(are(classified(as(“ciliary”(are(prime(candidates(
13/09/2014(
7(
Predic1ng$causal$genes$in$JBTS$pa1ents$
Five(pa,ents(with(JBTS(of(unknown(cause(and(no(family(history(
Marijn(Stokman,(Nephrology(dept.(UMC(Utrecht(
Filtering$and$ranking$of$causal$muta1ons$
Acknowledgements$• Radboudumc(
• John(van(Dam(• Robin(van(der(Lee(• Radek(Scklarczyk(
• Ronald(Roepman(• Dorus(Mans(• Minh(Nguyen(
• Hannie(Kremer(• Erik(de(Vrieze(
(• UCD(
• Oliver(Blacque(• Julie(Kennedy(
• UCL(• Phil(Beales(• Victor(Hernandez(• Miriam(Schmidts(
• Kouncil(WP1(• Marijn(Stokman(• Kirsten(Renkema(• M.R.(Lilien(• Heleen(Arts(• M.M.(Oud(• Ies(Nijman(• H.Y.(Kroes(• N.(Geijsen(• E.(Cuppen(• Rachel(Giles(• Nine(Knoers(
• WWU(• Heymut(Omran(• Gerry(Dougherty(
• UTUB(• Marius(Ueffing(• Karsten(Boldt(• Yves(Texier(
• TIGEM(• Brunella(Franco(
• LIMM(• Colin(Johnson(• Kasia(Szymanska(• Gabrielle(Wheway(
• Duke(• Erica(Davis(
• SYS(IT(• Grischa(Toedt((EMBL)(• Qianhao(Lu((UHEI)(• Konstan,nos(Koutroumpas((UEVE)(• Jeroen(van(Reeuwijk((RUNMC)(