pygopus amily PHD inge 1 (PYGO1) : Time beha iou al
s udy o 3 d o de combina ions in WNT3A s imula ed
HEK 293 cells
sh ip akash sinha
Independen Resea che ; O cid ID : o cid.o g/0000-0001-7027-5788
Add ess : 104-Madhu isha Heigh s Phase 1, Risali, Bhilai-490006, India
Co esponding au ho email : sinha.sh ip [email protected]
Abs ac
pygopus amily PHD inge 1 (PYGO1), ec ui men pe mi s β-ca enin o ansc ip-
ionally ac i a e WNT a ge genes. The p esence o a PHD domain implica es PYGO
p o eins in a ch oma in- ela ed unc ion. Guj al and MacBea h [1] p o ides a quan i-
a i e, and dynamic s udy o WNT3A-media ed s imula ion o HEK 293 cells, whe e
hey eco d ime based exp ession p o iles o se e al esponse genes which co ela ed
signi ican ly wi h p oli e a ion and mig a ion. By moni o ing he dynamics o gene
exp ession using sel -o ganizing maps, hey iden i ied clus e s o genes ha exhibi
simila exp ession dynamics and unco e ed p e iously un ecognized posi i e and neg-
a i e eedback loops. Howe e , hei s udy depic s/uses singula measu emen s o in-
di idual gene exp ession a di e en ime snapsho s/poin s o in e he sys em wide
analysis o he pa hway. A any pa icula ime poin , i is o en he case ha genes
a e wo king syne gis ically in combina ions, e en hough hei exp ession measu e-
men s a e singula in na u e. He e, I •enume a e and ank all 2415 PYGO1 ela ed
3 d o de combina ions in a o es o 71C3combina ions using ou di e en sensi i i y
me hods; •show he conse ed ankings o PYGO1-X-X combina ions, which poin
o exis ence o biological syne gy o some o hese combina ions ac oss he di e en
sensi i i y me hods; and •s udy he beha iou o some o hese combina ions ela ed o
WNT3A esponse genes ha a e anked by he machine lea ning sea ch engine (Sinha
[2]) in ime. Pa e ns o combina ions eme ge, some o which ha e been es ed in we
lab, while o he s equi e u he we lab analysis.
Keywo ds: Sensi i i y analysis, Suppo ec o anking, Hilbe Schmid
Independence C i e ion indices (HSIC) and Sobol indicies, WNT3A
ITime beha iou al s udy o 3-od PYGO1 comb. in WNT3A s imula ed cells
1Aspec s o unpublished wo k we e p esen ed in a pos e session a Cell Symposia: Technology. Biology.
Da a Science, 9-11 Oc obe 2016, Be keley, Cali o nia, USA.
P ep in submi ed o P ep in Ma ch 1, 2025
1. Signi icance
Sinha [2] ecen ly demons a ed he use o machine lea ning based sea ch engine o
ank/ e eal gene combina ions a 2nd o de o he ime se ies da a by Guj al and
MacBea h [1] and showed how i is possible o loca e combina ions o p io i y ha
migh be wo king syne gis ically, using sensi i i y me hods and powe ul suppo ec-
o anking algo i hm. Howe e , he p oblem explodes combina o ially wi h e en a
small se o 71 eco ded genes in he s udy by Guj al and MacBea h [1], when one
s eps o explo e 3 d o de combina ions. Wi h he o al numbe o 71C3(= 57155) com-
bina ions, i becomes nea ly impossible o any biologis o s udy he sys em wide dy-
namics o any pa hway. Also, he amoun o ime usually needed o sea ch o and es
a combina ion is a mo e han he sea ch down by he machine lea ning based sea ch
engine. He e, I ex end he esea ch wo k by Sinha [2] o conduc a beha io al s udy o
3 d o de PYGO1 ela ed combina ions using indi idual gene exp essions measu ed in
ime, in WNT3A s imula ed HEK 293 cells.
2. In oduc ion
The de ails o he machine lea ning based sea ch engine has been ecen ly published in
Sinha [2] and deployed o explo e he 2nd o de combina ions o genes in he da a se
p o ided by Guj al and MacBea h [1]. Ne e heless, he e, I poin o he undamen als
o he published wo k o comple eness.
2.1. A combina o ial p oblem
Sensi i i y analysis plays a majo ole in compu ing he s eng h o he in luence o
in ol ed ac o s in any phenomena unde in es iga ion. When applied o exp ession
p o iles o a ious in a/ex acellula ac o s ha o m an in eg al pa o a signaling
pa hway, he a iance and densi y based analysis yields a ange o sensi i i y indices
o indi idual as well as a ious combina ions o ac o s. These combina ions deno e
he highe o de in e ac ions among he in ol ed ac o s. Compu a ion o highe o -
de in e ac ions is o en ime consuming bu i gi es a chance o explo e he a ious
combina ions ha migh be o in e es in he wo king mechanism o he pa hway. Fo
example, in a ange o ou h o de combina ions among he a ious ac o s o he Wn
pa hway, i would be easy o assess he in luence o he des uc ion complex o med by
APC, AXIN, CSKI and GSK3 in e ac ion. Bu he e ec o hese combina ions a y
o e ime as measu emen s o old changes and de ia ions in old changes a y. So
i is impe a i e o know how an in e ac ion o a combina ion o he in ol ed ac o s
beha e in ime and Sinha [2] de elops a p ocedu e o ack he beha iou by exploi ing
he in luences o hese in ol ed ac o s.
2.2. A possible solu ion
In his wo k, a e es ima ing he indi idual e ec s o ac o s o a highe o de combi-
na ion, he indi idual indices a e conside ed as disc imina i e ea u es. A combina ion,
2
hen, is a ea u e se in highe o de (≥2 ,i.e mul i a ia e). Wi h an excessi ely la ge
numbe o ac o s in ol ed in he pa hway, i is di icul o sea ch o impo an com-
bina ions in a wide sea ch space o e di e en o de s. Exploi ing he analogy wi h
he issues o p io i izing webpages using anking algo i hms, o a pa icula o de , a
ull se o combina ions o in e ac ions can hen be p io i ized based on hese ea u es
using a powe ul anking algo i hm ia suppo ec o s Joachims [3]. Reco ding he
changing ankings o he combina ions o e ime e eals how highe o de in e ac ions
beha e wi hin he pa hway and when an in e en ion migh be necessa y o in luence
he in e ac ion wi hin he pa hway.
2.3. pygopus amily PHD inge 1 (PYGO1)
WNT/Wingless signaling con ols many undamen al p ocesses du ing animal de el-
opmen and WNT ansduc ion is media ed by he associa ion o β-ca enin wi h nu-
clea TCF DNA binding ac o s. K amps e al. [4] epo he iden i ica ion o wo
segmen pola i y genes in D osophila, i.e legless (LGS) and PYGO, and show ha
hey a e equi ed o WNT signal ansduc ion a he le el o nuclea β-ca enin. LGS
encodes BCL9, and hey p o ide gene ic/molecula e idence ha hese p o eins exe
hei unc ion by physically linking PYGO o β-ca enin. The PYGO-BCL9 complex
is a ch oma in eade , he unc ion o which elies on wo ligand-binding su aces o
PYGO’s PHD inge ha ancho he his one H3 ail me hyla ed a lysine 4 (H3K4me)
wi h assis ance om he BCL9 HD1 domain. Mille e al. [5] epo he i s use o
agmen -based sc eening by NMR o iden i y small molecules ha block p o einp o-
ein in e ac ions by a PHD inge . Thei esul s sugges ha he ec ui men o PYGO
pe mi s β-ca enin o ansc ip ionally ac i a e WNT a ge genes.
Thompson e al. [6] epo he disco e y o PYGO, whose mu an pheno ypes
speci ically mimic loss-o -Wingless (Wg) signalling. PYGO is equi ed o dTCF-
media ed ansc ip ion, bu no o Wg-induced s abiliza ion o A m and i is a nuclea
p o ein ha is ound in a complex wi h A m in i o. Humans possess wo PYGO p o-
eins, bo h o which a e equi ed o TCF-media ed ansc ip ion in colo ec al cance
cells. The p esence o a PHD domain implica es PYGO p o eins in a ch oma in- ela ed
unc ion, and he auh o s p opose ha hey media e ch oma in access o TCF o A m/β-
ca enin.
Homeodomain p o eins ha e been shown o play a majo ole in he de elopmen
o a ious o ganisms. Schindle e al. [7] isola ed a no el A abidopsis homeodomain
p o ein ha had he capabili y o in e ac wi h a DNA mo i . HAT3.1 does no con-
ain a leucine zippe mo i ollowing he homeodomain and is u he cha ac e ized by
an N- e minal egion ha sha es subs an ial sequence simila i y wi h he maize home-
odomain p o ein Zmhox1a. Wi hin his conse ed egion, he p esence o eigh egu-
la ly spaced cys eine/his idine esidues (Cys4-His-Cys3mo i ) was obse ed ha was
eminiscen o o he me al-binding domains. Being s ongly e olu iona y conse ed,
he au ho s p oposed ha his egion ep esen s a no el p o ein-mo i which is de-
no ed plan homeo-domain- inge (PHD- inge ). Fu he , in i o DNA binding s udies
demons a ed ha HAT3.1 was capable o in e ac ing wi h any DNA agmen la ge
han 100 bp and a dele ion o he N- e minal PHD- inge domain comple ely abolished
DNA binding, sugges ing ha his egion migh play an impo an unc ional ole in
3
p o ein-p o ein o p o ein-DNA in e ac ion. HAT3.1 mRNA was p ima ily de ec ed in
oo issue, implying a egula o y unc ion o his p o ein in oo de elopmen . The
implica ions o PHD inge in ch oma in-media ed ansc ip ional egula ion ha e been
documen ed in Aasland e al. [8].
In his esea ch wo k, I p esen 3 d o de combina ions o PYGO1 wi h o he genes,
ha he machine lea ning based sea ch engine poin s o, as possible syne gis ic combi-
na ions ha migh be wo king in ime.
3. Me hods
Please e e o sec ions o Sinha [2] o me hods, design o s udy and analysis o da a
o 2nd o de combina ions. The same me hod and design o s udy is used o gene a e
esul s o 3 d o de combina ions p esen ed in his s udy.
4. Time se ies da a
Guj al and MacBea h [1] p esen a se o 71 WNT- ela ed gene exp ession alues o 6
di e en imes poin s o e a ange o 24-hou pe iod using qPCR. The changes ep e-
sen he old-change in he exp ession le els o genes in 200 ng/mL WNT3A-s imula ed
HEK 293 cells in ime ela i e o hei le els in uns imula ed, se um-s a ed cells a 0-
hou . Guj al and MacBea h [1] s a e ha qPCR da a a e he means o h ee biological
eplica es. Only genes whose mean ansc ip le els changed by mo e han wo- old a
one o mo e ime poin s du ing he 24-hou ime cou se we e conside ed signi ican .
Posi i e (nega i e) numbe s ep esen up (down) - egula ion. We ha e al eady co e ed
he issues ela ed o hese da a se s in de ail in Sinha [9]. Reade s a e eques ed o go
h ough hem in he poin ed e e ence. The ools o s udy which a e used he e ha e
been published in ano he ounda ional wo k in Sinha [9].
5. Design o expe imen
5.1. Pipeline o ime se ies da a
Fo he case o ime se ies da a, in e ac ions among he con ibu ing ac o s a e s udied
by compa ing iple s o old-changes a single ime poin s. The p odecu e begins wi h
he gene a ion o dis ibu ion a ound measu emen s a single ime poin s wi h added
noise is done o es ima e he indices. A dis ibu ion is gene a ed o he old changes
a single ime poin s. Then o e e y gene, he e is a ec o o alues ep esen ing old
changes as well as de ia ions in old changes o di e en ime poin s and du a ions
be ween ime poin s, espec i ely. Nex a lis ing o all Cn
kcombina ions o knumbe
o genes om a o al o ngenes is gene a ed. kis ≥2 and ≤(n−1). Each o he com-
bina ion o o de k ep esen s a unique se o in e ac ion be ween he in ol ed gene ic
ac o s. A e his, he da ase s a e combined in a speci ed o ma which go as inpu
as pe he equi emen o a pa icula sensi i i y analysis me hod. Thus o each p h
combina ion in Cn
kcombina ions, he da ase is p epa ed in he equi ed o ma om
4
he dis ibu ions o wo sepa a e cases which ha e been discussed abo e. (See .R code
in mainSc ip -1-1.R). A e he da a has been ans o med, ec o ized p og amming
is employed o densi y based sensi i i y analysis and looping is employed o a i-
ance based sensi i i y analysis o compu e he equi ed sensi i i y indices o each o
he pcombina ions. This p ocedu e is done o di e en kinds o sensi i i y analysis
me hods.
A e he abo e sensi i i y indices ha e been s o ed o each o he p h combina-
ion, he nex s ep in he design o expe imen is conduc ed. Since he e is only one
eco ding o sensi i i y index pe combina ion, each combina ion o ms a aining ex-
ample which is allo ed a aining index and he sensi i i y indices o he indi idual
gene ic ac o s o m he aining example. Thus he e a e Cn
k aining examples o k h
o de in e ac ion. Using his aining se SVMRank
lea n Joachims [3] is used o gene a e a
model on de aul alue C alue o 20. In he cu en expe imen on oy model C alue
has no been unned. The aining se helps in he gene a ion o he model as he di -
e en gene combina ions a e numbe ed in o de which a e used as ank indices. The
model is hen used o gene a e sco e on he obse a ions in he es ing se using he
SV MRank
classi y Joachims [3]. No e ha due o a ailabili y o only one example pe com-
bina ion, a e he model has been buil , he same aining da a is used as es da a o
gene a es he sco es. This p ocedu e is execu ed o each and e e y sensi i i y analysis
me hod. This is ollowed by so ing o hese sco es along wi h he ank indices (i.e he
aining indices) al eady assigned o he gene combina ions. The end esul is a so ed
o de o he gene combina ions based on he anking sco e lea ned by he SV MRank
algo i hm. Finally, his en i e p ocedu e is compu ed o sensi i i y indices gene a ed
o each and e e y old change a ime poin and de ia ions in old change a di e en
du a ions. Obse ing he changing ank o a pa icula combina ion a di e en imes
and di e en ime pe iods will e eal how a combina ion is beha ing.
No e ha he ollowing is he o de in which he iles should be execu ed in R, in
o de , o ob aining he desi ed esul s (No e ha he code will no be explained he e) - •
use sou ce(”mainSc ip -1-1.R”) wi h a gumen s o Dynamic da a •sou ce(”SVMRank-
Resul s-D.R”), o ank he in e ac ions (again his needs o be done sepa a ely o
di e en kinds o SA me hods), •use sou ce(”Combine-Time- iles.R”), i compu -
ing indices sepa a ely ia p e ious ile, •sou ce(”So -n-Plo -D.R”) o so he in e -
ac ions. No e ha he so ing is chages he in e ac ion anking in ime. Thus •use
sou ce(”In e ac ion-P io i y-In ime.R”) o ind he p io i ized anking o each and e -
e y in e ac ion o e he di e en ime poin s and inally •use sou ce(”P in -Ranking-
AND-In e ac ion-Rank.R”) o p in indi idual anking o he equi ed inpu ac o wi h
o he in e ac ion ac o s.
6. Resul s & Discussion
6.1. Time se ies da a by Guj al and MacBea h [1]
NOTE - Ranking was assigned on sco es ha we e so ed in DECREASING alues.
So, 1 was assigned o highes sco e and ice e sa.
Resul s o he 3 d o de in e ac ions a e p esen ed he e. The esul s i s discuss
5
he beha iou o in e ac ions ac oss he snapsho s o ime using he compu ed sensi-
i i ies on old change measu emen s pe ime snapsho . The analysis was done us-
ing 4 di e en sensi i i y indices. Ou o he 71C3combina ions, I conside /p esen
only hose combina ions ha show a anking wi hin i s 10,000 ou o 57,155. This
choice is libe al and biologis s/oncologis s can ha e a mo e s ic e choice as pe need.
Two obse a ions a e made, • he anking o a pa icula combina ion is conse ed (i.e
wi hin he 10,000 ange) in a pa icula ime poin o in he ea ly phase o la e phase
o WNT3A s imula ion, ac oss he majo i y o he ou sensi i i y me hods, which is a
s ic c i e ia o assessmen o • he anking o a pa icula combina ion is conse ed
ac oss ime poin s/phase (i.e hey a e wi hin he 10,000 ange) and he majo i y o he
ou sensi i i y me hods, which is elaxed c i e ia o assessmen . Applying his il e
helps e eal impo an combina ions o in e es ha migh be wo king syne gis ically
a a highe o de le el in he cell.
Rega ding echnical poin s o implemen a ion, he ankings we e gene a ed wi h-
ou scaling/no malizing he ime se ies da a p o ided by Guj al and MacBea h [1].
Fo es ima ing he sensi i i y indices, a small gaussian dis ibu ion using he unc ion
no m ha gene a es a ec o o no mally dis ibu ed andom a iables gi en a ec o
leng h n (he e 9, he 10 h one is he mean/ eco ded gene egula ion i sel ), a popula ion
mean µand popula ion s anda d de ia ion σ. The syn ax o using no m is as ollows:
no m(n, mean, sd). Fu he , I use he ji e un ion o add a li le bi o noise o he
da a. This helps o see i he gene a ed ankings a e obus o no .
6.2. Enume a ion and anking o 2415 PYGO1-X-X combina ions
om Guj al and MacBea h [1]
In he supplemen a y sec ion, I p esen ou iles, each con aining he ankings o 3 d
o de combina ions, ha wa y in ime (shown o 5 ime poin s). Each ile ep esen s
he ankings compu ed using a pa icula sensi i i y me hod. The changing ankings
in ime o a pa icula combina ion ep esen s he impo ance o con ibu ion/ ole ha
combina ion plays in he cell s imula ed wi h WNT3A. The sensi i i y me hods used
a e Hilbe Schmid Independence C i e ion indices (HSIC) indices (wi h b and linea
ke nel in Da Veiga [10]) and Sobol indicies (wi h 2002 implemen a ion in Sal elli [11]
and ma inez implemen a ion in Ma inez [12] and Baudin e al. [13]).
6.3. Conse ed machine lea ning ankings o es ed PYGO1-X-X
combina ions
A o al o 2415, 3 d o de combina ions in ol ing PYGO1 we e ob ained om a ull
se o 71C3= 57155 combina ions. Fu he , om his selec ed se , using he abo e c i-
e ia o conse ed ankings, I epo / abula e he meaning ul combina ions ha migh
be wo king syne gis ically. Tables 2, 3 and 4 show he ankings o he same combi-
na ions as in able 1, bu using b ke nel o HSIC, 2002 implemen a ion o SOBOL
and ma inez implemen a ion o SOBOL, espec i ely. As one allies he ankings o
ac oss hese ables o a pa icula combina ion, one inds ha he ole o he combina-
ion o in e es is conse ed. This conse a ion poin s o he exis ence o he biological
6
RANKING @ iUSING HSIC - LINEAR
3 d o de comb. 1 3 6 12 24 3 d o de comb. 1 3 6 12 24
CCND1-PYGO1-WNT2 191 48833 33097 15727 49025 FBXW2-PYGO1-WNT2 214 45234 28842 7216 56378
DKK1-JUN-PYGO1 218 3236 51191 53120 2659 DAAM1-PYGO1-WNT2 228 35434 22217 8095 17961
CCND1-PYGO1-SFRP4 248 49907 20135 22408 46010 DKK1-PYGO1-SENP2 253 27760 37224 42160 22797
FBXW2-PYGO1-SENP2 295 47321 21813 9500 57018 FZD5-JUN-PYGO1 348 1924 32815 54426 36260
DAAM1-PYGO1-SENP2 365 46893 25207 11837 39100 JUN-PYGO1-WNT3A 570 14118 6423 19469 36536
DKK1-PYGO1-WNT2 590 20065 32145 31502 24399 FZD5-PYGO1-SENP2 593 19014 1524 25504 36744
LRP5-PYGO1-WNT2 598 6053 19908 2338 2607 DAAM1-PYGO1-SFRP4 632 42697 21965 12019 5353
PYGO1-WNT2-WNT2B 707 39634 53492 38813 24079 FRZB-PYGO1-WNT2 817 38567 8350 5946 10554
CCND1-PYGO1-TCF7 838 55341 21851 12115 40132 CCND1-PYGO1-WNT3A 859 46424 26699 23831 49470
FBXW2-PYGO1-WNT4 880 37565 28874 6673 56916 FBXW2-PYGO1-SFRP4 895 48164 18463 9004 56766
CTBP1-PYGO1-WNT2 902 24919 8428 7477 7761 CCND1-PYGO1-SLC9A3R1 906 40276 22490 8120 43281
DAAM1-PYGO1-TCF7L1 959 51225 30853 10497 25266 CCND1-PYGO1-RHOU 1040 56164 34165 35225 53648
DKK1-PYGO1-WNT2B 1041 14062 34049 32775 7169 KREMEN1-PYGO1-WNT3A 1064 30070 1340 20277 52737
FBXW2-PYGO1-FBXW4 1225 51095 49445 9156 56717 FBXW2-PYGO1-WNT3A 1243 46844 41378 10348 56667
FRZB-PYGO1-SENP2 1288 22336 2406 8695 20479 JUN-PYGO1-TLE2 1341 24994 40582 4657 37476
CCND3-JUN-PYGO1 1362 50934 48125 55357 25009 PYGO1-WNT2-WNT5A 1374 29615 29500 7763 11360
CTNNBIP1-PYGO1-WNT2 1475 25916 18667 8796 8023 CXXC4-JUN-PYGO1 1496 10929 38139 43065 17470
DKK1-PYGO1-WNT3A 1670 37660 30663 36831 4685 DKK1-PYGO1-TCF7L1 1693 11516 32448 36695 18729
DVL2-PYGO1-WNT2 1756 33028 17795 1917 9628 CTNNBIP1-JUN-PYGO1 1762 8717 41940 46022 26397
FZD8-PYGO1-WNT2 1765 28859 12717 10912 10009 FBXW2-PYGO1-TLE2 1777 51565 50865 5489 56420
FZD5-PYGO1-TLE2 1787 4926 20017 5465 20177 CTBP1-PYGO1-WNT4 1792 40412 9281 9970 26271
FZD6-JUN-PYGO1 1799 8152 37586 49539 6795 JUN-PYGO1-SLC9A3R1 1831 18378 16698 6395 19077
CTBP1-PYGO1-SFRP4 1847 16809 2399 14104 18544 FZD5-PYGO1-FBXW4 1870 26024 50283 20420 38032
FBXW11-PYGO1-WNT2B 1888 54480 51998 19913 52577 DKK1-PYGO1-FBXW4 1953 16029 50510 30614 10640
FBXW11-PYGO1-WNT2 1968 36688 49197 5995 43963 JUN-PYGO1-WNT2B 1971 11759 22992 23296 32538
CSNK1G1-JUN-PYGO1 2002 4879 55264 32395 18429 JUN-PYGO1-RHOU 2122 21105 6824 25993 11386
JUN-PYGO1-TCF7L1 2211 39190 16299 13477 38507 JUN-PYGO1-WNT5A 2273 30941 1607 4887 6388
FZD2-LEF1-PYGO1 2290 40489 23166 26380 24432 FOSL1-PYGO1-WNT2 2296 35937 10116 9410 31529
DKK1-PYGO1-RHOU 2300 4971 25047 37502 11114 CCND1-PYGO1-SENP2 2354 25381 28894 27333 44018
DKK1-PYGO1-TLE2 2419 23834 49694 20745 27128 FBXW11-PYGO1-SENP2 2510 31734 49998 10856 40231
LEF1-PYGO1-WNT2 2594 6119 13281 49 48852 FZD1-PYGO1-SENP2 2614 7568 813 3128 19562
FRZB-PYGO1-SFRP4 2652 36390 1403 9283 23896 BTRC-GSK3A-PYGO1 2771 52085 53096 35408 47985
FZD5-PYGO1-TCF7L1 2870 23621 13839 25788 23143 CCND1-PYGO1-WNT2B 2903 53947 46315 34453 44275
KREMEN1-PYGO1-WNT2B 2904 37451 19332 29917 48436 FBXW11-PYGO1-TLE2 2906 44334 53969 2181 52786
DVL2-PYGO1-WNT3A 2944 11528 11111 13103 36306 EP300-PYGO1-WNT2 2948 43158 15314 8724 20816
CSNK1D-FGF4-PYGO1 2967 23307 41918 28164 3925 FZD5-PYGO1-WNT4 2980 4229 8238 13260 24709
CTBP1-PYGO1-TCF7 3004 9210 11537 8220 11898 CCND1-PYGO1-TLE2 3079 50182 49938 8787 45301
CTBP1-PYGO1-SENP2 3081 8819 2492 14648 22660 FRAT1-JUN-PYGO1 3094 24225 44587 43882 21321
DKK1-PYGO1-SFRP4 3105 19240 32988 43051 11763 DIXDC1-PYGO1-WNT2 3118 7736 11350 8945 27577
LEF1-PYGO1-WNT3 3128 434 1488 29 43589 FBXW11-PYGO1-SFRP4 3151 40355 44072 9884 48388
DIXDC1-PYGO1-TCF7L1 3193 33168 18183 17378 30931 PYGO1-WIF1-WNT4 3238 2703 55318 32252 32949
CCND1-PYGO1-FBXW4 3286 26986 41890 24897 52371 AES-FOXN1-PYGO1 3305 25455 24093 28905 5012
LEF1-PYGO1-WNT3A 3330 4637 9706 492 45999 FZD1-PYGO1-WNT2 3364 25302 3943 1660 5188
FZD1-JUN-PYGO1 3370 32879 29253 28580 37224 FRZB-PYGO1-WNT2B 3417 26749 22590 20939 16614
CCND3-PYGO1-WNT3A 3454 44362 41213 28500 14947 CSNK2A1-PYGO1-WNT2 3524 34440 54530 10724 18467
DVL2-PYGO1-TLE2 3562 38778 46616 3007 4918 CSNK1G1-PYGO1-TLE2 3565 24569 56894 5227 16188
FOSL1-JUN-PYGO1 3590 27459 38794 51790 55928 DVL2-PYGO1-FBXW4 3595 20330 55028 6420 42020
FZD7-PYGO1-WNT3A 3597 13080 6229 10048 9938 LRP5-PYGO1-SFRP4 3644 1525 13977 5563 18801
CTNNBIP1-PYGO1-SENP2 3739 6879 9216 12253 26261 CCND1-PYGO1-TCF7L1 3805 54303 46961 29812 46238
FRZB-PYGO1-TLE2 3809 42613 27606 3105 9575 DVL2-PYGO1-SENP2 3834 9313 17069 10011 29581
GSK3B-PYGO1-TLE2 3889 10232 51499 31294 33743 CSNK1D-JUN-PYGO1 3934 1849 39438 23725 46933
AXIN1-MYC-PYGO1 3954 3444 10994 17667 24234 FBXW11-PYGO1-WNT3A 3985 37584 56193 14716 37160
LEF1-PYGO1-SFRP1 3992 3352 7797 117 11186 FOSL1-PYGO1-SFRP4 4009 35642 2304 16916 49861
FRAT1-PYGO1-WNT3A 4132 14097 12408 18181 22475 FOSL1-PYGO1-WNT2B 4188 9498 24044 24964 53151
NLK-PYGO1-WNT2 4229 27445 40800 13988 7354 AES-JUN-PYGO1 4280 27713 29918 47174 22758
FZD8-PYGO1-WNT2B 4284 33894 27397 24043 46041 LRP6-PYGO1-WIF1 4293 17363 23754 6785 4390
KREMEN1-PYGO1-WNT2 4388 35884 6984 16968 53392 DVL2-PYGO1-TCF7L1 4435 40655 29622 11516 29181
FOSL1-PYGO1-SENP2 4461 29888 2317 13025 53543 DAAM1-PYGO1-TCF7 4470 39048 22767 7968 11799
FZD5-PYGO1-RHOU 4574 8615 5925 32341 7083 PITX2-PYGO1-TLE2 4588 2003 47249 8274 54767
FZD5-PYGO1-SLC9A3R1 4599 10851 8326 6893 16260 DAAM1-PYGO1-TLE1 4607 51419 32265 15738 19668
CCND1-PYGO1-SFRP1 4616 49034 18235 22959 42446 FBXW11-PYGO1-TCF7L1 4731 49531 56943 14286 52062
DAAM1-JUN-PYGO1 4756 47637 25776 47368 4345 CSNK2A1-PYGO1-TCF7 4808 27076 33334 7691 16030
FBXW2-PYGO1-TLE1 4855 13029 28477 2137 56462 GSK3B-PYGO1-WNT2 4907 10086 14344 28902 33359
Table 1: Rankings o PYGO1-X-X. A lis o app oxima ely i s 125 combina ions wi h ankings below
10,000 ou o 57,155. SA - HSIC; Ke nel - linea
syne gy, whe he he combina ion has been es ed o unexplo ed/un es ed.
7
RANKING @ iUSING HSIC - RBF
3 d o de comb. 1 3 6 12 24 3 d o de comb. 1 3 6 12 24
CCND1-PYGO1-WNT2 7685 48218 45186 9095 12535 FBXW2-PYGO1-WNT2 6632 47000 10921 10825 10660
DKK1-JUN-PYGO1 35611 92 8244 34065 42310 DAAM1-PYGO1-WNT2 13220 45816 8644 1196 14447
CCND1-PYGO1-SFRP4 7901 42981 32796 28660 4288 DKK1-PYGO1-SENP2 403 23098 9845 24707 36625
FBXW2-PYGO1-SENP2 5592 54522 7624 18559 15058 FZD5-JUN-PYGO1 13399 522 43506 36219 51123
DAAM1-PYGO1-SENP2 7009 46420 6762 21944 29682 JUN-PYGO1-WNT3A 10181 579 35737 33610 43746
DKK1-PYGO1-WNT2 3282 13742 6775 127 50868 FZD5-PYGO1-SENP2 864 25856 32614 26549 46981
LRP5-PYGO1-WNT2 24259 12625 15087 18812 33964 DAAM1-PYGO1-SFRP4 10758 46433 7217 48291 9239
PYGO1-WNT2-WNT2B 42713 30786 13433 45795 1718 FRZB-PYGO1-WNT2 2310 29711 53695 989 52194
CCND1-PYGO1-TCF7 3994 53634 8088 23990 6794 CCND1-PYGO1-WNT3A 2014 38925 42108 33199 4343
FBXW2-PYGO1-WNT4 7669 38818 19627 43015 3643 FBXW2-PYGO1-SFRP4 6016 52622 9354 20433 4893
CTBP1-PYGO1-WNT2 7914 40190 53737 1881 54646 CCND1-PYGO1-SLC9A3R1 8501 30855 32431 9221 12132
DAAM1-PYGO1-TCF7L1 6113 49053 11325 35525 9242 CCND1-PYGO1-RHOU 5175 54648 29737 46772 20403
DKK1-PYGO1-WNT2B 2010 10992 36338 13891 29481 KREMEN1-PYGO1-WNT3A 7864 26771 50128 43544 15239
FBXW2-PYGO1-FBXW4 5992 55287 26603 3782 9116 FBXW2-PYGO1-WNT3A 7391 48320 9248 34651 2524
FRZB-PYGO1-SENP2 334 16496 42795 31671 51686 JUN-PYGO1-TLE2 2091 9953 10078 26860 57045
CCND3-JUN-PYGO1 15052 38950 15637 52389 3265 PYGO1-WNT2-WNT5A 36384 23157 25066 7716 16105
CTNNBIP1-PYGO1-WNT2 20950 23080 54816 1243 54421 CXXC4-JUN-PYGO1 15268 3842 31367 43633 53074
DKK1-PYGO1-WNT3A 1315 30284 30530 24495 19085 DKK1-PYGO1-TCF7L1 2316 814 35496 27049 25451
DVL2-PYGO1-WNT2 6832 31920 27128 7953 48179 CTNNBIP1-JUN-PYGO1 41135 2896 47437 45143 51706
FZD8-PYGO1-WNT2 3462 16415 43751 3201 35160 FBXW2-PYGO1-TLE2 6212 55715 1956 16260 24385
FZD5-PYGO1-TLE2 4104 5653 37820 25448 57153 CTBP1-PYGO1-WNT4 22742 44655 41099 39815 48059
FZD6-JUN-PYGO1 35697 277 641 50698 33247 JUN-PYGO1-SLC9A3R1 3487 15500 27418 13894 51869
CTBP1-PYGO1-SFRP4 22918 27814 51439 38327 43332 FZD5-PYGO1-FBXW4 8568 40214 29428 9395 56877
FBXW11-PYGO1-WNT2B 7490 53715 924 4743 16840 DKK1-PYGO1-FBXW4 4015 24648 4205 73 53593
FBXW11-PYGO1-WNT2 13085 22818 21507 4205 23661 JUN-PYGO1-WNT2B 2703 20164 23828 23294 38517
CSNK1G1-JUN-PYGO1 17775 1546 36260 46060 22759 JUN-PYGO1-RHOU 3507 2626 38630 48273 44356
JUN-PYGO1-TCF7L1 2998 25012 36064 35492 46418 JUN-PYGO1-WNT5A 9776 20912 9445 6655 56641
FZD2-LEF1-PYGO1 19320 30354 33523 35934 37423 FOSL1-PYGO1-WNT2 4646 17601 54904 1115 45855
DKK1-PYGO1-RHOU 594 8755 12396 40458 43195 CCND1-PYGO1-SENP2 2887 16723 33585 4220 12776
DKK1-PYGO1-TLE2 1493 17701 42910 17294 57025 FBXW11-PYGO1-SENP2 882 19979 26917 15962 25523
LEF1-PYGO1-WNT2 4130 36760 36544 4175 29233 FZD1-PYGO1-SENP2 2237 14486 13479 4706 37678
FRZB-PYGO1-SFRP4 2953 27146 15142 30999 39478 BTRC-GSK3A-PYGO1 16077 51303 28870 23885 34429
FZD5-PYGO1-TCF7L1 552 22883 43339 19418 51934 CCND1-PYGO1-WNT2B 4500 49840 12312 25574 14068
KREMEN1-PYGO1-WNT2B 2480 27771 5674 47549 30280 FBXW11-PYGO1-TLE2 3570 44556 20858 19771 39583
DVL2-PYGO1-WNT3A 18087 2223 36690 1889 26351 EP300-PYGO1-WNT2 5709 43420 44821 441 34191
CSNK1D-FGF4-PYGO1 35224 24194 35705 42726 4974 FZD5-PYGO1-WNT4 12619 9688 49504 15069 50337
CTBP1-PYGO1-TCF7 12120 36550 3309 22441 35094 CCND1-PYGO1-TLE2 2708 46475 14041 11551 36187
CTBP1-PYGO1-SENP2 14535 11602 42803 9594 47507 FRAT1-JUN-PYGO1 33906 17235 3856 45519 18398
DKK1-PYGO1-SFRP4 3408 7708 23449 34970 34048 DIXDC1-PYGO1-WNT2 9260 4892 51202 277 17819
LEF1-PYGO1-WNT3 8116 6747 37915 10868 26227 FBXW11-PYGO1-SFRP4 3371 25915 424 42974 31841
DIXDC1-PYGO1-TCF7L1 9179 35935 48383 28960 14143 PYGO1-WIF1-WNT4 30659 11937 27320 45398 3025
CCND1-PYGO1-FBXW4 5824 27098 17674 13615 16815 AES-FOXN1-PYGO1 4657 24703 43459 42184 24604
LEF1-PYGO1-WNT3A 7948 34410 45811 27590 17447 FZD1-PYGO1-WNT2 8801 28775 15560 1399 50819
FZD1-JUN-PYGO1 46076 19069 9867 46865 48594 FRZB-PYGO1-WNT2B 3505 12360 4064 28390 55537
CCND3-PYGO1-WNT3A 782 47470 16204 30209 9636 CSNK2A1-PYGO1-WNT2 6052 32583 28770 2461 39536
DVL2-PYGO1-TLE2 8563 43181 37961 17796 56882 CSNK1G1-PYGO1-TLE2 21495 24434 29013 7562 53728
FOSL1-JUN-PYGO1 39670 8443 35133 55401 48823 DVL2-PYGO1-FBXW4 15883 30156 604 9016 45692
FZD7-PYGO1-WNT3A 2200 3030 44689 22869 39525 LRP5-PYGO1-SFRP4 14664 1251 37823 41590 26535
CTNNBIP1-PYGO1-SENP2 8077 5298 52208 9813 41948 CCND1-PYGO1-TCF7L1 10623 47122 16194 21979 4501
FRZB-PYGO1-TLE2 3510 45630 22284 32164 57136 DVL2-PYGO1-SENP2 3166 806 29432 11396 29074
GSK3B-PYGO1-TLE2 1511 1087 42710 14437 57121 CSNK1D-JUN-PYGO1 46732 424 46278 41608 30058
AXIN1-MYC-PYGO1 33645 8460 51922 49242 34352 FBXW11-PYGO1-WNT3A 5958 23716 24819 17645 5707
LEF1-PYGO1-SFRP1 8248 25313 16956 27035 35434 FOSL1-PYGO1-SFRP4 3428 32371 44049 49180 33552
FRAT1-PYGO1-WNT3A 6165 13829 40997 17174 24514 FOSL1-PYGO1-WNT2B 4892 4674 16682 35879 52932
NLK-PYGO1-WNT2 7759 3583 13307 7257 24124 AES-JUN-PYGO1 25084 9408 7447 50510 20969
FZD8-PYGO1-WNT2B 5074 27177 16095 15562 27187 LRP6-PYGO1-WIF1 40589 27373 31407 1058 42246
KREMEN1-PYGO1-WNT2 2834 38294 45106 1581 41784 DVL2-PYGO1-TCF7L1 4022 45893 41755 25284 37685
FOSL1-PYGO1-SENP2 977 13985 43848 13391 28612 DAAM1-PYGO1-TCF7 1729 42769 4631 5543 14266
FZD5-PYGO1-RHOU 543 3213 11602 49189 47416 PITX2-PYGO1-TLE2 1782 35079 27236 39307 56772
FZD5-PYGO1-SLC9A3R1 13164 6884 38119 28521 55716 DAAM1-PYGO1-TLE1 9367 40988 3088 5163 19658
CCND1-PYGO1-SFRP1 7909 44664 37002 6160 22267 FBXW11-PYGO1-TCF7L1 2771 39588 19054 11993 11919
DAAM1-JUN-PYGO1 35403 45052 14876 50189 5673 CSNK2A1-PYGO1-TCF7 4497 22038 26026 17251 48738
FBXW2-PYGO1-TLE1 11243 2157 3112 11981 5521 GSK3B-PYGO1-WNT2 4395 4042 49269 2154 52107
Table 2: Rankings o PYGO1-X-X. A lis o app oxima ely i s 125 combina ions wi h ankings below
10,000 ou o 57,155. SA - HSIC; Ke nel - b
6.3.1. Examining he beha iou o WNT-PYGO1-X combina ions
K amps e al. [4] indica e ha TCF and β-ca enin unc ion in a e apa i e complex
wi h LGS/BCL9 and PYGO o ansc ip ionally ac i a e a ge genes in esponse o
8
RANKING @ iUSING SOBOL - 2002
3 d o de comb. 1 3 6 12 24 3 d o de comb. 1 3 6 12 24
CCND1-PYGO1-WNT2 23571 36410 15616 2053 54902 FBXW2-PYGO1-WNT2 22809 29745 16419 7178 38040
DKK1-JUN-PYGO1 20453 50366 17707 8188 42484 DAAM1-PYGO1-WNT2 11655 42330 15885 7995 42320
CCND1-PYGO1-SFRP4 6028 42645 2289 1001 55845 DKK1-PYGO1-SENP2 5320 8965 25659 16677 34823
FBXW2-PYGO1-SENP2 19176 47463 19297 20507 39832 FZD5-JUN-PYGO1 22701 20169 18383 5021 52969
DAAM1-PYGO1-SENP2 10224 2416 14099 12136 24858 JUN-PYGO1-WNT3A 1610 17601 24757 13846 37085
DKK1-PYGO1-WNT2 3006 20828 19259 10946 32234 FZD5-PYGO1-SENP2 10582 54199 15471 9662 50406
LRP5-PYGO1-WNT2 6676 12359 19432 21720 43580 DAAM1-PYGO1-SFRP4 12825 5259 14739 27176 39944
PYGO1-WNT2-WNT2B 35098 23545 34784 49087 46216 FRZB-PYGO1-WNT2 12262 22169 15532 4607 50768
CCND1-PYGO1-TCF7 18122 45572 2446 2027 55938 CCND1-PYGO1-WNT3A 39080 19013 56205 51729 13572
FBXW2-PYGO1-WNT4 6048 35274 8914 13383 40180 FBXW2-PYGO1-SFRP4 22052 24720 4613 5903 54788
CTBP1-PYGO1-WNT2 20153 27678 2537 9397 33838 CCND1-PYGO1-SLC9A3R1 5279 1741 18699 16570 29343
DAAM1-PYGO1-TCF7L1 37114 50690 39761 33545 3030 CCND1-PYGO1-RHOU 43775 56261 50329 56095 7953
DKK1-PYGO1-WNT2B 54188 35764 37921 46266 24833 KREMEN1-PYGO1-WNT3A 52932 56977 53771 37472 14726
FBXW2-PYGO1-FBXW4 35105 32258 52561 51292 2367 FBXW2-PYGO1-WNT3A 52031 31884 46340 31823 2792
FRZB-PYGO1-SENP2 11932 6986 25367 12616 52422 JUN-PYGO1-TLE2 3800 11209 18229 255 32111
CCND3-JUN-PYGO1 25910 36673 28134 15173 40843 PYGO1-WNT2-WNT5A 38189 30159 29176 50070 44803
CTNNBIP1-PYGO1-WNT2 11459 19288 18921 4735 9712 CXXC4-JUN-PYGO1 13879 27252 8058 10543 55180
DKK1-PYGO1-WNT3A 49429 22863 28934 47297 24196 DKK1-PYGO1-TCF7L1 53013 53567 39949 56715 29970
DVL2-PYGO1-WNT2 24731 1948 7444 10308 48870 CTNNBIP1-JUN-PYGO1 14511 20205 9590 1997 56057
FZD8-PYGO1-WNT2 1313 6988 26283 14105 56504 FBXW2-PYGO1-TLE2 32326 13530 50413 46764 9323
FZD5-PYGO1-TLE2 43027 104 46448 43876 24737 CTBP1-PYGO1-WNT4 27649 26968 22166 2301 29150
FZD6-JUN-PYGO1 660 283 18899 19152 55094 JUN-PYGO1-SLC9A3R1 39968 7155 33471 32607 20833
CTBP1-PYGO1-SFRP4 18530 26668 2815 1299 30332 FZD5-PYGO1-FBXW4 39183 2104 46072 43193 2921
FBXW11-PYGO1-WNT2B 33541 53563 42544 44866 11547 DKK1-PYGO1-FBXW4 51614 42906 37164 38683 33819
FBXW11-PYGO1-WNT2 23628 3600 14621 12236 45387 JUN-PYGO1-WNT2B 784 29200 21789 1189 45268
CSNK1G1-JUN-PYGO1 39719 47416 54841 47973 10949 JUN-PYGO1-RHOU 3266 37143 23419 11201 38259
JUN-PYGO1-TCF7L1 4196 41759 27014 26374 34930 JUN-PYGO1-WNT5A 2460 21997 22687 2953 49677
FZD2-LEF1-PYGO1 39966 1605 48301 38150 15979 FOSL1-PYGO1-WNT2 47297 50999 30260 50806 4015
DKK1-PYGO1-RHOU 52268 16276 32787 34998 26390 CCND1-PYGO1-SENP2 9363 55936 5775 22711 48701
DKK1-PYGO1-TLE2 53371 13490 36793 44944 18357 FBXW11-PYGO1-SENP2 23322 1344 12785 8511 39644
LEF1-PYGO1-WNT2 17290 10437 14509 19069 34734 FZD1-PYGO1-SENP2 17605 57130 8602 23919 43562
FRZB-PYGO1-SFRP4 19646 20211 10524 5314 31613 BTRC-GSK3A-PYGO1 40306 12238 50108 55258 7556
FZD5-PYGO1-TCF7L1 39929 19214 31569 49425 9668 CCND1-PYGO1-WNT2B 33592 20824 41585 55101 2315
KREMEN1-PYGO1-WNT2B 49601 24278 44028 43212 12272 FBXW11-PYGO1-TLE2 33225 36548 35122 52970 13629
DVL2-PYGO1-WNT3A 35451 32587 46161 49351 14826 EP300-PYGO1-WNT2 31940 56694 45844 45742 12882
CSNK1D-FGF4-PYGO1 41090 20740 34158 34168 20857 FZD5-PYGO1-WNT4 15278 10463 12217 3358 53540
CTBP1-PYGO1-TCF7 19167 24468 5531 3011 33571 CCND1-PYGO1-TLE2 38969 17016 52496 38112 384
CTBP1-PYGO1-SENP2 10240 25052 9151 4681 32852 FRAT1-JUN-PYGO1 17301 5414 8956 6791 9863
DKK1-PYGO1-SFRP4 5555 14281 20084 18507 23471 DIXDC1-PYGO1-WNT2 38631 6548 43841 56224 38207
LEF1-PYGO1-WNT3 16493 40318 11960 27230 25603 FBXW11-PYGO1-SFRP4 19547 42729 11935 6272 45795
DIXDC1-PYGO1-TCF7L1 2188 42426 19263 4130 25766 PYGO1-WIF1-WNT4 35126 29183 31527 51724 48035
CCND1-PYGO1-FBXW4 51130 15106 54885 56151 1320 AES-FOXN1-PYGO1 13627 21324 3166 21211 26154
LEF1-PYGO1-WNT3A 40621 17131 45161 29919 31440 FZD1-PYGO1-WNT2 18743 55194 11811 3018 31667
FZD1-JUN-PYGO1 10842 9463 23552 17495 34735 FRZB-PYGO1-WNT2B 44906 34928 41610 52546 6348
CCND3-PYGO1-WNT3A 51433 27320 30354 36390 39641 CSNK2A1-PYGO1-WNT2 27842 10862 10937 6962 53007
DVL2-PYGO1-TLE2 34256 44308 42625 42719 3422 CSNK1G1-PYGO1-TLE2 17136 9155 11518 17890 32040
FOSL1-JUN-PYGO1 35100 56491 39744 42645 28344 DVL2-PYGO1-FBXW4 29729 33444 33783 33618 40149
FZD7-PYGO1-WNT3A 1165 50482 26897 25768 53340 LRP5-PYGO1-SFRP4 2300 55858 7523 22759 51715
CTNNBIP1-PYGO1-SENP2 21191 36983 19971 3965 11871 CCND1-PYGO1-TCF7L1 39036 11286 54703 55124 1216
FRZB-PYGO1-TLE2 45842 21383 45458 51288 1668 DVL2-PYGO1-SENP2 26270 14793 24368 13556 8825
GSK3B-PYGO1-TLE2 55303 43455 31052 48879 8231 CSNK1D-JUN-PYGO1 24772 39437 27678 10274 47704
AXIN1-MYC-PYGO1 4066 39692 3553 5774 48217 FBXW11-PYGO1-WNT3A 39947 53781 36004 52889 15695
LEF1-PYGO1-SFRP1 53359 2338 48778 30849 55971 FOSL1-PYGO1-SFRP4 39720 40936 40202 47288 2631
FRAT1-PYGO1-WNT3A 37686 56136 39369 37595 23150 FOSL1-PYGO1-WNT2B 12441 11961 5832 1148 30998
NLK-PYGO1-WNT2 7875 21489 20941 18370 48652 AES-JUN-PYGO1 20909 15200 3792 28252 30883
FZD8-PYGO1-WNT2B 55854 50198 30897 43016 651 LRP6-PYGO1-WIF1 44508 17042 34865 29362 9716
KREMEN1-PYGO1-WNT2 7561 33096 13153 14050 44943 DVL2-PYGO1-TCF7L1 30408 40479 30117 35926 43261
FOSL1-PYGO1-SENP2 48594 56124 49849 56650 18878 DAAM1-PYGO1-TCF7 20090 6405 17388 23697 54109
FZD5-PYGO1-RHOU 43941 53488 41361 43989 27808 PITX2-PYGO1-TLE2 19850 10379 18180 6093 40940
FZD5-PYGO1-SLC9A3R1 10744 49408 12251 7758 51542 DAAM1-PYGO1-TLE1 11821 6611 9646 28349 35623
CCND1-PYGO1-SFRP1 47797 1211 51402 34391 8554 FBXW11-PYGO1-TCF7L1 34205 54669 45848 48836 12237
DAAM1-JUN-PYGO1 10734 4256 13652 16493 54555 CSNK2A1-PYGO1-TCF7 15569 3364 11248 16573 53352
FBXW2-PYGO1-TLE1 24866 43447 6766 10408 47911 GSK3B-PYGO1-WNT2 7165 34491 27181 12830 44756
Table 3: Rankings o PYGO1-X-X. A lis o app oxima ely i s 125 combina ions wi h ankings below
10,000 ou o 57,155. SA - SOBOL; Implemen a ion - 2002
WNT signals. Using in si u hyb idiza ion, Schwab e al. [14] examined exp ession
o h ee WNT7B (Kispe e al. [15]), WNT11 (Majumda e al. [16]) and WNT9B
(Ca oll e al. [17]) genes in PYGO mu an s. They ound ha all h ee genes showed
simila exp ession pa e ns in PYGO2+/+and PYGO1/PYGO2 double-mu an kid-
9
