pai ed-like homeodomain ansc ip ion ac o 2 (PITX2) :
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
104-Madhu isha Heigh s Phase 1, Risali, Bhilai-490006, India
Abs ac
PITX2 (also known as pi ui a y homeobox 2), encodes a membe o he RIEG/PITX
homeobox amily (a bicoid class o homeodomain p o eins), ha ac s as a ansc ip-
ion ac o and egula es p ocollagen lysyl hyd oxylase gene exp ession. I is known o
play an impo an ole in pi ui a y, hea , b ain, lungs, spleen, wis ing o he gu and
s omach, as well as he de elopmen o he eyes. 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 ex-
hibi simila exp ession dynamics and unco e ed p e iously un ecognized posi i e and
nega i e eedback loops. Howe e , hei s udy depic s/uses singula measu emen s o
indi 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 emen s
a e singula in na u e. He e, I •enume a e and ank all 2415 PITX2 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 PITX2-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 PITX2 comb. in WNT3A s imula ed cells
Email add ess: [email p o ec ed] (sh ip akash sinha)
1Aspec s o unpublished wo k we e p esen ed in a pos e session a Cell Symposia: Technology.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 Feb ua y 22, 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 PITX2 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. pai ed-like homeodomain ansc ip ion ac o 2 (PITX2)
PITX2, encodes a membe o he RIEG/PITX homeobox amily (a bicoid class o
homeodomain p o eins (Holland e al. [4])). A homeobox is an app oxima ely 180
base pai s long DNA sequence, ha egula es ana omical ea u es in he ea ly s ages o
emb yonic de elopmen (Wikipedia con ibu o s [5]). Mu a ions in a homeobox may
change la ge-scale ana omical ea u es o he ull-g own o ganism. Homeobox genes
(Geh ing [6] and Geh ing [7]) encode homeodomain p o ein p oduc s a e ansc ip ion
ac o s wi h impo an oles in emb yonic pa e ning and cell di e en ia ion, and se -
e al ha e been implica ed in human diseases and congeni al abno mali ies (Boncinelli
[8]).
ALL1 (human homologue o D osophila i ho ax), is di ec ly in ol ed in human
acu e leukemias associa ed wi h abno mali ies a 11q23. A akawa e al. [9] isola ed a
gene ha is down- egula ed in All1 double-knockou mouse emb yonic s em (ES) cells
and designa ed i ARP1 (also e med RIEG, PITX2, o OTLX2) belonging o he PRD
class homeoboxes and pseudogenes. Logan e al. [10] show ha PITX2 encodes a an-
sc ip ion ac o exp essed h ough-ou he le la e al pla e mesode m and subsequen -
lyon he le side o asymme ic o gans such as he hea and gu du ing o ganogenesis
in he chick emb yo. Misexp ession o PITX2 on he igh side o he emb yo is su i-
cien o p oduce e e sed hea looping and hea isome isms, e e sed body o a ion,
and e e sed gu si us (Campione e al. [11], Shi a o i e al. [12], Zacha ias e al. [13]).
Finally, di e en iso o ms o he ansc ip ion ac o : PITX2-A/B/C, each wi h dis inc
and non-o e lapping unc ions exis .
In his esea ch wo k, I p esen 3 d o de combina ions o PITX2 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.
3
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 [14]. 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 [14].
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
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
4
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
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].
5
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 PITX2-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 [15]) and Sobol indicies (wi h 2002 implemen a ion in Sal elli [16]
and ma inez implemen a ion in Ma inez [17] and Baudin e al. [18]).
6.3. Conse ed machine lea ning ankings o es ed PITX2-X-X
combina ions
A o al o 2415, 3 d o de combina ions in ol ing PITX2 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 combina-
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
syne gy, whe he he combina ion has been es ed o unexplo ed/un es ed.
6.3.1. Examining he beha iou o WNT-PITX2-X combina ions
Kioussi e al. [19] epo ha PITX2 is apidly induced by he WNT/DVL/β-ca enin
pa hway and is equi ed o e ec i e cell- ype-speci ic p oli e a ion by di ec ly ac-
i a ing speci ic g ow h- egula ing genes. Regula ed exchange o HDAC1/β-ca enin
con e s PITX2 om ep esso o ac i a o (analogous o con ol o TCF/LEF1) which
hen se es as a compe ence ac o equi ed o he empo ally o de ed and g ow h
ac o -dependen ec ui men o a se ies o speci ic coac i a o complexes ha p o e
necessa y o CCND2 gene induc ion. Since he WNT pa hway is s ongly in ol ed
in o a ian de elopmen and cance , Basu and Roy [20] ocused on he possible asso-
cia ion be ween PITX2 and WNT pa hway in o a ian ca cinoma cells and ound ha
PITX2 in e ac s and egula es WNT2/5A/9A/6/2B genes o he canonical, noncanoni-
cal, o o he pa hways in he human o a ian adenoca cinoma cell SKOV-3. Ch oma in
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
APC-PITX2-SFRP4 14 26861 33433 10197 18249 APC-PITX2-SENP2 37 14630 40719 18877 15582
APC-PITX2-TCF7 130 34995 20653 15851 17861 PITX2-PORCN-TLE2 169 22676 35351 44094 14423
PITX2-PORCN-WNT4 177 16891 32175 32123 27627 PITX2-PORCN-SENP2 270 20159 27718 25665 18827
DVL2-JUN-PITX2 293 27343 32163 42607 21387 FZD5-PITX2-SENP2 328 35236 22541 1156 14803
APC-PITX2-PPP2CA 334 14753 2900 19245 30663 APC-PITX2-RHOU 397 38592 32191 27698 15148
PITX2-PORCN-WNT5A 460 36097 17787 53210 23094 PITX2-PORCN-FBXW4 499 12554 20396 26439 17842
DIXDC1-PITX2-SENP2 526 28707 7274 5676 35235 CTNNBIP1-JUN-PITX2 546 6389 45937 54346 11119
CCND1-FGF4-PITX2 554 54983 49474 23953 29838 PITX2-PORCN-TCF7L1 572 42893 22702 25919 13429
DIXDC1-PITX2-SFRP4 614 29602 6543 3868 43507 PITX2-PORCN-WNT2B 630 42884 44910 50269 15186
APC-PITX2-WNT4 634 38339 50192 23301 18227 FRAT1-JUN-PITX2 748 50033 38113 50412 44660
PITX2-PORCN-TLE1 813 43605 21513 22845 15786 DKK1-JUN-PITX2 913 6407 42538 52798 634
AES-AXIN1-PITX2 978 46773 17137 47484 7382 PITX2-PORCN-PPP2R1A 1003 8071 39783 44256 5481
PITX2-PORCN-RHOU 1220 38816 23756 29958 13324 CCND1-JUN-PITX2 1260 51205 42011 18763 52917
FOSL1-JUN-PITX2 1479 1306 37107 54515 12375 FZD6-PITX2-WNT2B 1504 10725 14634 54318 17213
FZD5-PITX2-SFRP4 1559 51198 27058 788 14497 PITX2-PORCN-SFRP4 1607 32189 32121 34063 21972
CCND1-CTBP1-PITX2 1644 56351 33831 12174 34580 FSHB-FZD2-PITX2 1681 25060 56319 33266 14323
PITX2-WNT3-WNT5A 1726 32979 5136 41370 17012 CCND2-LRP6-PITX2 1887 36449 16637 55064 12492
PITX2-PORCN-SFRP1 1905 39236 31192 37387 9110 AES-EP300-PITX2 2125 50901 43504 52709 17083
FZD6-PITX2-SFRP4 2136 44403 22523 5027 25162 PITX2-WNT3-WNT3A 2155 50776 27010 46543 38273
DAAM1-FOXN1-PITX2 2183 45577 23419 17738 1022 CSNK2A1-JUN-PITX2 2295 11006 48321 42767 19026
FBXW11-LRP6-PITX2 2307 48503 41757 51594 35405 CCND1-CTNNBIP1-PITX2 2315 54797 33975 42838 34145
CSNK2A1-FOXN1-PITX2 2356 10265 14777 12628 14201 FOXN1-GSK3A-PITX2 2381 48019 32781 32844 21255
AXIN1-FZD6-PITX2 2480 44926 6159 53367 9917 FRZB-GSK3A-PITX2 2482 10817 2209 668 13402
PITX2-WNT1-WNT5A 2609 5926 14837 51671 5692 BTRC-GSK3A-PITX2 2613 49655 52881 6241 50065
PITX2-SFRP4-WNT2B 2621 47714 20847 52301 32634 DAAM1-JUN-PITX2 2739 53719 47964 55644 657
CXXC4-JUN-PITX2 2757 3424 36286 40345 36376 APC-PITX2-WNT3 2778 27077 34088 34710 13819
PITX2-PORCN-PPP2CA 2847 24874 33030 43172 21878 DIXDC1-PITX2-WNT2B 2863 14619 3889 45440 40568
FBXW11-FOXN1-PITX2 2886 3843 4957 17704 23607 CSNK1D-FGF4-PITX2 3093 11417 24874 53815 30793
LRP5-PITX2-SFRP4 3210 16171 31459 8145 26086 DIXDC1-PITX2-WNT4 3214 52681 19702 7189 37575
FOSL1-GSK3A-PITX2 3246 8961 918 7353 12922 FBXW11-JUN-PITX2 3374 35385 45082 55257 41153
CCND3-PITX2-WNT3A 3484 43666 17937 5868 27350 CSNK2A1-MYC-PITX2 3758 35967 33879 27313 16543
EP300-FZD2-PITX2 3771 12315 30396 16799 22718 CSNK1D-FOXN1-PITX2 3778 6280 4678 21772 36345
FZD1-GSK3A-PITX2 3798 30685 3123 6034 15265 CCND3-PITX2-SFRP4 3846 53365 17972 862 38266
DVL1-EP300-PITX2 3886 37126 11319 53194 36735 DAAM1-GSK3A-PITX2 3913 55523 36819 2364 4273
DKK1-PITX2-SFRP4 3919 36216 54630 36550 651 GSK3B-PITX2-SFRP4 3969 51591 11242 44587 45162
FBXW2-JUN-PITX2 4023 15973 52731 42180 49503 FRZB-FZD2-PITX2 4081 5620 20625 16286 21997
DIXDC1-PITX2-PPP2CA 4123 13781 1911 14325 45522 DAAM1-LRP6-PITX2 4134 55453 49218 56874 3254
FOSL1-FOXN1-PITX2 4149 3704 3591 20411 19698 CTNNB1-FOXN1-PITX2 4154 3460 4095 19749 19696
PITX2-WNT1-WNT2B 4158 13925 1462 19718 12422 CCND3-PITX2-SENP2 4176 37072 17505 1404 25804
FZD5-PITX2-WNT2B 4182 39042 16284 52876 11251 FZD7-GSK3A-PITX2 4302 20752 736 4869 12197
AXIN1-EP300-PITX2 4304 11293 12887 53659 13073 FRAT1-GSK3A-PITX2 4363 56251 2247 18588 44926
CSNK1G1-FZD2-PITX2 4407 7002 19144 19885 20203 FOSL1-FZD2-PITX2 4478 4516 12555 16266 17423
FBXW11-PITX2-SFRP4 4547 50563 47219 11287 28318 CSNK1G1-PITX2-SENP2 4579 38292 32335 49857 17717
PITX2-PYGO1-TLE2 4588 2003 47249 8274 54767 CTBP2-FOXN1-PITX2 4611 37603 5195 14491 24302
DVL1-FOXN1-PITX2 4624 42047 4932 11016 38406 CTBP1-JUN-PITX2 4720 2611 40385 42676 12432
PITX2-WNT1-WNT4 4724 908 3272 33901 10244 DIXDC1-PITX2-RHOU 4739 31170 5147 8935 19768
FZD6-PITX2-TCF7 4754 25933 18851 11368 22426 DKK1-GSK3A-PITX2 4759 23623 33694 50522 6502
FZD6-PITX2-SENP2 4827 47413 31625 7317 23700 APC-PITX2-SLC9A3R1 4832 13233 25730 31544 21292
FBXW11-PITX2-WNT2B 4991 55437 27760 53601 30191 DIXDC1-PITX2-TCF7 5005 35931 12459 6681 32860
CSNK1D-PITX2-SFRP4 5096 37446 11233 10582 41257 FZD5-CCND3-PITX2 5106 9271 1305 14097 20152
FZD6-PITX2-WNT2 5113 30267 46069 12925 22168 AES-FOXN1-PITX2 5216 17207 8200 21874 15128
CSNK1G1-PITX2-RHOU 5258 16813 32518 47070 15599 FZD7-NKD1-PITX2 5310 12439 25127 40994 7486
FGF4-JUN-PITX2 5343 2634 32672 53764 32133 CCND1-NLK-PITX2 5399 39293 9478 4030 37469
FZD6-PITX2-SFRP1 5615 37130 25262 13890 9233 CXXC4-FGF4-PITX2 5669 13032 45784 50894 54142
AXIN1-FOXN1-PITX2 5682 1318 3400 19758 15375 FOSL1-PITX2-SFRP4 5766 29791 9666 5926 21290
FOSL1-NKD1-PITX2 5819 8607 19299 34454 29628 DKK1-PITX2-WNT2B 5967 20722 56765 57114 736
FZD5-PITX2-RHOU 5980 46930 21039 1256 11863 GSK3B-PITX2-SENP2 6007 41103 11874 39352 36153
FZD6-PITX2-TCF7L1 6019 6483 34081 5505 18404 CTBP1-FOXN1-PITX2 6078 7952 6550 21251 24027
FBXW2-GSK3A-PITX2 6113 23145 5408 17937 19874 EP300-GSK3B-PITX2 6131 11194 7972 26156 25577
CTBP1-FGF4-PITX2 6132 3149 40895 31072 24686 FRAT1-FZD2-PITX2 6168 56131 24624 14249 36492
AES-FZD2-PITX2 6222 26972 33042 43053 11722 FBXW11-GSK3A-PITX2 6249 47414 27591 23081 13112
FZD8-JUN-PITX2 6263 42057 38010 54749 2980 CXXC4-FZD2-PITX2 6270 7825 20641 14923 45220
PITX2-SFRP4-TLE2 6342 43420 38012 10662 35267 DIXDC1-FZD2-PITX2 6448 5815 17969 18298 39281
Table 1: Rankings o PITX2-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
immunop ecipi a ion and p omo e - epo e assays u he indica ed he signi ican as-
socia ion o PITX2 wi h WNT2 and WNT5A p omo e s. Looking a he ables abo e,
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
APC-PITX2-SFRP4 6639 22831 16274 5172 51942 APC-PITX2-SENP2 2251 31790 24604 4114 39754
APC-PITX2-TCF7 5809 43852 29659 2608 54162 PITX2-PORCN-TLE2 13743 33091 10461 36155 54286
PITX2-PORCN-WNT4 9247 2492 36647 13332 52478 PITX2-PORCN-SENP2 9499 29209 4986 29421 35908
DVL2-JUN-PITX2 22623 16521 37051 52719 15020 FZD5-PITX2-SENP2 10435 42695 24116 3954 33353
APC-PITX2-PPP2CA 4042 33446 8698 18579 52910 APC-PITX2-RHOU 4699 39874 24475 1820 49384
PITX2-PORCN-WNT5A 11335 32674 6548 15458 54809 PITX2-PORCN-FBXW4 2782 19946 19676 35972 50951
DIXDC1-PITX2-SENP2 3537 43865 15972 13329 19669 CTNNBIP1-JUN-PITX2 55688 6273 51543 54725 26305
CCND1-FGF4-PITX2 55688 6273 51543 54725 26305 PITX2-PORCN-TCF7L1 13222 48332 9570 21227 51753
DIXDC1-PITX2-SFRP4 9005 33898 35036 1595 29334 PITX2-PORCN-WNT2B 6802 25276 16938 13140 54357
APC-PITX2-WNT4 6441 34135 28508 16023 43494 FRAT1-JUN-PITX2 28349 55075 50998 56838 16856
PITX2-PORCN-TLE1 13872 39019 23792 7146 28106 DKK1-JUN-PITX2 24208 3989 4050 56945 15274
AES-AXIN1-PITX2 37489 47814 10383 53313 22724 PITX2-PORCN-PPP2R1A 11355 50277 26063 28070 55628
PITX2-PORCN-RHOU 11795 30595 6426 8297 54722 CCND1-JUN-PITX2 44312 43519 25962 49672 2955
FOSL1-JUN-PITX2 42099 14135 52254 57017 24935 FZD6-PITX2-WNT2B 8135 7300 14987 7418 3456
FZD5-PITX2-SFRP4 8368 50438 38825 1203 52857 PITX2-PORCN-SFRP4 7652 26224 19994 9630 41059
CCND1-CTBP1-PITX2 7652 26224 19994 9630 41059 FSHB-FZD2-PITX2 11301 38653 1373 55358 36056
PITX2-WNT3-WNT5A 50071 36976 3198 23809 43096 CCND2-LRP6-PITX2 20931 44333 44558 56656 20738
PITX2-PORCN-SFRP1 20244 35446 37738 24843 55456 AES-EP300-PITX2 46927 53837 1549 50756 32506
FZD6-PITX2-SFRP4 7135 52408 16969 864 34132 PITX2-WNT3-WNT3A 36927 41437 49172 9526 14468
DAAM1-FOXN1-PITX2 1685 41262 42245 51064 684 CSNK2A1-JUN-PITX2 25321 4747 44298 56844 22732
FBXW11-LRP6-PITX2 29615 47349 39438 51084 16601 CCND1-CTNNBIP1-PITX2 17845 54257 2633 44741 19765
CSNK2A1-FOXN1-PITX2 1130 13168 44037 50356 32422 FOXN1-GSK3A-PITX2 35488 50787 34525 56639 52918
AXIN1-FZD6-PITX2 33737 44557 47717 57090 46710 FRZB-GSK3A-PITX2 5619 18480 55073 54146 46967
PITX2-WNT1-WNT5A 29674 16693 38632 17220 34621 BTRC-GSK3A-PITX2 2406 44730 43962 54217 10925
PITX2-SFRP4-WNT2B 13474 47608 14111 19231 2624 DAAM1-JUN-PITX2 35874 49308 2846 56449 4164
CXXC4-JUN-PITX2 5093 2871 54929 54382 25342 APC-PITX2-WNT3 18467 40797 31945 31204 47530
PITX2-PORCN-PPP2CA 9603 23189 295 19554 39138 DIXDC1-PITX2-WNT2B 12649 21037 9654 4112 8993
FBXW11-FOXN1-PITX2 661 3042 46901 46989 62 CSNK1D-FGF4-PITX2 21808 22200 7826 55044 47261
LRP5-PITX2-SFRP4 1850 34385 2623 13213 34281 DIXDC1-PITX2-WNT4 11062 47027 42885 4269 14148
FOSL1-GSK3A-PITX2 22118 26574 53921 57069 41599 FBXW11-JUN-PITX2 17945 27019 14279 54975 2329
CCND3-PITX2-WNT3A 14471 39233 2731 800 1147 CSNK2A1-MYC-PITX2 21651 30519 49254 52766 16814
EP300-FZD2-PITX2 21066 1261 6278 52068 50788 CSNK1D-FOXN1-PITX2 11143 1695 49037 55776 7145
FZD1-GSK3A-PITX2 6732 30000 53910 56520 34560 CCND3-PITX2-SFRP4 4452 50957 21986 3532 9719
DVL1-EP300-PITX2 56689 28520 27891 57038 12113 DAAM1-GSK3A-PITX2 1726 56240 42341 56628 12934
DKK1-PITX2-SFRP4 9818 37503 3735 2305 35936 GSK3B-PITX2-SFRP4 5309 55194 34830 18305 53111
FBXW2-JUN-PITX2 42532 23223 17767 54368 980 FRZB-FZD2-PITX2 22096 10208 42171 51487 54482
DIXDC1-PITX2-PPP2CA 5166 50281 13595 5982 18374 DAAM1-LRP6-PITX2 25969 54325 26609 56867 3176
FOSL1-FOXN1-PITX2 10188 3461 55891 51582 2726 CTNNB1-FOXN1-PITX2 7149 4991 55880 53565 3414
PITX2-WNT1-WNT2B 26078 12206 23538 5721 30071 CCND3-PITX2-SENP2 10221 40135 22112 6819 7030
FZD5-PITX2-WNT2B 22980 38933 9787 4035 21144 FZD7-GSK3A-PITX2 3127 14890 47575 57093 5868
AXIN1-EP300-PITX2 56749 23129 25963 57120 24869 FRAT1-GSK3A-PITX2 4495 57021 56892 56898 45139
CSNK1G1-FZD2-PITX2 45412 7430 10137 54602 39686 FOSL1-FZD2-PITX2 12121 784 39252 52428 46106
FBXW11-PITX2-SFRP4 8480 50287 2608 4536 27519 CSNK1G1-PITX2-SENP2 8978 31266 11421 7149 23975
PITX2-PYGO1-TLE2 1782 35079 27236 39307 56772 CTBP2-FOXN1-PITX2 18240 37720 54077 51182 7949
DVL1-FOXN1-PITX2 7131 54107 45985 52139 6298 CTBP1-JUN-PITX2 33380 1220 50964 56252 25801
PITX2-WNT1-WNT4 32926 5300 37141 2973 19943 DIXDC1-PITX2-RHOU 6079 35454 22214 14529 24323
FZD6-PITX2-TCF7 5172 16188 13820 2800 28962 DKK1-GSK3A-PITX2 605 17271 39751 55202 27215
FZD6-PITX2-SENP2 3363 52204 8127 5114 16199 APC-PITX2-SLC9A3R1 6853 5291 25242 18176 16828
FBXW11-PITX2-WNT2B 30130 56623 1327 7485 2572 DIXDC1-PITX2-TCF7 4373 37581 13658 8585 32638
CSNK1D-PITX2-SFRP4 4175 45358 33459 4847 37993 FZD5-CCND3-PITX2 40000 10437 46397 54792 20187
FZD6-PITX2-WNT2 10709 28286 1099 25993 43215 AES-FOXN1-PITX2 3577 24818 43679 53324 2571
CSNK1G1-PITX2-RHOU 11965 12955 21369 13669 45243 FZD7-NKD1-PITX2 25856 8298 48445 57028 4035
FGF4-JUN-PITX2 24971 2275 32263 54277 17002 CCND1-NLK-PITX2 30491 29337 45690 39544 1454
FZD6-PITX2-SFRP1 18690 52116 9763 7836 11500 CXXC4-FGF4-PITX2 3350 14873 39377 55805 47209
AXIN1-FOXN1-PITX2 17522 6988 53149 55878 9496 FOSL1-PITX2-SFRP4 7348 5890 31972 1744 49355
FOSL1-NKD1-PITX2 29467 27288 56470 57104 13679 DKK1-PITX2-WNT2B 17111 13223 40502 378 15000
FZD5-PITX2-RHOU 12500 51905 7871 1962 44656 GSK3B-PITX2-SENP2 915 34496 33084 22723 32507
FZD6-PITX2-TCF7L1 4289 24535 11228 6141 16611 CTBP1-FOXN1-PITX2 8239 9962 54422 50573 10742
FBXW2-GSK3A-PITX2 13991 18085 38156 55688 24970 EP300-GSK3B-PITX2 22813 7384 28301 49131 16578
CTBP1-FGF4-PITX2 24027 10442 12158 56632 50733 FRAT1-FZD2-PITX2 19475 56951 30336 56309 51026
AES-FZD2-PITX2 56089 10520 10321 45652 34757 FBXW11-GSK3A-PITX2 6618 46116 51128 49797 28514
FZD8-JUN-PITX2 40592 40343 33795 57114 7221 CXXC4-FZD2-PITX2 28941 8301 39688 54135 51166
PITX2-SFRP4-TLE2 23954 41848 2647 36476 20017 DIXDC1-FZD2-PITX2 15533 7397 34069 54461 17799
Table 2: Rankings o PITX2-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
one inds he ollowing combina ions o membe o WNT amily along wi h PITX2,
o be p ominen a 3 d o de le el - PITX2-PORCN-WNT4, PITX2-PORCN-WNT5A,
APC-PITX2-WNT4, PITX2-WNT3-WNT5A, PITX2-WNT1-WNT5A, PITX2-SFRP4-
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
APC-PITX2-SFRP4 17003 19660 7752 22464 43680 APC-PITX2-SENP2 16777 21369 5264 21757 43696
APC-PITX2-TCF7 19670 34554 8057 26773 38941 PITX2-PORCN-TLE2 14738 34228 17868 7003 24689
PITX2-PORCN-WNT4 38340 23219 45866 44679 30684 PITX2-PORCN-SENP2 49351 13297 36355 31422 8016
DVL2-JUN-PITX2 23671 1038 12434 27999 54466 FZD5-PITX2-SENP2 521 47250 5679 3657 50275
APC-PITX2-PPP2CA 24251 10201 10127 26604 27627 APC-PITX2-RHOU 39285 21142 46215 32608 9407
PITX2-PORCN-WNT5A 4366 18564 14041 19978 22471 PITX2-PORCN-FBXW4 8808 13705 22864 22147 31748
DIXDC1-PITX2-SENP2 55978 6920 46363 55209 16814 CTNNBIP1-JUN-PITX2 11329 1937 10201 6114 40219
CCND1-FGF4-PITX2 43513 2928 44305 52341 16554 PITX2-PORCN-TCF7L1 7523 29695 12381 16214 18408
DIXDC1-PITX2-SFRP4 46369 10535 43650 52909 13347 PITX2-PORCN-WNT2B 9536 5794 12824 18961 17911
APC-PITX2-WNT4 24477 25329 3199 26367 36930 FRAT1-JUN-PITX2 19630 54721 9834 10848 22067
PITX2-PORCN-TLE1 49615 27631 44758 40897 38885 DKK1-JUN-PITX2 2939 11664 23494 19389 44598
AES-AXIN1-PITX2 47687 38614 55070 30813 9874 PITX2-PORCN-PPP2R1A 10644 13926 20214 8562 45301
PITX2-PORCN-RHOU 7818 43749 20769 25705 49200 CCND1-JUN-PITX2 17592 41812 11511 4995 53682
FOSL1-JUN-PITX2 29544 47341 44123 52018 7644 FZD6-PITX2-WNT2B 42547 47083 32974 29428 5335
FZD5-PITX2-SFRP4 2340 11183 13226 1835 33599 PITX2-PORCN-SFRP4 52035 24948 36616 49778 2115
CCND1-CTBP1-PITX2 47450 37599 38364 47623 15808 FSHB-FZD2-PITX2 21425 53181 12066 7803 49923
PITX2-WNT3-WNT5A 3631 37403 19670 4320 50237 CCND2-LRP6-PITX2 45303 45054 55240 46174 9660
PITX2-PORCN-SFRP1 5118 32289 20516 7388 55055 AES-EP300-PITX2 27429 13782 22774 24073 54041
FZD6-PITX2-SFRP4 2191 34024 23773 8678 52481 PITX2-WNT3-WNT3A 1072 29636 4611 11649 49643
DAAM1-FOXN1-PITX2 23402 15789 15327 5498 42400 CSNK2A1-JUN-PITX2 4226 47140 759 5831 41428
FBXW11-LRP6-PITX2 19674 55920 14183 7236 52984 CCND1-CTNNBIP1-PITX2 23479 13649 20332 26184 43389
CSNK2A1-FOXN1-PITX2 484 10646 14993 25101 56105 FOXN1-GSK3A-PITX2 55917 8017 43971 38630 16813
AXIN1-FZD6-PITX2 45899 20191 43656 43529 4707 FRZB-GSK3A-PITX2 42536 49066 41808 51663 3945
PITX2-WNT1-WNT5A 34 29476 27919 8439 25717 BTRC-GSK3A-PITX2 53128 50908 30242 52659 1281
PITX2-SFRP4-WNT2B 7927 31067 16874 24744 41200 DAAM1-JUN-PITX2 24371 14681 11567 17251 47118
CXXC4-JUN-PITX2 7081 3972 8713 3640 32183 APC-PITX2-WNT3 14234 12446 706 24449 41729
PITX2-PORCN-PPP2CA 48624 10701 36145 29228 15037 DIXDC1-PITX2-WNT2B 3174 55687 15258 13757 29932
FBXW11-FOXN1-PITX2 13141 28980 22463 8005 34303 CSNK1D-FGF4-PITX2 45761 16034 33072 48233 21295
LRP5-PITX2-SFRP4 315 27436 8203 2763 45221 DIXDC1-PITX2-WNT4 44341 8459 33406 35610 21988
FOSL1-GSK3A-PITX2 3336 36071 6692 8964 29002 FBXW11-JUN-PITX2 27953 2910 15908 22 55248
CCND3-PITX2-WNT3A 34394 29164 30661 48295 16862 CSNK2A1-MYC-PITX2 9129 7731 935 12301 36666
EP300-FZD2-PITX2 24012 15242 9973 25729 44122 CSNK1D-FOXN1-PITX2 18384 31000 27911 15993 34137
FZD1-GSK3A-PITX2 49460 16018 43507 46060 9217 CCND3-PITX2-SFRP4 22350 19459 24189 16717 42599
DVL1-EP300-PITX2 548 36053 11918 25848 49131 DAAM1-GSK3A-PITX2 40753 11421 43484 37867 5004
DKK1-PITX2-SFRP4 694 29442 14079 22614 54046 GSK3B-PITX2-SFRP4 14898 11432 9059 22640 47560
FBXW2-JUN-PITX2 12579 4618 17745 364 46971 FRZB-FZD2-PITX2 35229 41345 36872 51138 49950
DIXDC1-PITX2-PPP2CA 53041 37364 46121 33605 10550 DAAM1-LRP6-PITX2 16446 14829 15814 16673 38836
FOSL1-FOXN1-PITX2 54512 5376 32845 52595 9349 CTNNB1-FOXN1-PITX2 29063 19206 49013 56402 27258
PITX2-WNT1-WNT2B 3768 50433 24960 15608 43963 CCND3-PITX2-SENP2 22323 19475 21774 24829 24809
FZD5-PITX2-WNT2B 45840 6879 31831 55937 11210 FZD7-GSK3A-PITX2 8237 8741 24973 13187 10820
AXIN1-EP300-PITX2 1837 53367 4684 5496 23397 FRAT1-GSK3A-PITX2 44421 52054 29767 47337 1646
CSNK1G1-FZD2-PITX2 4443 9218 19882 21920 41937 FOSL1-FZD2-PITX2 18628 35229 27305 27474 41987
FBXW11-PITX2-SFRP4 22007 13276 18287 6972 23461 CSNK1G1-PITX2-SENP2 42929 46890 54375 42726 15165
PITX2-PYGO1-TLE2 19850 10379 18180 6093 40940 CTBP2-FOXN1-PITX2 12690 32085 14231 16779 36765
DVL1-FOXN1-PITX2 13716 11451 12180 2594 46581 CTBP1-JUN-PITX2 24353 53693 13796 3746 36415
PITX2-WNT1-WNT4 55244 8137 33549 51863 7332 DIXDC1-PITX2-RHOU 1183 50113 10813 1973 40351
FZD6-PITX2-TCF7 58 54592 26110 20224 55626 DKK1-GSK3A-PITX2 51992 863 44516 56355 32287
FZD6-PITX2-SENP2 432 817 17937 22896 56866 APC-PITX2-SLC9A3R1 17574 24386 9813 27607 44761
FBXW11-PITX2-WNT2B 29169 24449 32207 29680 32687 DIXDC1-PITX2-TCF7 53422 53152 36175 42927 22338
CSNK1D-PITX2-SFRP4 26609 44292 24260 17256 33381 FZD5-CCND3-PITX2 19393 32161 13355 11683 5330
FZD6-PITX2-WNT2 14637 10126 24195 27724 51796 AES-FOXN1-PITX2 17914 21798 703 10325 20162
CSNK1G1-PITX2-RHOU 14243 10287 2792 14397 41970 FZD7-NKD1-PITX2 1376 4501 11215 16178 40845
FGF4-JUN-PITX2 18457 36152 1440 2969 44768 CCND1-NLK-PITX2 48102 11112 41884 52866 812
FZD6-PITX2-SFRP1 56728 56346 39190 34205 286 CXXC4-FGF4-PITX2 38294 56427 50581 38505 7533
AXIN1-FOXN1-PITX2 4350 36135 20767 6550 49005 FOSL1-PITX2-SFRP4 48140 54056 50405 43960 31198
FOSL1-NKD1-PITX2 9221 29087 12816 22289 29068 DKK1-PITX2-WNT2B 54626 45844 39615 30595 28144
FZD5-PITX2-RHOU 55488 4203 52989 44916 13289 GSK3B-PITX2-SENP2 16336 29784 11616 16048 44166
FZD6-PITX2-TCF7L1 57098 2574 31025 36904 1546 CTBP1-FOXN1-PITX2 8046 12489 6826 22448 30443
FBXW2-GSK3A-PITX2 36714 12730 37356 49810 4808 EP300-GSK3B-PITX2 26038 11762 5959 1368 52250
CTBP1-FGF4-PITX2 41855 4408 56680 38243 15293 FRAT1-FZD2-PITX2 55832 24730 44050 40886 5856
AES-FZD2-PITX2 29806 34809 44098 30337 5206 FBXW11-GSK3A-PITX2 35583 52800 48903 44758 21694
FZD8-JUN-PITX2 14227 28361 22535 6380 50233 CXXC4-FZD2-PITX2 49278 49869 33082 55290 10056
PITX2-SFRP4-TLE2 16042 46434 18733 25251 47254 DIXDC1-FZD2-PITX2 7897 43672 18332 7315 28673
Table 3: Rankings o PITX2-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
WNT2B, CCND3-PITX2-WNT3A, PITX2-WNT1-WNT2B, FZD5-PITX2-WNT2B,
PITX2-WNT1-WNT4, FBXW11-PITX2-WNT2B, FZD6-PITX2-WNT2, PITX2-PORCN-
WNT2B, FZD6-PITX2-WNT2B, PITX2-WNT3-WNT3A, APC-PITX2-WNT3, DIXDC1-
9
