-jun a ian sa coma i us 17 oncogene homolog / Jun
p o o-oncogene, AP-1 ansc ip ion ac o subuni (JUN) :
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
JUN encode he ansc ip ion ac o Jun. The s uc u e o ac i a o p o ein (AP1) is a
he e odime composed o p o eins belonging o he c-FOS, c-JUN, ATF and JDP ami-
lies. Bo h JUN and i s he e odime iza ion pa ne s in AP1 o ma ion, a e subjec o eg-
ula ion by di e se ex acellula s imuli, like pep ide g ow h ac o s, p o-in lamma o y
cy okines, oxida i e and o he o ms o cellula s ess, and UV i adia 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 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
JUN 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 JUN-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
ITime beha iou al s udy o 3-od JUN 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1Aspec s o unpublished wo k we e p esen ed in a pos e session a Cell Symposia: Technology.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 9, 2025
Independence C i e ion indices (HSIC) and Sobol indicies, WNT3A
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)
combina ions, i becomes nea ly impossible o any biologis o s udy he sys em wide
dynamics 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 JUN ela ed combina ions using indi idual gene exp essions mea-
su 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
2.2. 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,
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. -jun a ian sa coma i us 17 oncogene homolog / Jun p o o-
oncogene, AP-1 ansc ip ion ac o subuni (JUN)
Maki e al. [4] show ha biologically ac i e molecula clones o a ian sa coma i us
17 (ASV 17) con ain a eplica ion-de ec i e p o i al genome o 3.5 kilobases (kb), ha
e ains pa ial gag and en sequences, which lank a cell-de i ed pu a i e oncogene o
0.93 kb, e med jun. jun lacks p ese ed coding domains o y osine-speci ic p o ein ki-
nases and he p obable ans o ma ion-speci ic p o ein in ASV 17- ans o med cells is
a 55-kDa gag-jun usion p oduc . Angel e al. [5] show ha binding o he human an-
sc ip ion ac o JUN/AP1 o a conse ed 8 bp nucleo ide sequence (TRE) is esponsible
o inc eased ansc ip ion o di e en cellula genes in esponse o umo p omo e s,
such as TPA, and se um ac o s. Enhanced JUN/AP1 ac i i y in TPA-s imula ed cells
is egula ed by wo di e en mechanisms: a pos ansla ional e en ac ing on p eexis -
ing JUN/AP1 molecules, and ansc ip ional ac i a ion o JUN gene exp ession leading
o an inc ease in he o al amoun o JUN/AP1. Thei esul s demons a ed ha JUN
ansc ip ion was di ec ly s imula ed by i s own gene p oduc .
c-JUN was o iginally isola ed as he cellula homolog o -JUN, as s a ed in Wis-
dom e al. [6]. They s a e ha c-JUN is a componen o AP1, which is ac i a ed by a
wide a ie y o ex acellula s imuli. The egula ion o c-JUN is complex and in ol es
bo h inc eases in he le els o c-JUN p o ein as well as phospho yla ion o speci ic se -
ines (63 and 73) by JUN N- e minal kinase (JNK). They used ib oblas s de i ed om
c-JUN null emb yos o de ine he ole o c-JUN in wo sepa a e p ocesses, namely, cell
g ow h and apop osis.
Hess e al. [7] s a e ha The AP1 ansc ip ion ac o is mainly composed o JUN,
FOS and ATF p o ein dime s. I media es gene egula ion in esponse o a many phys-
iological and pa hological s imuli, including cy okines, g ow h ac o s, s ess signals,
bac e ial and i al in ec ions, as well as oncogenic s imuli.
I p esen 3 d o de combina ions o JUN wi h o he genes, ha he machine lea n-
ing based sea ch engine poin s o, as possible syne gis ic combina ions ha migh be
wo king in ime.
3
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 [8]. 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 [8].
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
4
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
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
5
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 JUN-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 [9]) and Sobol indicies (wi h 2002 implemen a ion in Sal elli [10]
and ma inez implemen a ion in Ma inez [11] and Baudin e al. [12]).
6.3. Conse ed machine lea ning ankings o es ed JUN-X-X com-
bina ions
A o al o 2415, 3 d o de combina ions in ol ing JUN 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 k-
ing 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 FOSL1-JUN-X combina ions
The FOS and JUN amilies o euka yo ic ansc ip ion ac o s he e odime ize o o m
complexes capable o binding 5’-TGAGTCA-3’ DNA elemen s. Glo e and Ha ison
[13] de e mined he X- ay c ys al s uc u e o a he e odime o he bZIP egions o
c-FOS and c-JUN bound o DNA. Bo h subuni s o med con inuous α-helices. The
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
DKK1-JUN-SENP2 3 13146 45291 34681 42815 FZD5-JUN-TLE2 33 2696 2557 55511 23342
DVL2-JUN-SENP2 40 7571 46728 17699 4151 DKK1-JUN-LRP5 48 627 56508 43115 37922
DVL2-JUN-TCF7L1 50 34646 43296 24980 5047 DKK1-JUN-SFRP4 53 1275 44566 37789 42099
DKK1-JUN-TLE2 55 1113 26078 37591 53405 DKK1-JUN-WNT2B 56 5221 53944 55625 44612
DKK1-JUN-WNT2 58 1936 43652 35916 55311 DKK1-JUN-TCF7L1 67 33647 49514 43728 45939
FZD5-JUN-WNT2 71 4401 38543 47328 33190 FZD5-JUN-WNT4 79 8704 42294 52216 15657
DVL2-JUN-TLE2 87 42627 5285 18305 31287 FZD5-JUN-KREMEN1 96 24278 50801 44288 10781
FZD5-JUN-FBXW4 97 13039 25922 48154 41051 FZD5-JUN-RHOU 105 12942 56571 25707 3718
FRZB-JUN-SENP2 109 12704 33983 8160 2937 CTNNBIP1-JUN-RHOU 119 11333 54553 37428 52492
DKK1-JUN-FBXW4 129 11896 24951 31819 36572 DVL2-JUN-FBXW4 135 26880 12581 12861 8554
DVL2-JUN-KREMEN1 178 24966 37758 33223 9256 DVL2-JUN-WNT4 203 51324 39998 14159 3459
CTNNBIP1-JUN-WNT2 206 3834 45427 33327 6238 DKK1-JUN-RHOU 210 3670 42736 27568 47765
DKK1-JUN-PYGO1 218 3236 51191 53120 2659 CTNNBIP1-JUN-PPP2R1A 222 17930 41664 52992 44848
CTNNBIP1-JUN-KREMEN1 241 6571 55388 29592 14471 DVL2-JUN-WNT2 252 43710 32464 4614 34311
DKK1-JUN-PPP2R1A 267 12255 56230 48454 54215 CTNNBIP1-JUN-SFRP4 282 1303 44436 32290 6337
FZD5-JUN-WNT5A 284 6962 43259 53869 10481 DKK1-JUN-WNT3A 289 6120 44241 52638 12402
DVL2-JUN-PITX2 293 27343 32163 42607 21387 DVL2-JUN-TLE1 306 36089 30012 17193 22195
DKK1-JUN-WNT4 316 926 46570 28834 28365 DVL2-JUN-WNT3A 322 31939 32028 29113 4760
DKK1-JUN-TCF7 337 19121 54724 39273 38745 FZD5-JUN-PYGO1 348 1924 32815 54426 36260
DVL2-JUN-WNT2B 350 44599 38931 37935 16078 FZD5-JUN-TCF7 367 15943 33055 41015 49415
DKK1-JUN-PPP2CA 408 27394 50340 28816 37806 DKK1-JUN-TLE1 417 16467 28915 26740 32148
EP300-JUN-KREMEN1 445 18545 22047 46724 18178 FZD5-JUN-TLE1 448 23448 36104 54093 45715
CTNNBIP1-JUN-SENP2 451 45193 55478 33408 6827 FOSL1-JUN-SENP2 458 25557 50663 10786 1378
FRZB-JUN-WNT2 478 2085 21294 5583 18722 DVL2-JUN-PPP2CA 504 43997 37157 2445 3613
DVL2-JUN-TCF7 544 30871 41999 26197 8304 CTNNBIP1-JUN-PITX2 546 6389 45937 54346 11119
DVL2-JUN-PPP2R1A 555 45651 45029 17035 30388 JUN-PYGO1-WNT3A 570 14118 6423 19469 36536
DKK1-JUN-LRP6 617 2273 42860 25885 1791 FZD5-JUN-SLC9A3R1 625 5957 34667 52133 12374
CTNNBIP1-JUN-TCF7 674 24941 33229 25262 14247 FRAT1-JUN-WNT2 676 35197 23478 4515 23224
DVL1-JUN-WNT2B 696 13741 48925 47783 49704 CTNNBIP1-JUN-LRP5 697 1403 32050 45845 13691
FRAT1-JUN-PITX2 748 50033 38113 50412 44660 CXXC4-JUN-TLE2 764 1040 837 11709 30876
DVL2-JUN-WIF1 788 37918 18319 11932 34672 FBXW11-JUN-WNT2B 795 49304 52661 53929 54016
FBXW2-JUN-WNT4 806 26430 56620 37987 55952 FZD8-JUN-WNT2B 814 45692 37162 26955 54126
CSNK1D-JUN-LEF1 823 6859 47543 11454 29458 CXXC4-JUN-SENP2 843 21048 50347 6595 9660
CSNK1G1-JUN-SENP2 855 23957 55112 11084 3256 DVL2-JUN-WNT5A 871 37929 52159 30056 48277
FRAT1-JUN-SENP2 876 27146 34459 14780 13983 CXXC4-JUN-RHOU 881 1789 43156 41625 53423
FBXW11-JUN-LRP5 885 23634 45785 27639 45330 FZD1-JUN-WNT2 886 20644 39928 4484 8707
BCL9-JUN-TCF7L1 889 18626 55169 44931 2804 DKK1-JUN-PITX2 913 6407 42538 52798 634
FRZB-JUN-TCF7L1 932 26973 39828 31513 3210 DKK1-JUN-SFRP1 961 1935 26970 33204 47981
FBXW11-JUN-SFRP4 964 21656 28145 38980 53582 CXXC4-JUN-LRP5 971 1353 33173 6860 26690
FOSL1-JUN-TCF7 996 16387 31797 50574 23742 FOSL1-JUN-TCF7L1 1000 17132 38816 29827 1621
FZD1-JUN-TLE2 1075 7630 4614 20083 41713 FBXW11-JUN-SENP2 1078 21114 46139 41233 39344
CSNK1G1-JUN-WNT2 1087 1305 52762 5894 31347 CTNNBIP1-JUN-WNT3A 1110 7698 46119 54862 31125
CSNK1G1-JUN-TCF7L1 1134 10933 47748 13909 25134 CXXC4-JUN-FBXW4 1138 13192 17971 6537 17461
DVL2-JUN-SFRP1 1150 43616 21933 13995 37221 DAAM1-JUN-WNT2B 1152 51606 37010 32012 42370
CTNNBIP1-JUN-PPP2CA 1160 17287 25595 27276 6859 FRAT1-JUN-SFRP4 1177 38200 26202 16198 34929
CCND1-JUN-SFRP4 1212 40676 36610 54521 54939 CTNNBIP1-JUN-SLC9A3R1 1227 27649 37384 31940 7577
FZD7-JUN-WNT3A 1250 17078 32637 21100 43381 FBXW2-JUN-SENP2 1253 32758 51631 42897 55749
CCND1-JUN-PITX2 1260 51205 42011 18763 52917 GSK3B-JUN-TLE1 1271 40084 30251 26212 40565
FBXW11-JUN-TLE2 1285 29879 11548 36884 55887 CXXC4-JUN-SFRP4 1308 4014 25558 7639 24982
CTNNBIP1-JUN-FBXW4 1320 15975 24670 37193 14093 DIXDC1-JUN-FBXW4 1340 27624 17009 49472 50306
JUN-PYGO1-TLE2 1341 24994 40582 4657 37476 BCL9-JUN-TLE2 1353 1767 14711 51640 5284
CCND3-JUN-PYGO1 1362 50934 48125 55357 25009 CSNK1G1-JUN-TLE2 1365 2675 25875 17296 52928
CXXC4-JUN-TCF7 1379 21025 34076 34379 37036 FRZB-JUN-PPP2CA 1383 13534 31714 933 1610
DIXDC1-JUN-TCF7L1 1397 18158 39442 41838 15514 DKK1-JUN-LEF1 1407 345 39847 38268 15426
FRZB-JUN-SLC9A3R1 1409 6059 10710 25355 42497 FOSL1-JUN-PITX2 1479 1306 37107 54515 12375
FZD1-JUN-SFRP4 1493 24355 34044 13135 44616 CXXC4-JUN-PYGO1 1496 10929 38139 43065 17470
CTNNBIP1-JUN-LRP6 1499 4675 42275 26834 8608 CCND1-JUN-PPP2CA 1525 10554 35624 43398 54711
FBXW11-JUN-TCF7L1 1526 26632 46567 48986 51386 FOSL1-JUN-WNT3A 1542 4890 20754 29478 5342
CXXC4-JUN-TCF7L1 1561 16594 47993 27451 26786 CSNK1G1-JUN-KREMEN1 1571 14622 52390 27682 7035
CTBP2-JUN-TLE2 1591 46301 4452 35914 13872 CSNK1A1-JUN-TCF7L1 1599 2840 51813 45452 5619
CTNNBIP1-JUN-WNT4 1605 6379 54822 28948 10594 FRAT1-JUN-TCF7L1 1626 39276 40450 30425 12391
DVL1-JUN-SENP2 1636 27735 49539 26023 46168 FZD1-JUN-WNT2B 1648 10746 47422 41725 9336
BCL9-JUN-RHOU 1653 6622 56085 44704 6331 DAAM1-JUN-SENP2 1658 50069 38838 15921 52559
Table 1: Rankings o JUN-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
ca boxy- e minal egions o m an asymme ic coiled-coil, and he amino- e minal e-
gions make base-speci ic con ac s wi h DNA in he majo g oo e. Compa ison o he
wo c ys allog aphically dis inc p o ein-DNA complexes showed ha he coiled-coil
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
DKK1-JUN-SENP2 2445 33296 14766 10519 28087 FZD5-JUN-TLE2 18001 2899 23629 42402 48124
DVL2-JUN-SENP2 4462 28972 39208 37581 33219 DKK1-JUN-LRP5 44363 59 7957 31836 12454
DVL2-JUN-TCF7L1 6692 37689 42883 22632 2352 DKK1-JUN-SFRP4 23984 4600 8391 10229 3109
DKK1-JUN-TLE2 10461 10318 33449 28447 40483 DKK1-JUN-WNT2B 23590 25652 38169 27335 22936
DKK1-JUN-WNT2 15958 5371 10742 16568 29068 DKK1-JUN-TCF7L1 11024 26336 26266 38929 770
FZD5-JUN-WNT2 45453 4822 16137 45403 40375 FZD5-JUN-WNT4 28544 6905 45357 25315 15393
DVL2-JUN-TLE2 6307 41319 43004 40157 41574 FZD5-JUN-KREMEN1 14265 7847 49848 50707 31671
FZD5-JUN-FBXW4 32588 11252 24483 51441 24264 FZD5-JUN-RHOU 21830 6512 2365 50002 36411
FRZB-JUN-SENP2 1481 18469 38799 27435 28090 CTNNBIP1-JUN-RHOU 47402 8172 34623 51918 36687
DKK1-JUN-FBXW4 36904 13586 2374 17612 12759 DVL2-JUN-FBXW4 26608 26616 1444 12875 15856
DVL2-JUN-KREMEN1 13103 30709 32743 48017 27397 DVL2-JUN-WNT4 21779 49475 22727 20132 5205
CTNNBIP1-JUN-WNT2 42601 8719 44457 18821 49158 DKK1-JUN-RHOU 4232 243 27543 37520 46403
DKK1-JUN-PYGO1 35611 92 8244 34065 42310 CTNNBIP1-JUN-PPP2R1A 42662 30325 24952 5762 37211
CTNNBIP1-JUN-KREMEN1 49360 1571 43983 55548 30690 DVL2-JUN-WNT2 26710 49937 37500 33239 22813
DKK1-JUN-PPP2R1A 19726 15606 1085 29841 36603 CTNNBIP1-JUN-SFRP4 50889 5964 52857 23302 32122
FZD5-JUN-WNT5A 8780 13834 37545 56636 50500 DKK1-JUN-WNT3A 10634 402 10397 46299 3270
DVL2-JUN-PITX2 22623 16521 37051 52719 15020 DVL2-JUN-TLE1 36629 36442 30000 29148 8644
DKK1-JUN-WNT4 8806 34 14859 3085 9371 DVL2-JUN-WNT3A 25797 23440 31750 21568 1485
DKK1-JUN-TCF7 4445 24804 23041 23852 12528 FZD5-JUN-PYGO1 13399 522 43506 36219 51123
DVL2-JUN-WNT2B 35904 41234 30718 47010 18770 FZD5-JUN-TCF7 13732 2579 35094 27662 15659
DKK1-JUN-PPP2CA 872 31181 33266 21737 30431 DKK1-JUN-TLE1 12635 18611 25341 28339 18891
EP300-JUN-KREMEN1 4639 142 27356 54948 19911 FZD5-JUN-TLE1 12747 18718 33701 39270 7010
CTNNBIP1-JUN-SENP2 42259 50095 40291 21021 34386 FOSL1-JUN-SENP2 15271 29847 41615 22047 48378
FRZB-JUN-WNT2 21990 24790 40993 7840 23831 DVL2-JUN-PPP2CA 12969 55374 21200 14940 32734
DVL2-JUN-TCF7 4906 31956 33672 6793 3595 CTNNBIP1-JUN-PITX2 55688 6273 51543 54725 26305
DVL2-JUN-PPP2R1A 7790 48428 6855 9490 40633 JUN-PYGO1-WNT3A 10181 579 35737 33610 43746
DKK1-JUN-LRP6 26624 5079 13887 46906 27696 FZD5-JUN-SLC9A3R1 39777 3230 20742 34190 14401
CTNNBIP1-JUN-TCF7 41380 25473 25735 11678 41440 FRAT1-JUN-WNT2 21621 33205 53792 38197 10593
DVL1-JUN-WNT2B 44382 6721 23326 56398 1235 CTNNBIP1-JUN-LRP5 51735 260 35013 50048 34138
FRAT1-JUN-PITX2 28349 55075 50998 56838 16856 CXXC4-JUN-TLE2 2337 10507 41107 15818 46481
DVL2-JUN-WIF1 6060 40504 15256 30934 48673 FBXW11-JUN-WNT2B 30736 52338 1286 29236 8641
FBXW2-JUN-WNT4 49067 9843 17416 10298 1111 FZD8-JUN-WNT2B 30397 45061 16498 26458 9398
CSNK1D-JUN-LEF1 20901 8723 38740 12713 27687 CXXC4-JUN-SENP2 1851 32478 55139 8858 43014
CSNK1G1-JUN-SENP2 2057 43819 27038 14070 17638 DVL2-JUN-WNT5A 16054 35608 35930 17120 44512
FRAT1-JUN-SENP2 8350 32873 42151 5535 36295 CXXC4-JUN-RHOU 7270 2533 37155 46380 45935
FBXW11-JUN-LRP5 46395 5177 29373 22491 4533 FZD1-JUN-WNT2 52622 26168 48248 5592 27125
BCL9-JUN-TCF7L1 24868 22870 36874 19513 1734 DKK1-JUN-PITX2 24208 3989 4050 56945 15274
FRZB-JUN-TCF7L1 9523 51903 25105 20905 20981 DKK1-JUN-SFRP1 15339 875 11803 12204 51585
FBXW11-JUN-SFRP4 17788 9290 3063 40195 1563 CXXC4-JUN-LRP5 15539 1611 30933 39991 28813
FOSL1-JUN-TCF7 18538 5069 12539 42545 29271 FOSL1-JUN-TCF7L1 5134 9823 24616 52464 27691
FZD1-JUN-TLE2 24688 27343 11195 3015 35208 FBXW11-JUN-SENP2 2521 15541 36470 25945 625
CSNK1G1-JUN-WNT2 10147 5580 32401 33214 37290 CTNNBIP1-JUN-WNT3A 24780 5944 45858 50803 13854
CSNK1G1-JUN-TCF7L1 827 11310 28918 27910 3674 CXXC4-JUN-FBXW4 14457 30498 1036 36322 33651
DVL2-JUN-SFRP1 10995 46091 46482 41358 52216 DAAM1-JUN-WNT2B 34628 49211 11090 56716 4721
CTNNBIP1-JUN-PPP2CA 38196 36635 20141 33944 38991 FRAT1-JUN-SFRP4 35091 47221 26189 44148 8318
CCND1-JUN-SFRP4 38237 25562 52223 10931 1352 CTNNBIP1-JUN-SLC9A3R1 45992 37553 52601 42751 21986
FZD7-JUN-WNT3A 6578 3907 45657 38691 15081 FBXW2-JUN-SENP2 25289 51870 3972 20307 1098
CCND1-JUN-PITX2 44312 43519 25962 49672 2955 GSK3B-JUN-TLE1 21678 43855 47673 19227 7501
FBXW11-JUN-TLE2 12624 12939 27323 12238 19853 CXXC4-JUN-SFRP4 8489 12877 51306 12233 26552
CTNNBIP1-JUN-FBXW4 38357 26382 25017 7941 22281 DIXDC1-JUN-FBXW4 53699 39669 27414 16988 3729
JUN-PYGO1-TLE2 2091 9953 10078 26860 57045 BCL9-JUN-TLE2 25015 9243 46821 14241 21549
CCND3-JUN-PYGO1 15052 38950 15637 52389 3265 CSNK1G1-JUN-TLE2 3633 15390 39684 2796 27092
CXXC4-JUN-TCF7 2896 16477 22826 15141 21339 FRZB-JUN-PPP2CA 5873 49297 3845 20493 26715
DIXDC1-JUN-TCF7L1 36423 32144 31736 45585 462 DKK1-JUN-LEF1 15292 323 25256 25883 39611
FRZB-JUN-SLC9A3R1 34630 15041 47309 7650 16482 FOSL1-JUN-PITX2 42099 14135 52254 57017 24935
FZD1-JUN-SFRP4 44475 27425 5889 28115 8946 CXXC4-JUN-PYGO1 15268 3842 31367 43633 53074
CTNNBIP1-JUN-LRP6 41800 8865 4751 4915 48545 CCND1-JUN-PPP2CA 40595 14062 3971 9088 1571
FBXW11-JUN-TCF7L1 5238 17905 26014 9636 4500 FOSL1-JUN-WNT3A 37744 27522 48171 53533 24924
CXXC4-JUN-TCF7L1 5069 16399 50288 29076 25853 CSNK1G1-JUN-KREMEN1 6491 2240 25073 41585 18357
CTBP2-JUN-TLE2 2322 40968 49108 5379 22516 CSNK1A1-JUN-TCF7L1 22985 1629 17522 50991 17011
CTNNBIP1-JUN-WNT4 39260 388 36889 17634 53412 FRAT1-JUN-TCF7L1 25869 45197 28755 12226 10788
DVL1-JUN-SENP2 17916 15346 45039 2157 2784 FZD1-JUN-WNT2B 49546 6083 15156 54576 28886
BCL9-JUN-RHOU 27668 3483 7713 42147 31193 DAAM1-JUN-SENP2 28479 51658 5776 9253 6064
Table 2: Rankings o JUN-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
is lexibly joined o he basic egions and ha he FOS-JUN he e odime did no ecog-
nize he asymme ic 5’-TGAGTCA-3’ ecogni ion elemen in a unique o ien a ion.
He e odime iza ion among he basic-leucine zippe (bZIP) p o eins o among he
basic-helixloophelix-leucine zippe (bHLHZip) p o eins con e s a mul i ude o combi-
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
DKK1-JUN-SENP2 754 9372 11904 1076 33385 FZD5-JUN-TLE2 41573 48046 35361 54860 7334
DVL2-JUN-SENP2 13471 27115 21910 19735 56433 DKK1-JUN-LRP5 48850 11594 39630 41378 23052
DVL2-JUN-TCF7L1 31682 50919 38373 29294 10924 DKK1-JUN-SFRP4 2159 46694 9618 10954 53360
DKK1-JUN-TLE2 51285 19906 29994 46512 5324 DKK1-JUN-WNT2B 55363 40120 46822 38204 3471
DKK1-JUN-WNT2 1793 17173 10372 18936 53659 DKK1-JUN-TCF7L1 54195 13967 44741 41708 6796
FZD5-JUN-WNT2 5734 51317 12612 516 52072 FZD5-JUN-WNT4 10431 52363 25511 23980 54325
DVL2-JUN-TLE2 37775 50662 42118 36079 5151 FZD5-JUN-KREMEN1 40950 45002 28772 48247 9854
FZD5-JUN-FBXW4 38132 1495 38878 55550 15245 FZD5-JUN-RHOU 34450 36279 38790 52137 4153
FRZB-JUN-SENP2 12004 43333 28019 17227 24299 CTNNBIP1-JUN-RHOU 42622 37205 47514 55158 1108
DKK1-JUN-FBXW4 55004 10538 47561 46222 3807 DVL2-JUN-FBXW4 37995 50015 47979 42759 40551
DVL2-JUN-KREMEN1 30808 34898 33501 52840 6443 DVL2-JUN-WNT4 16345 10299 13394 21121 55056
CTNNBIP1-JUN-WNT2 11911 1262 6891 21217 41714 DKK1-JUN-RHOU 36718 6757 39502 48964 14725
DKK1-JUN-PYGO1 20453 50366 17707 8188 42484 CTNNBIP1-JUN-PPP2R1A 55954 42703 38291 39039 13787
CTNNBIP1-JUN-KREMEN1 42377 46499 47863 48423 9908 DVL2-JUN-WNT2 16689 9003 10414 13155 52714
DKK1-JUN-PPP2R1A 57057 38094 40889 35645 26618 CTNNBIP1-JUN-SFRP4 16570 24584 750 1029 26322
FZD5-JUN-WNT5A 46652 4870 31639 33192 2907 DKK1-JUN-WNT3A 51531 12300 46409 53700 3796
DVL2-JUN-PITX2 23671 1038 12434 27999 54466 DVL2-JUN-TLE1 19381 6455 15016 21061 52059
DKK1-JUN-WNT4 2160 6260 8008 12977 38088 DVL2-JUN-WNT3A 35727 49416 40820 44967 1537
DKK1-JUN-TCF7 2980 42733 12423 15481 50307 FZD5-JUN-PYGO1 22701 20169 18383 5021 52969
DVL2-JUN-WNT2B 40463 48141 46668 43995 4463 FZD5-JUN-TCF7 21221 30391 25873 1692 44909
DKK1-JUN-PPP2CA 98 19110 16349 21529 30627 DKK1-JUN-TLE1 5879 37800 27150 10679 51804
EP300-JUN-KREMEN1 11605 22398 6349 16123 55238 FZD5-JUN-TLE1 15555 9196 21799 2297 49839
CTNNBIP1-JUN-SENP2 15007 40205 17451 2535 40407 FOSL1-JUN-SENP2 51877 55400 52885 34018 3734
FRZB-JUN-WNT2 18619 2162 17358 26907 22142 DVL2-JUN-PPP2CA 24259 24689 15208 7592 48248
DVL2-JUN-TCF7 25445 6256 18825 27882 46274 CTNNBIP1-JUN-PITX2 11329 1937 10201 6114 40219
DVL2-JUN-PPP2R1A 32860 32858 41918 49574 8979 JUN-PYGO1-WNT3A 1610 17601 24757 13846 37085
DKK1-JUN-LRP6 889 49598 15333 19395 28530 FZD5-JUN-SLC9A3R1 19660 8438 20540 9033 50969
CTNNBIP1-JUN-TCF7 6098 7522 16157 8323 52544 FRAT1-JUN-WNT2 8549 16613 14863 6104 42450
DVL1-JUN-WNT2B 55129 18060 30213 42553 11665 CTNNBIP1-JUN-LRP5 52013 50795 51766 42741 16689
FRAT1-JUN-PITX2 19630 54721 9834 10848 22067 CXXC4-JUN-TLE2 47401 2576 46879 53365 10616
DVL2-JUN-WIF1 15451 4813 8548 10559 52944 FBXW11-JUN-WNT2B 29211 26070 34684 38249 15818
FBXW2-JUN-WNT4 6945 6699 21970 13741 36379 FZD8-JUN-WNT2B 53721 5288 39583 33282 9288
CSNK1D-JUN-LEF1 25658 26247 28008 9195 29514 CXXC4-JUN-SENP2 8825 8401 12932 7871 27023
CSNK1G1-JUN-SENP2 49955 51075 54767 43231 2180 DVL2-JUN-WNT5A 40789 46792 43770 36077 2170
FRAT1-JUN-SENP2 10491 7985 11945 814 56174 CXXC4-JUN-RHOU 43332 26892 49071 46614 1986
FBXW11-JUN-LRP5 29144 55408 44273 48284 18254 FZD1-JUN-WNT2 22633 26709 17508 18911 53158
BCL9-JUN-TCF7L1 10128 54591 19355 15272 44855 DKK1-JUN-PITX2 2939 11664 23494 19389 44598
FRZB-JUN-TCF7L1 43224 53526 30364 36886 9096 DKK1-JUN-SFRP1 56402 47506 45269 56075 23790
FBXW11-JUN-SFRP4 26958 10929 24740 8133 48845 CXXC4-JUN-LRP5 33040 7608 43755 38567 13643
FOSL1-JUN-TCF7 49067 35754 42264 40921 9123 FOSL1-JUN-TCF7L1 4213 41297 24152 15364 47738
FZD1-JUN-TLE2 44268 55094 42586 33377 6664 FBXW11-JUN-SENP2 27619 7495 18431 13804 56624
CSNK1G1-JUN-WNT2 43215 54139 50622 48011 1250 CTNNBIP1-JUN-WNT3A 47078 14611 51686 48296 5008
CSNK1G1-JUN-TCF7L1 6017 14337 20145 21380 48232 CXXC4-JUN-FBXW4 49484 2831 37278 49282 3845
DVL2-JUN-SFRP1 43635 30176 35239 37444 736 DAAM1-JUN-WNT2B 52248 43893 48230 50763 39740
CTNNBIP1-JUN-PPP2CA 1200 14488 18797 18145 43428 FRAT1-JUN-SFRP4 21027 9576 20703 24611 44741
CCND1-JUN-SFRP4 20932 24429 19033 3104 41522 CTNNBIP1-JUN-SLC9A3R1 22481 35127 9927 24826 33487
FZD7-JUN-WNT3A 1386 53880 25449 20632 45908 FBXW2-JUN-SENP2 10475 4057 14601 2973 45279
CCND1-JUN-PITX2 17592 41812 11511 4995 53682 GSK3B-JUN-TLE1 8247 452 12235 2187 37512
FBXW11-JUN-TLE2 28842 50663 44769 54898 3936 CXXC4-JUN-SFRP4 7686 54368 19771 7870 53296
CTNNBIP1-JUN-FBXW4 40631 32924 56400 56117 31184 DIXDC1-JUN-FBXW4 23517 23502 15073 18409 44877
JUN-PYGO1-TLE2 3800 11209 18229 255 32111 BCL9-JUN-TLE2 3148 9305 24717 4483 54658
CCND3-JUN-PYGO1 25910 36673 28134 15173 40843 CSNK1G1-JUN-TLE2 6833 1066 4928 11587 56004
CXXC4-JUN-TCF7 1115 20606 6634 9559 44618 FRZB-JUN-PPP2CA 7300 14667 7614 23131 10832
DIXDC1-JUN-TCF7L1 9143 45568 15142 2009 35636 DKK1-JUN-LEF1 8362 45451 17549 15763 34051
FRZB-JUN-SLC9A3R1 21722 36003 21129 27499 26935 FOSL1-JUN-PITX2 29544 47341 44123 52018 7644
FZD1-JUN-SFRP4 9479 1152 20120 8827 46342 CXXC4-JUN-PYGO1 13879 27252 8058 10543 55180
CTNNBIP1-JUN-LRP6 16607 13114 2882 13685 40184 CCND1-JUN-PPP2CA 23078 52819 9647 2403 50956
FBXW11-JUN-TCF7L1 29061 55371 38660 56451 2416 FOSL1-JUN-WNT3A 10579 5753 10670 19639 21636
CXXC4-JUN-TCF7L1 56040 37157 50497 47616 12624 CSNK1G1-JUN-KREMEN1 6923 4884 26325 13483 55790
CTBP2-JUN-TLE2 45238 40887 44003 32561 49556 CSNK1A1-JUN-TCF7L1 48736 28417 29151 42720 4277
CTNNBIP1-JUN-WNT4 18138 13025 5771 2388 39991 FRAT1-JUN-TCF7L1 43935 48115 50408 53433 8865
DVL1-JUN-SENP2 1057 37410 27437 23571 26894 FZD1-JUN-WNT2B 34517 30754 39634 38168 4014
BCL9-JUN-RHOU 13091 32869 21205 19079 54663 DAAM1-JUN-SENP2 17437 48030 18701 23987 45424
Table 3: Rankings o JUN-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
na ional ac i i ies o hese ansc ip ion ac o s. To u he examine he unc ion o he
bHLHZip p o ein, USF, Pognonec e al. [14] ound cellula bZip p o ein, FRA1/FOSL1
o di ec ly in e ac wi h USF using he yeas wo-hyb id sys em. Exp ession o ex-
ogenous USF led o a dec ease in AP1-dependen ansc ip ion in F9 cells and co-
9
