be a- ansducin epea con aining E3 ubiqui in p o ein
ligase o F-box/WD epea -con aining p o ein 1A
(BTRC/FBXW1A) : 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
βT CP (BTRC/FBXW1A) encodes a membe o he F-box p o ein amily, cha ac-
e ized by an app oxima ely 40 esidue s uc u al mo i , he F-box. The F-box p o-
eins o m one o he ou subuni s o ubiqui in p o ein ligase complex called Skp1-
Cul1-F-box (SCF) p o ein, which o en, bu no always, ecognize subs a es in a
phospho yla ion-dependen manne . BTRC associa es wi h phospho yla ed β-ca enin
des uc ion mo i s, p obably unc ioning in mul iple ansc ip ional p og ams by eg-
ula ing he WNT pa hways. 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 us-
ing sel -o ganizing maps, hey iden i ied clus e s o genes ha exhibi simila exp es-
sion 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 BTRC 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 con-
se ed ankings o BTRC-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
ITime beha iou al s udy o 3-od BTRC 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1Aspec s o unpublished wo k we e p esen ed in a pos e session a Cell Symposia: Technology.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 18, 2025
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
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 BTRC 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
2
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,
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. be a- ansducin epea con aining E3 ubiqui in p o ein ligase
(BTRC)
Lin oo e al. [4] measu ed cell-cycle, phase-speci ic cell kill caused by ca mus ine
(BCNU), in 4 cell lines wi h di e en sensi i i ies o he d ug. En iched subpopula ions
in a ious phases o he cell cycle we e ob ained and assayed o cell su i al, a e he
cells we e ea ed wi h BCNU o 1 hou . They measu ed le els o ac i i y o guanine
O6-alkyl ans e ase o each line and and ound ha only BTRC-19, a clone o he
9L line, had signi ican le els o alkyl ans e ase ac i i y and exhibi ed a ela i ely la
age- esponse cu e o BCNU. Spe ak e al. [5] cons uc ed a Xenopus oocy e cDNA
lib a y in a Saccha omyces ce e isiae exp ession ec o and used i o isola e genes ha
could unc ion in yeas cells o supp ess he empe a u e sensi i e (co ec ed) de ec o
he CDC15 mu a ion. Two ma e nally exp essed Xenopus cDNAs which ul illed hese
condi ions we e isola ed. One o hese clones encoded Xenopus N- as. They obse ed
ha o e exp ession o Xenopus N- as in S. ce e isiae did no ac i a e he RAS-cyclic
AMP (cAMP) pa hway, bu esul ed in dec eased le els o in acellula cAMP in bo h
mu an CDC15 and wild- ype cells. Thei esul s sugges ed ha a key s ep o he
cell cycle was dependen upon a phospho yla ion e en ca alyzed by cAMP-dependen
p o ein kinase. Addi ionally, hey obse ed ha he second clone, be a T CP (BTRC),
encoded a p o ein o 518 amino acids ha showed signi ican homology o he be a
subuni s o G p o eins in i s C- e minal hal . In his egion, BTRC was composed o
se en be a- ansducin epea s.
HIV-1 Vpu in e ac s wi h CD4 in he endoplasmic e iculum and igge s CD4
deg ada ion, p esumably by p o easomes. Human BTRC iden i ied by in e ac ion wi h
Vpu connec ed CD4 o his p o eoly ic machine y, and Ma go in e al. [6] ia coim-
munop ecipi a ion de ec ed CD4-Vpu-be a T CP (BTRC) e na y complexes. They ob-
se ed ha BTRC binding o Vpu and i s ec ui men o memb anes equi ed wo phos-
phose ine esidues in Vpu essen ial o CD4 deg ada ion. In BTRC, WD epea s a he
C e minus media ed binding o Vpu, and an F-box nea he N e minus was in ol ed in
3
in e ac ion wi h Skp1p (a a ge ing ac o o ubiqui in-media ed p o eolysis). Fu he ,
an F-box dele ion mu an o BTRC had a dominan -nega i e e ec on Vpu-media ed
CD4 deg ada ion. Thei da a sugges ed ha BTRC and Skp1p ep esen ed componen s
o a no el ER-associa ed p o ein deg ada ion pa hway ha media ed CD4 p o eolysis.
Membe s o he WNT/Wingless (Wg) amilies o sec e ed p o eins con ol many
aspec s o g ow h and pa e ning du ing animal de elopmen . Wg signal ansduc ion
causes inc eased s abili y o A madillo (A m/β-ca enin), a possible co- ac o o he
ansc ip ional egula o LEF1/TCF. Jiang and S uhl [7] desc ibed a new gene, slimb
( o supe nume a y limbs), which nega i ely egula ed he Wg pa hway along wi h he
Hedgehog (Hh) pa hway. They ound ha loss o unc ion o slimb esul ed in a cell-
au onomous accumula ion o high le els o bo h Ci and A m, and he ec opic exp es-
sion o bo h Hh- and Wg- esponsi e genes. They obse ed ha he slimb gene encoded
a conse ed F-box/WD40- epea p o ein ela ed o CDC4P (a p o ein in budding yeas )
ha a ge s cell-cycle egula o s o deg ada ion by he ubiqui in/p o easome pa hway.
They u he p oposed ha slimb p o ein a ge ed Ci and A m/β-ca enin o p ocessing
o deg ada ion by he ubiqui in/p o easome pa hway, and ha Wg egula e gene ex-
p ession a leas in pa by inducing changes in Ci and A m, which p o ec hem om
slimb-media ed p o eolysis.
Based on he abo e indings o Jiang and S uhl [7], Ma ikawa and Elinson [8]
examined he ole o e eb a e homolog be aT Cp (BTRC) in he WNT/β-ca enin sig-
naling and do sal axis o ma ion in Xenopus emb yos. They obse ed ha co-injec ion
o BTRC mRNA diminished Xwn 8 mRNA-induced axis o ma ion and exp ession
o Siamois and Xn 3, hus sugges ing ha BTRC was a nega i e egula o o he
WNT/β-ca enin signaling pa hway. An mRNA o a BTRC mu an cons uc (Del aF),
which lacked he F-box domain, was ound o induce an ec opic axis and exp ession o
Siamois and Xn 3. Because his ac i i y o Del aF was supp essed by co-injec ion o
Del aF T CP mRNA, Del aF was hough o ac in a dominan nega i e ashion. Fu -
he , he ac i i y o Del aF was diminished by C-cadhe in, GSK3 and AXIN, bu no by
a dominan nega i e dishe elled (DSH/DVL) . Thei esul s poin ed ha BTRC could
ac as a nega i e egula o o do sal axis o ma ion in Xenopus emb yos.
While sc eening a ma e nal Xenopus exp ession lib a y o ac i i ies ha syne -
gize wi h low le els o injec ed β-ca enin, Lagna e al. [9] isola ed a clone encod-
ing he C- e minal end o x-be a T CP-2, a highly conse ed p o ein belonging o he
F-box/WD40 amily o ubiqui in-ligase speci ici y ac o s. They showed ha x-be a
T CP-2 exp ession educed do sal axis o ma ion in Xenopus emb yos, while a dom-
inan nega i e mu an lacking he F-box igge ed he opposi e e ec , hus inducing
seconda y axes and ac i a ion o he exp ession o WNT esponsi e genes in ec ode -
mal explan s. In ligh o he exis ence o BTRC ansc ip s associa ed wi h he ege al
co ex, hey p oposed ha BTRC played an impo an ole in he es ablishmen o he
do sal de e minan s du ing co ical o a ion in Xenopus.
I p esen 3 d o de combina ions o BTRC 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.
4
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 [10]. 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 [10].
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
5
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
6
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 BTRC-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 [11]) and Sobol indicies (wi h 2002 implemen a ion in Sal elli [12]
and ma inez implemen a ion in Ma inez [13] and Baudin e al. [14]).
6.3. Conse ed machine lea ning ankings o es ed BTRC-X-X com-
bina ions
A o al o 2415, 3 d o de combina ions in ol ing BTRC 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 FBXW-BTRC-X combina ions
Suzuki e al. [15] had ound ha o e exp ession o an F-box p o ein βT CP1 (BTRC)
and he s uc u ally ela ed βT CP2 (FBXW11) augmen ed ubiqui ina ion o phospho-
yla ed IκBα(pIκBα) induced by umo nec osis ac o -α(TNF-α), bu he ela ion-
ship o he wo homologous βT CP p o eins emained unknown. Suzuki e al. [15]
e ealed ha dele ion mu an s o βT CP-1/2 lacking he F-box domain supp essed
7
RANKING @ iUSING HSIC - LINEAR
3 d o de comb. 1 3 6 12 24 3 d o de comb. 1 3 6 12 24
BTRC-GSK3A-FBXW4 399 54756 38346 27654 17743 BTRC-GSK3A-WNT4 427 51118 43719 18596 24127
BTRC-GSK3A-T 433 50464 41129 26608 19994 BTRC-GSK3A-NLK 685 15173 52709 30778 8565
APC-BTRC-WNT2 692 20019 7270 10948 40790 BTRC-FOXN1-FZD8 853 10860 9974 20227 10455
APC-BTRC-DAAM1 945 6005 22642 3587 1629 BTRC-GSK3A-SENP2 1269 31571 50158 24473 28342
BTRC-FOXN1-WNT4 1304 7824 14183 18230 50189 BTRC-PPP2CA-WNT4 1638 35087 32587 22025 51750
BTRC-GSK3A-SLC9A3R1 1828 47182 56469 39060 32535 BTRC-GSK3A-WNT2 1829 55217 56594 37274 29011
APC-BTRC-PPP2CA 1858 9257 27876 20401 45249 BTRC-GSK3A-WNT2B 1933 56634 52638 55432 37508
BTRC-GSK3A-WNT3A 1946 45085 56552 39291 36207 APC-BTRC-FRAT1 1993 4234 3760 19668 20988
BTRC-GSK3A-LRP5 2279 50637 43268 46359 31488 BTRC-GSK3A-GSK3B 2331 27348 55816 23870 24273
BTRC-GSK3A-LEF1 2442 51793 56318 34877 43742 APC-BTRC-TCF7 2478 22925 18323 15264 35151
AXIN1-BTRC-DVL2 2500 11477 16610 53250 27050 APC-BTRC-FBXW2 2597 1236 21415 35908 27898
BTRC-GSK3A-PITX2 2613 49655 52881 6241 50065 APC-BTRC-FBXW11 2628 10595 8869 37962 22016
APC-BTRC-FZD5 2646 23572 8780 23130 34875 BTRC-PPP2CA-SFRP4 2649 45450 55216 41081 48220
APC-BTRC-KREMEN1 2657 22730 7427 25126 52825 BTRC-CCND3-WNT4 2710 41552 34711 18962 45323
BTRC-FOXN1-KREMEN1 2723 18292 12669 9692 56435 APC-BTRC-SENP2 2742 2914 7275 11614 33253
BTRC-GSK3A-PYGO1 2771 52085 53096 35408 47985 BTRC-FOXN1-SENP2 2819 7789 14297 7017 45881
BTRC-GSK3A-KREMEN1 3010 54393 56679 45265 52103 BTRC-WNT1-WNT4 3062 33354 28305 33341 21189
APC-BTRC-FZD1 3143 5688 12315 18344 2535 BTRC-RHOU-TCF7 3204 41293 40925 4745 45576
BTRC-RHOU-FBXW4 3368 55758 50831 743 47356 BTRC-GSK3A-SFRP4 3507 52295 54637 26019 28662
APC-BTRC-SFRP4 3513 8753 5458 12692 40484 BTRC-GSK3A-MYC 3555 32516 35803 30274 45481
BTRC-FOXN1-LRP5 3607 18421 14993 15012 46079 BTRC-WNT2-WNT3A 3667 53111 52839 51977 37208
BTRC-FOXN1-FRAT1 3675 26618 6170 28752 42168 BTRC-FOXN1-WNT5A 3687 8784 26520 30828 49695
BTRC-PPP2CA-T 3795 43011 19216 31904 44253 BTRC-FOXN1-SLC9A3R1 3990 7530 9458 10316 38446
AXIN1-BTRC-WNT2 4161 12843 11806 22233 22893 BTRC-NKD1-WIF1 4240 14000 50136 49384 38469
BTRC-GSK3A-TCF7L1 4309 38628 56805 38238 43590 BTRC-PPP2CA-TCF7L1 4339 35835 48253 44827 55748
BTRC-NKD1-WNT2B 4358 53865 52313 44893 36090 BTRC-CCND3-CXXC4 4501 45355 32569 38665 55920
BTRC-GSK3A-TCF7 4603 42589 43515 30573 20648 AES-BTRC-FZD5 4783 25177 12006 24403 11782
BTRC-GSK3A-WNT5A 4818 34238 56433 32265 47742 BTRC-GSK3A-TLE2 4865 53235 52059 36282 26238
BTRC-JUN-TCF7L1 4943 27837 47631 38349 49359 APC-BTRC-CTBP1 4950 9391 8017 9007 18060
BTRC-CCND2-WNT4 4953 52895 11943 23405 44507 BTRC-PPP2CA-TCF7 4981 40531 13651 43079 34410
AES-BTRC-DVL2 5030 12391 15984 50914 7881 APC-BTRC-FZD7 5075 17371 3368 9728 6810
BTRC-FBXW2-WNT3A 5104 43223 55035 20534 48314 BTRC-FGF4-NLK 5183 27874 56631 56082 16933
BTRC-LRP6-FBXW4 5274 56436 32556 47321 9771 BTRC-GSK3A-PPP2R1A 5299 26077 46257 26921 27177
BTRC-GSK3A-RHOU 5363 54749 48852 32167 37017 BTRC-RHOU-SFRP1 5398 50476 50062 18513 5471
APC-BTRC-CSNK1D 5442 10883 12646 22647 6204 BTRC-CCND3-FZD7 5447 42687 17440 28743 32509
BTRC-NKD1-WNT4 5567 46251 34634 38788 50581 APC-BTRC-WNT5A 5579 11328 15422 26429 56417
BTRC-FOXN1-FZD1 5639 18624 18170 16160 42888 BTRC-GSK3A-LRP6 5724 52749 52308 52281 23121
BTRC-JUN-LRP5 5888 37770 52457 34858 25652 APC-BTRC-WNT4 5911 19764 3320 4428 21935
BTRC-JUN-WNT3A 5912 40253 46236 41851 33745 BTRC-FOXN1-TLE2 6027 19449 11379 20626 38897
BTRC-GSK3A-SFRP1 6145 52112 56467 35900 15415 APC-BTRC-SLC9A3R1 6177 4157 5216 19065 21916
AXIN1-BTRC-FOSL1 6468 1244 4919 15349 38579 BTRC-CCND3-WNT2B 6518 55220 31850 31279 47240
BTRC-NKD1-TCF7L1 6683 39081 40177 56129 51053 BTRC-FOXN1-T 6737 27226 10335 37112 39462
BTRC-PPP2CA-SENP2 6802 36289 41584 41333 55323 APC-BTRC-LRP6 6911 28737 4615 26743 4691
AES-BTRC-DKK1 6960 18725 17933 22340 4842 BTRC-CCND3-FOSL1 6965 37686 28713 22894 44780
BTRC-RHOU-SENP2 6988 41062 38132 3821 50210 BTRC-JUN-WNT4 7215 48917 43726 14501 33278
BTRC-NKD1-SENP2 7336 41049 43342 47787 47593 BTRC-NKD1-SFRP1 7350 47032 49057 53468 51
BTRC-CCND3-EP300 7453 26437 27976 19877 47477 BTRC-PPP2CA-RHOU 7476 44384 28579 43147 51079
BTRC-RHOU-WNT4 7503 46563 30678 9407 48820 AES-BTRC-GSK3A 7511 649 23146 16630 18979
BTRC-GSK3A-WIF1 7516 12519 55092 29987 46937 BTRC-CCND3-DKK1 7699 42444 50739 46484 28100
BTRC-CCND3-TLE1 7893 37196 27722 17214 40980 BTRC-WNT1-WNT3A 7906 35272 32813 57093 19625
BTRC-PPP2CA-WNT5A 7995 31259 42194 31725 49589 AES-BTRC-PPP2CA 8064 4470 33612 23597 26264
BTRC-FOXN1-FSHB 8118 14636 21198 33671 46291 AXIN1-BTRC-SENP2 8132 4244 7993 9640 29213
AES-BTRC-RHOU 8185 12590 5280 21149 27171 BTRC-JUN-PYGO1 8263 45096 50540 41901 54741
BTRC-JUN-SENP2 8343 24938 49553 30325 39253 AES-BTRC-JUN 8360 1344 7172 18393 10377
BTRC-CCND3-FZD2 8456 51566 34885 26667 37022 BTRC-PYGO1-WNT3A 8510 43352 44095 22599 55396
BTRC-CCND3-TCF7 8538 37690 31426 32017 39147 APC-BTRC-PYGO1 8570 7302 8530 26749 53032
AES-BTRC-FOSL1 8607 22428 10525 19327 9562 AXIN1-BTRC-FZD5 8616 22820 8767 21827 26979
BTRC-KREMEN1-WNT4 8701 48514 48223 13062 56268 AXIN1-BTRC-CTBP1 8711 1472 9482 8596 26250
BTRC-LRP6-SLC9A3R1 8815 46754 26428 52824 19745 AXIN1-BTRC-CCND3 8836 8488 11500 25054 29424
BTRC-NKD1-PPP2CA 8947 49640 54091 53329 49700 BTRC-JUN-LEF1 8950 36031 39447 23517 51208
BTRC-NKD1-SFRP4 8970 49955 44941 50705 38441 BTRC-NKD1-TLE2 9050 49909 47985 49811 37145
AXIN1-BTRC-PPP2CA 9123 8661 42188 25227 33335 BTRC-FGF4-WNT4 9178 41675 44825 23499 35812
BTRC-JUN-WNT2 9191 44964 44172 23594 37421 AES-BTRC-WIF1 9239 22292 6516 14211 26584
Table 1: Rankings o BTRC-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
ubiqui ina ion and des uc ion o pIκBαas well as ansc ip ional ac i a ion o NF-
κB. They obse ed ha he ec opically exp essed βT CP-1/2 o med bo h homodime
8
RANKING @ iUSING HSIC - RBF
3 d o de comb. 1 3 6 12 24 3 d o de comb. 1 3 6 12 24
BTRC-GSK3A-FBXW4 8681 52442 37486 14095 29234 BTRC-GSK3A-WNT4 12669 46498 44112 7495 6959
BTRC-GSK3A-T 6821 52908 40648 46892 3130 BTRC-GSK3A-NLK 8965 2718 45185 45189 49869
APC-BTRC-WNT2 9163 27280 43155 9369 48381 BTRC-FOXN1-FZD8 2072 28008 41249 39653 29423
APC-BTRC-DAAM1 3743 41091 17783 36176 30576 BTRC-GSK3A-SENP2 9345 20665 52714 11947 10821
BTRC-FOXN1-WNT4 5283 22892 41659 11506 604 BTRC-PPP2CA-WNT4 13782 33555 41012 5416 5243
BTRC-GSK3A-SLC9A3R1 6194 38474 43825 24758 45073 BTRC-GSK3A-WNT2 14070 55767 42437 51240 22931
APC-BTRC-PPP2CA 4261 9237 29395 1410 54704 BTRC-GSK3A-WNT2B 9987 55898 47839 49215 4144
BTRC-GSK3A-WNT3A 12858 35286 25253 43232 19671 APC-BTRC-FRAT1 3582 6272 14221 27977 50407
BTRC-GSK3A-LRP5 11198 46675 45351 47368 14161 BTRC-GSK3A-GSK3B 4319 5498 47667 46132 13660
BTRC-GSK3A-LEF1 12346 48661 30805 40415 35765 APC-BTRC-TCF7 4705 13188 32095 8871 53791
AXIN1-BTRC-DVL2 29410 5833 29596 43603 19764 APC-BTRC-FBXW2 4038 1370 25809 43009 39049
BTRC-GSK3A-PITX2 2406 44730 43962 54217 10925 APC-BTRC-FBXW11 3206 17373 31138 32376 35753
APC-BTRC-FZD5 5256 25681 42284 19958 53619 BTRC-PPP2CA-SFRP4 23701 47316 54163 28713 3631
APC-BTRC-KREMEN1 8265 27604 37492 52851 54717 BTRC-CCND3-WNT4 19497 48624 44461 7332 36428
BTRC-FOXN1-KREMEN1 2135 23342 46688 49929 2379 APC-BTRC-SENP2 2108 12223 33937 28608 53523
BTRC-GSK3A-PYGO1 16077 51303 28870 23885 34429 BTRC-FOXN1-SENP2 1453 25844 25117 25651 4578
BTRC-GSK3A-KREMEN1 5140 53781 54895 53606 19024 BTRC-WNT1-WNT4 32427 25452 32871 49 5853
APC-BTRC-FZD1 2781 7110 14462 30867 34404 BTRC-RHOU-TCF7 35804 52449 25786 16725 22733
BTRC-RHOU-FBXW4 46779 55127 25525 2995 35190 BTRC-GSK3A-SFRP4 1874 50406 40267 35344 14342
APC-BTRC-SFRP4 9796 14815 39313 5822 33503 BTRC-GSK3A-MYC 14888 25284 36568 2248 10669
BTRC-FOXN1-LRP5 3273 30198 36284 23532 241 BTRC-WNT2-WNT3A 31077 52852 5122 22613 1175
BTRC-FOXN1-FRAT1 4169 16478 20795 41841 6870 BTRC-FOXN1-WNT5A 4629 21553 49380 16835 38205
BTRC-PPP2CA-T 42753 54274 45558 42965 26218 BTRC-FOXN1-SLC9A3R1 987 5633 38486 32921 3284
AXIN1-BTRC-WNT2 19940 34779 51998 35294 51645 BTRC-NKD1-WIF1 40459 4738 29145 25508 9960
BTRC-GSK3A-TCF7L1 5048 35224 47809 21415 23988 BTRC-PPP2CA-TCF7L1 12141 39746 46412 24183 1736
BTRC-NKD1-WNT2B 34862 55446 41413 44594 7779 BTRC-CCND3-CXXC4 28171 55841 38665 6723 12780
BTRC-GSK3A-TCF7 2679 48529 45973 20262 37169 AES-BTRC-FZD5 23300 36671 28933 1416 44834
BTRC-GSK3A-WNT5A 1331 34656 55842 14294 39235 BTRC-GSK3A-TLE2 7469 51695 40008 8656 21337
BTRC-JUN-TCF7L1 24959 20714 12947 17672 1488 APC-BTRC-CTBP1 22415 7521 32949 1572 36489
BTRC-CCND2-WNT4 37807 46204 36799 15309 3667 BTRC-PPP2CA-TCF7 21166 51448 42519 19099 5073
AES-BTRC-DVL2 35795 3432 49151 31669 27699 APC-BTRC-FZD7 1697 7624 29404 31973 44902
BTRC-FBXW2-WNT3A 39006 39760 21198 32505 45697 BTRC-FGF4-NLK 3757 10635 33258 31931 37107
BTRC-LRP6-FBXW4 48827 55300 26952 16212 26925 BTRC-GSK3A-PPP2R1A 8962 30689 29658 9241 43973
BTRC-GSK3A-RHOU 7245 54116 45531 48716 24019 BTRC-RHOU-SFRP1 54868 48925 28543 13342 47372
APC-BTRC-CSNK1D 2236 12178 36083 5029 45903 BTRC-CCND3-FZD7 20781 46050 30736 40377 49178
BTRC-NKD1-WNT4 42581 46731 36629 5396 4774 APC-BTRC-WNT5A 7886 8328 47613 21423 25207
BTRC-FOXN1-FZD1 4693 25266 25295 4116 67 BTRC-GSK3A-LRP6 13109 51610 41523 24298 23690
BTRC-JUN-LRP5 47907 24894 17380 45629 3061 APC-BTRC-WNT4 2113 8450 42844 16 50560
BTRC-JUN-WNT3A 37538 33079 9131 18895 7570 BTRC-FOXN1-TLE2 5417 15877 5904 34425 5486
BTRC-GSK3A-SFRP1 1664 48238 36146 6688 23640 APC-BTRC-SLC9A3R1 37704 1044 33896 21821 50112
AXIN1-BTRC-FOSL1 28079 9720 56293 17570 50701 BTRC-CCND3-WNT2B 24254 53442 21428 39052 47106
BTRC-NKD1-TCF7L1 35223 44544 35152 5915 7887 BTRC-FOXN1-T 2442 42400 34919 11494 2235
BTRC-PPP2CA-SENP2 8723 30185 52002 10650 31985 APC-BTRC-LRP6 49993 33786 30995 2710 30279
AES-BTRC-DKK1 29312 29441 54503 3484 32873 BTRC-CCND3-FOSL1 29635 39122 40121 26979 11246
BTRC-RHOU-SENP2 22351 33999 30105 8174 21370 BTRC-JUN-WNT4 35141 42866 20608 11140 4126
BTRC-NKD1-SENP2 37667 27919 25558 3108 6916 BTRC-NKD1-SFRP1 33217 47597 38470 24028 21787
BTRC-CCND3-EP300 16838 27677 31497 24519 27349 BTRC-PPP2CA-RHOU 21096 51135 35466 34465 21156
BTRC-RHOU-WNT4 17430 46737 39943 11925 27423 AES-BTRC-GSK3A 27622 3812 52880 9059 31196
BTRC-GSK3A-WIF1 8879 9323 38999 19148 14457 BTRC-CCND3-DKK1 16607 38588 8857 2264 31589
BTRC-CCND3-TLE1 34230 30664 34418 21643 23378 BTRC-WNT1-WNT3A 35227 22898 11695 12947 7878
BTRC-PPP2CA-WNT5A 32549 27398 48062 6477 24654 AES-BTRC-PPP2CA 30718 1100 13573 25518 52217
BTRC-FOXN1-FSHB 6670 20222 33570 45284 22 AXIN1-BTRC-SENP2 18487 9499 51352 5160 50244
AES-BTRC-RHOU 31407 10383 39972 41558 56068 BTRC-JUN-PYGO1 37910 44163 1594 29208 42451
BTRC-JUN-SENP2 29065 17777 6958 20628 6213 AES-BTRC-JUN 35333 7469 37870 8738 37322
BTRC-CCND3-FZD2 34361 45890 35141 11003 14788 BTRC-PYGO1-WNT3A 30112 36444 28501 12063 23745
BTRC-CCND3-TCF7 23555 47574 37247 26531 11513 APC-BTRC-PYGO1 51355 9058 6097 3990 45511
AES-BTRC-FOSL1 33302 22693 50032 1890 51445 AXIN1-BTRC-FZD5 13481 19478 52911 4874 45918
BTRC-KREMEN1-WNT4 47643 49959 34801 36178 5914 AXIN1-BTRC-CTBP1 33060 3616 54070 3 28263
BTRC-LRP6-SLC9A3R1 44418 44933 17317 32739 29397 AXIN1-BTRC-CCND3 14456 12461 46193 30484 34903
BTRC-NKD1-PPP2CA 29398 53369 45941 27262 4730 BTRC-JUN-LEF1 30590 27148 1645 43584 10953
BTRC-NKD1-SFRP4 43680 50931 6721 32883 11764 BTRC-NKD1-TLE2 39341 53201 13833 15359 18076
AXIN1-BTRC-PPP2CA 18230 23560 32745 6358 50799 BTRC-FGF4-WNT4 3320 45404 24731 7888 11796
BTRC-JUN-WNT2 41795 46313 18106 41395 6164 AES-BTRC-WIF1 31157 16922 12256 24179 30618
Table 2: Rankings o BTRC-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
and he e odime complexes wi hou displaying he ime complex. In iguingly, hey
u he obse ed ha he βT CP homodime , bu no he he e odime , was selec i ely
ec ui ed o pIκBαinduced by TNF-α. Thei esul s indica ed ha no only βT CP1
9
