ollicle s imula ing ho mone subuni be a (FSHB) : 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
The pi ui a y glycop o ein ho mone amily includes ollicle-s imula ing ho mone (FSH),
lu einizing ho mone (LH), cho ionic gonado opin (CG), and hy oid-s imula ing ho -
mone (TSH). They all consis o an iden ical alpha subuni and a ho mone-speci ic
be a subuni . FSHB encodes he be a subuni o FSH. Along wi h LH, FSH induces
egg and spe m p oduc ion. Guj al and MacBea h [1] p o ides a quan i a i e, and dy-
namic 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 FSHB 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 FSHB-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 FSHB 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1Aspec s o unpublished wo k we e p esen ed in a pos e session a Cell Symposia: Technology.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 17, 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 FRAT1 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
2.2. 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. ollicle s imula ing ho mone subuni be a (FSHB)
As eco ded in S and ing [4], he pi ui a y gland o hypophysis is an endoc ine gland in
e eb a es. I has wo main lobes, namely, an an e io lobe, and a pos e io lobe joined
and sepa a ed by a small in e media e lobe. The an e io lobe (adenohypophysis) is he
glandula pa ha p oduces and sec e es se e al ho mones. The pos e io lobe (neu o-
hypophysis) sec e es neu ohypophysial ho mones p oduced in he hypo halamus. Bo h
lobes ha e di e en o igins and hey a e bo h con olled by he hypo halamus. Ho -
mones sec e ed om he pi ui a y gland help o con ol g ow h, ene gy managemen ,
all unc ions o he sex o gans, blood p essu e, hy oid gland, me abolism, as well as
some aspec s o p egnancy, childbi h, b eas eeding, empe a u e egula ion, wa e /sal
concen a ion a he kidneys and pain elie . Glycop o ein ho mones (GPHs) a e he
mos complex molecules wi h ho monal ac i i y. They exis only in e eb a es bu he
genes encoding hei subuni s’ ances o s a e ound in mos e eb a e and in e eb a e
species al hough hei oles a e s ill unknown. Caho eau e al. [5] e iew he a ailable
s uc u al and unc ional da a conce ning GPHs and hei subuni s’ ances o s. Glyco-
p o ein ho mones (GPHs) a e he mos complex molecules wi h ho monal ac i i y. The
pi ui a y glycop o ein ho mone amily includes ollicle-s imula ing ho mone (FSH),
lu einizing ho mone (LH), cho ionic gonado opin (CG), and hy oid-s imula ing ho -
mone (TSH). They all consis o an iden ical alpha subuni and a ho mone-speci ic be a
subuni . FSHB encodes he be a subuni o FSH and along wi h LH, FSH induces egg
and spe m p oduc ion.
[6] analyzed a human genomic DNA agmen in phage λcon aining FSHB, and
de e mined he nucleo ide sequence o he egion o he clone encoding FSHβ. Thei
analysis o a se o cell hyb ids con aining ansloca ed de i a i es o ch omosome
11 u he localized FSHB o he human ch omosome egion llpll.2→11p e . A Hind
III es ic ion agmen leng h polymo phism (RFLP) de ec ed by ano he subclone o
he λphage con aining FSHB p o ided a gene ic ma ke o his egion o he human
genome.
FSH, and he FSH ecep o (FSHR), a G p o ein-coupled ecep o , play cen al
oles in human ep oduc ion. Jiang e al. [7] epo ed he c ys al s uc u e o FSH in
complex wi h he en i e ex acellula domain o FSHR (FSHRED), including he enig-
3
ma ic hinge egion ha is esponsible o signal speci ici y. Su p isingly, hey ound
ha he hinge egion did no o m a sepa a e s uc u al uni , bu was pa o he in eg al
s uc u e o FSHRED. In addi ion o he known ho mone-binding si e, FSHRED p o-
ided in e ac ion si es wi h he ho mone: a sul o y osine (sTy ) si e in he hinge egion
consis en wi h p e ious s udies and a po en ial exosi e esul ing om pu a i e ecep o
ime iza ion. Thei s uc u e, in compa ison o o he s, sugges ed ha FSHR in e ac ed
wi h i s ligand in wo s eps: ligand ec ui men ollowed by sTy ecogni ion. •FSH
i s binds o he high-a ini y ho mone-binding subdomain o FSHR and eshapes he
ligand con o ma ion o o m a sTy -binding pocke . •Nex , FSHR inse s i s sTy (i.e.,
sul a ed Ty 335) in o he FSH nascen pocke , e en ually leading o ecep o ac i a ion.
Summa izing Ulloa-Agui e e al. [8], Tao and Segalo [9] and Simoni e al. [10],
FSH (a gonado opin) s imula es s e oidogenesis and game ogenesis in he gonads,
while being sec e ed by he an e io pi ui a y gland. I egula es he mens ual cycle
and o a ian ollicula ma u a ion in women and suppo s spe m p oduc ion in men, by
binding o he FSH ecep o (FSHR) on he g anulosa cell su ace in o a ies and he
Se oli cell su ace in es es. The s imula ed ecep o leads o he dissocia ion o α-
and βγ- subuni s o G p o ein he e o ime inside he cell. The α-subuni ac i a es
adenylyl cyclase, esul ing in an inc ease o cAMP le els, and ul ima ely leads o he
inc eased s e oid p oduc ion ha is necessa y o ollicula g ow h and o ula ion in
women, while he ee βγ dime s ec ui G p o ein-coupled ecep o (GPCR) kinases
o he ecep o , which, in u n, lead o he ec ui men o β-a es in o he ecep o .
I p esen 3 d o de combina ions o FSHB 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. 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 [11]. 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 [11].
4
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
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
5
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].
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 FSHB-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
6
a e Hilbe Schmid Independence C i e ion indices (HSIC) indices (wi h b and linea
ke nel in Da Veiga [12]) and Sobol indicies (wi h 2002 implemen a ion in Sal elli [13]
and ma inez implemen a ion in Ma inez [14] and Baudin e al. [15]).
6.3. Conse ed machine lea ning ankings o es ed FSHB-X-X com-
bina ions
A o al o 2415, 3 d o de combina ions in ol ing FSHB 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. GnRH egula es FSH
Gonado opin- eleasing ho mone (GnRH) a decapep ide ha is sec e ed in pulsa ile
ashion om he hypo halamus in o he pi ui a y po al ascula u e, egula es FSH syn-
hesis and sec e ion (Be na d e al. [16]). Mason e al. [17] obse e ha he dele ional
mu a ion o a leas 33.5 kilobases encompassing he dis al hal o he gene o he
common biosyn he ic p ecu so o GnRH and GnRH-associa ed pep ide (GAP) causes
he edi a y hypogonadism in he hypogonadal (hpg) mouse. The pa ially dele ed gene
was ound o be ansc ip ionally ac i e as e ealed by in si u hyb idiza ion his ochem-
is y o hpg hypo halamic issue sec ions, bu immunocy ochemical analysis ailed o
show he p esence o an igen co esponding o any pa o he p ecu so p o ein.
6.3.2. Examining he beha iou o JUN / TCF / LEF / CTNNB1 -FSHB-X combi-
na ions
GnRH is known o egula e gonado ope unc ion h ough a complex ansc ip ional
ne wo k ha includes h ee membe s o he immedia e ea ly gene amily: EGR1, JUN,
and ATF3. These DNA-binding p o eins ac alone o in pai s o con e ho monal e-
sponsi eness o CGA, LHB, FSHB, and GnRH . Salisbu y e al. [18] sugges ed ha
he ansc ip ional esponse o JUN equi ed a unc ional in e ac ion be ween he T-cell
ac o (TCF)/lymphoid enhance ac o (LEF) amily o DNA-binding p o eins and β-
ca enin (CTNNB1), a coac i a o o TCF/LEF. Thei suppo ing da a include demon-
s a ion ha GnRH inc eases ac i i y o TOP lash, a TCF/LEF-dependen luci e ase
epo e , in LβT2 cells (a gonado ope-de i ed cell line). Thei addi ional co ans ec-
ion expe imen s indica ed ha a dominan -nega i e o m o TCF7L2 (TCFDN) ha
bound DNA, bu no β-ca enin, blocked GnRH induc ion o TOP lash. O e exp es-
sion o AXIN, an inhibi o o β-ca enin, also educed GnRH s imula ion o TOP lash.
T ansduc ion o LβT2 cells wi h TCFDN adeno i uses diminished GnRH s imula ion
o JUN mRNA wi hou al e ing exp ession o EGR1 and ATF3, wo o he immedia e
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
FSHB-T-WNT5A 122 29061 57139 24091 2613 FSHB-NKD1-PPP2R1A 134 11978 48147 4744 11658
FSHB-FZD2-SENP2 155 15792 56338 24860 16086 FSHB-FZD2-WNT4 180 18252 43713 35344 29882
FSHB-FZD2-PPP2R1A 216 20883 30530 35125 19903 FSHB-FZD2-FZD7 245 31877 48883 39603 50115
FSHB-T-WNT4 285 39661 52682 15922 9372 FSHB-FZD2-KREMEN1 286 34277 55693 24595 31187
FSHB-FZD2-TLE2 364 13058 45957 41222 23515 FSHB-FZD2-LRP5 366 26369 56367 45627 15311
FSHB-T-TLE2 387 33919 53269 21476 12396 FSHB-FZD2-SFRP4 413 14029 55048 31393 43835
FSHB-NKD1-TLE2 425 30274 24343 8644 12849 CSNK2A1-FSHB-SFRP4 494 33797 31035 11392 7911
CSNK1G1-FSHB-LEF1 506 19332 10676 684 44215 FSHB-LEF1-TCF7L1 545 41739 49954 24854 32409
CSNK1D-FSHB-LEF1 553 21733 36403 3295 16135 FSHB-NKD1-SENP2 574 7025 14205 7566 35992
CSNK1G1-FSHB-SFRP4 577 21679 6780 1575 25615 FSHB-T-TCF7 594 43385 15195 24841 22963
DKK1-FSHB-SENP2 641 24808 11118 11901 46995 FSHB-FZD2-FZD6 659 6508 55230 34443 24422
FSHB-NKD1-WNT2B 699 45757 26960 8191 17214 FSHB-LEF1-TLE2 716 51374 47443 17350 25939
DIXDC1-FSHB-LEF1 777 45272 10940 6689 22555 FSHB-GSK3A-KREMEN1 921 38254 26495 28443 3064
FSHB-FZD2-PPP2CA 922 15170 15248 37534 18116 FSHB-FZD2-TCF7L1 938 32749 50626 39797 16705
FSHB-NKD1-RHOU 974 40540 14067 9128 35039 FSHB-NKD1-SFRP1 1011 27987 21471 7065 1983
FRZB-FSHB-SENP2 1016 13136 7480 5202 23820 FSHB-FZD2-TCF7 1018 40516 45622 34770 44337
FSHB-T-TCF7L1 1029 40997 50899 17756 3833 CSNK1G1-FSHB-SENP2 1054 47479 13640 1228 8996
FSHB-GSK3A-SENP2 1073 16288 25162 18111 11005 FSHB-FZD2-LEF1 1118 14309 53366 39173 16692
FSHB-NKD1-WNT5A 1146 8921 29165 19619 2206 FSHB-FZD2-WNT5A 1190 10906 56570 47332 4072
FRZB-FSHB-SFRP4 1223 1819 11388 6342 54806 FSHB-LEF1-SENP2 1229 12578 50301 24457 7777
FSHB-FZD2-GSK3B 1266 6938 48999 24583 37158 FSHB-NKD1-TCF7L1 1387 37613 10650 8338 29279
FSHB-T-WNT2 1426 29977 55062 12965 5468 CSNK1G1-FSHB-PPP2CA 1518 8523 49087 1235 27660
DKK1-FSHB-LRP5 1519 2594 16842 22232 37962 FSHB-NKD1-TCF7 1528 46050 40792 9733 32939
CSNK2A1-FSHB-PPP2CA 1544 21794 56458 12205 17377 FSHB-FZD2-FBXW4 1577 20522 32605 35591 25593
CSNK2A1-FSHB-WNT2B 1655 8809 48217 20614 27385 FSHB-MYC-SENP2 1661 3841 29219 24971 7457
FSHB-FZD2-FZD8 1671 15948 53024 35438 29190 FSHB-FZD2-PITX2 1681 25060 56319 33266 14323
FSHB-FZD2-SLC9A3R1 1795 39694 55787 33593 41890 FSHB-FZD2-TLE1 2027 28569 54198 36983 37820
FSHB-LEF1-WNT4 2075 48049 40576 21210 20847 FSHB-FZD2-RHOU 2193 41442 55751 28895 48790
FSHB-T-WNT3A 2242 34175 56516 9226 7363 FSHB-GSK3A-PPP2R1A 2267 11196 31968 18565 24240
FSHB-FZD2-WNT3A 2298 5294 54044 44316 3638 CSNK1D-FGF4-FSHB 2441 17927 45150 34061 11599
FSHB-NKD1-WNT2 2558 23961 6398 3570 16109 FSHB-FZD2-SFRP1 2559 7953 55268 36582 36554
CSNK2A1-FSHB-FZD7 2601 20741 19320 11749 49197 FSHB-GSK3A-FBXW4 2618 22592 44503 22209 12645
FZD5-FSHB-SENP2 2620 37348 11372 8592 28360 FSHB-GSK3A-RHOU 2698 43462 34330 18933 10698
FSHB-FZD2-PORCN 2817 7691 52867 46696 28509 FSHB-MYC-TLE2 2923 22109 41704 26993 3530
AES-FOXN1-FSHB 2965 25071 19677 24885 18445 FSHB-MYC-FBXW4 2969 20328 49942 19643 3752
CSNK1G1-FSHB-MYC 2987 47541 16749 1201 23454 FSHB-FZD2-NLK 3007 15229 33503 42544 8088
DIXDC1-FSHB-FZD1 3136 45375 11211 11425 29794 FBXW11-FOXN1-FSHB 3234 11903 14136 23118 30110
DIXDC1-FSHB-T 3255 34040 47003 6560 49035 CSNK1G1-FSHB-WNT2B 3357 9060 28997 6593 37362
FSHB-PORCN-PPP2R1A 3420 9611 38143 42456 10626 CSNK2A1-FSHB-FZD1 3426 27759 33533 13224 35693
FSHB-GSK3A-WNT2B 3606 48867 25838 31124 32847 CSNK2A1-FSHB-SENP2 3609 22209 11628 8401 7649
CSNK1G1-FSHB-T 3636 19798 35345 312 15515 FOSL1-FOXN1-FSHB 3654 9548 17000 26396 39466
FSHB-NKD1-FBXW4 3657 26327 16817 8776 23172 CSNK1G1-FSHB-TLE2 3685 24521 6309 2390 53267
FRZB-FSHB-FZD1 3720 4368 14235 6831 35610 FSHB-FZD2-WNT2 3865 8188 47829 31525 37626
FSHB-GSK3A-TCF7 3904 42280 17499 22970 14161 FSHB-FZD2-MYC 3937 20248 51571 40207 23081
BCL9-FGF4-FSHB 3946 21353 21679 27526 39205 FSHB-LEF1-SFRP4 4021 45839 49234 24757 19506
FSHB-T-TLE1 4039 47213 53727 15401 29351 FRZB-FSHB-LEF1 4117 679 11864 4333 15549
CSNK1G1-FSHB-TCF7L1 4143 12682 12075 1247 30342 CSNK2A1-FSHB-TCF7L1 4195 17520 18854 11543 18761
CSNK1G1-FSHB-FZD1 4236 6426 15848 1850 51462 FSHB-JUN-SENP2 4244 14780 24139 39454 6681
CSNK2A1-FSHB-LEF1 4295 17954 29380 8714 37682 CTBP2-FOXN1-FSHB 4321 40069 14063 16057 55165
FSHB-LEF1-SLC9A3R1 4432 52834 53254 14595 12082 APC-FSHB-LEF1 4475 29086 10632 356 11132
DIXDC1-FSHB-TCF7L1 4549 31863 7056 10277 4669 FRZB-FSHB-LRP5 4578 8579 23418 10147 14329
FSHB-NKD1-WIF1 4621 8153 24750 5910 428 FSHB-GSK3A-WNT5A 4722 7262 52632 23091 38927
DIXDC1-FSHB-SFRP4 4856 39200 10508 10415 29896 CCND1-CTNNBIP1-FSHB 5004 56520 36248 44136 53863
FSHB-MYC-WNT4 5012 22216 28105 26520 25995 DVL1-FSHB-FZD1 5035 53508 16024 470 29060
FRZB-FSHB-PPP2CA 5204 19049 54010 6133 27813 EP300-FOXN1-FSHB 5265 2613 14431 12535 3811
FSHB-JUN-WNT4 5288 7708 26793 41418 20711 CSNK1G1-FSHB-LRP5 5322 17826 30783 6398 41762
CSNK1G1-FSHB-RHOU 5346 18138 12773 776 39643 FSHB-JUN-TCF7 5360 34303 50964 37006 36402
FSHB-FZD2-NKD1 5427 29500 40214 25746 8412 FSHB-PYGO1-WNT2 5434 16740 51092 5393 7521
CSNK1G1-FSHB-WNT2 5533 14900 6829 467 30316 FSHB-LEF1-MYC 5538 29950 53848 14480 13768
FSHB-MYC-RHOU 5540 30302 25407 22566 36470 FSHB-NKD1-NLK 5645 9268 38001 6547 21186
FSHB-PYGO1-WNT3A 5662 9149 57045 8437 25486 DAAM1-FOXN1-FSHB 5694 41648 33798 14726 32752
FBXW2-FSHB-FZD2 5712 39025 17476 10127 28195 CCND1-CTBP1-FSHB 5725 56748 29592 50558 40456
FSHB-JUN-LRP5 5767 3832 55208 46575 2660 CSNK1D-CTNNBIP1-FSHB 5796 33605 55368 17392 32696
Table 1: Rankings o FSHB-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
ea ly genes ha con e GnRH esponsi eness. Reduc ion o β-ca enin in LβT2 cells,
h ough s able exp ession o sho hai pin RNA, also selec i ely comp omised GnRH
egula ion o JUN exp ession and le els o JUN p o ein. Finally, o e exp ession o
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
FSHB-T-WNT5A 38699 15943 1093 25527 28563 FSHB-NKD1-PPP2R1A 25236 3206 25555 29398 54336
FSHB-FZD2-SENP2 722 17184 1804 8318 40473 FSHB-FZD2-WNT4 6816 11813 3438 4079 43595
FSHB-FZD2-PPP2R1A 10535 30086 26859 11239 24594 FSHB-FZD2-FZD7 4675 7640 5535 26749 20732
FSHB-T-WNT4 34987 32997 6654 22504 22222 FSHB-FZD2-KREMEN1 5444 42081 8565 25988 38407
FSHB-FZD2-TLE2 7557 9982 1122 7042 3191 FSHB-FZD2-LRP5 21769 12398 268 13884 19062
FSHB-T-TLE2 47144 33788 2346 14889 40413 FSHB-FZD2-SFRP4 4323 24559 5053 16811 10612
FSHB-NKD1-TLE2 17403 24824 7167 5397 44661 CSNK2A1-FSHB-SFRP4 17159 7039 55890 17829 49642
CSNK1G1-FSHB-LEF1 46458 5148 44690 1362 34271 FSHB-LEF1-TCF7L1 9166 53185 6306 4873 46442
CSNK1D-FSHB-LEF1 55374 14244 35230 8216 18460 FSHB-NKD1-SENP2 14879 5325 16207 3306 6877
CSNK1G1-FSHB-SFRP4 49673 9604 39425 16774 51741 FSHB-T-TCF7 15963 48634 1342 15324 3240
DKK1-FSHB-SENP2 13659 23744 54431 4021 33075 FSHB-FZD2-FZD6 983 15277 13285 3027 53066
FSHB-NKD1-WNT2B 24792 50507 25909 42911 36692 FSHB-LEF1-TLE2 17915 43860 1667 9288 40897
DIXDC1-FSHB-LEF1 56469 45507 52727 16153 8570 FSHB-GSK3A-KREMEN1 9218 45608 28535 37479 51895
FSHB-FZD2-PPP2CA 3195 15838 9446 3827 55246 FSHB-FZD2-TCF7L1 6008 50769 1291 19412 38941
FSHB-NKD1-RHOU 13620 39126 15440 38560 46234 FSHB-NKD1-SFRP1 28217 31095 6105 12696 47885
FRZB-FSHB-SENP2 5870 14328 41622 369 53440 FSHB-FZD2-TCF7 1081 40554 555 14927 53924
FSHB-T-TCF7L1 18421 44307 4524 7756 18690 CSNK1G1-FSHB-SENP2 41253 35360 30966 2443 33408
FSHB-GSK3A-SENP2 6015 32214 13032 15982 30339 FSHB-FZD2-LEF1 31623 27017 5219 9724 12888
FSHB-NKD1-WNT5A 45339 3228 1592 43013 28219 FSHB-FZD2-WNT5A 4908 10915 532 36402 27866
FRZB-FSHB-SFRP4 29574 11603 47383 3209 39274 FSHB-LEF1-SENP2 16486 20092 5835 4015 10968
FSHB-FZD2-GSK3B 1138 11606 9514 35386 46428 FSHB-NKD1-TCF7L1 29187 51071 22006 21070 31900
FSHB-T-WNT2 50362 19754 5809 18153 37120 CSNK1G1-FSHB-PPP2CA 50070 6934 23453 5427 21042
DKK1-FSHB-LRP5 17832 17573 35336 9068 54014 FSHB-NKD1-TCF7 16167 43862 3127 18402 9845
CSNK2A1-FSHB-PPP2CA 14473 24470 49869 6189 28547 FSHB-FZD2-FBXW4 7085 29857 30431 35123 20442
CSNK2A1-FSHB-WNT2B 14373 4327 52378 10457 6308 FSHB-MYC-SENP2 11261 1740 20872 3892 32896
FSHB-FZD2-FZD8 2194 10044 3079 38866 42600 FSHB-FZD2-PITX2 11301 38653 1373 55358 36056
FSHB-FZD2-SLC9A3R1 10325 37190 5991 25853 20686 FSHB-FZD2-TLE1 15519 25551 3634 38322 51551
FSHB-LEF1-WNT4 41497 39387 3625 9699 38711 FSHB-FZD2-RHOU 1282 37769 4155 7339 22181
FSHB-T-WNT3A 37189 29725 873 25128 8181 FSHB-GSK3A-PPP2R1A 2994 22740 47062 16574 48029
FSHB-FZD2-WNT3A 10002 6702 2404 44003 56141 CSNK1D-FGF4-FSHB 8876 10878 93 40281 25736
FSHB-NKD1-WNT2 18619 15105 23935 22810 37921 FSHB-FZD2-SFRP1 4118 5933 771 28595 38823
CSNK2A1-FSHB-FZD7 32789 11231 55533 29970 24990 FSHB-GSK3A-FBXW4 6890 34487 49297 34455 41976
FZD5-FSHB-SENP2 9883 31485 55203 2031 44565 FSHB-GSK3A-RHOU 10474 45128 43164 20106 41469
FSHB-FZD2-PORCN 2992 1901 452 19552 44544 FSHB-MYC-TLE2 13836 3400 17675 6625 31499
AES-FOXN1-FSHB 1721 19780 28544 48157 1579 FSHB-MYC-FBXW4 20928 25297 33253 38049 15480
CSNK1G1-FSHB-MYC 43747 46283 35580 1289 17413 FSHB-FZD2-NLK 17474 18226 16777 16181 28351
DIXDC1-FSHB-FZD1 3527 44586 55840 4528 2895 FBXW11-FOXN1-FSHB 6061 13017 20945 50606 93
DIXDC1-FSHB-T 36455 36262 54691 1183 12572 CSNK1G1-FSHB-WNT2B 43414 19138 41449 6694 6668
FSHB-PORCN-PPP2R1A 16061 9508 29505 10633 56512 CSNK2A1-FSHB-FZD1 14124 9142 52833 6616 46703
FSHB-GSK3A-WNT2B 7286 43548 39375 36993 48203 CSNK2A1-FSHB-SENP2 11122 26033 53443 288 43776
CSNK1G1-FSHB-T 51659 22786 44198 2191 56498 FOSL1-FOXN1-FSHB 11030 8091 27557 40910 3512
FSHB-NKD1-FBXW4 37869 19703 30672 38958 40988 CSNK1G1-FSHB-TLE2 37782 18462 30690 1164 44080
FRZB-FSHB-FZD1 9269 6036 46659 4923 47946 FSHB-FZD2-WNT2 7061 8112 4177 36695 20465
FSHB-GSK3A-TCF7 3290 46377 13521 12930 56685 FSHB-FZD2-MYC 29737 23746 2961 11852 18465
BCL9-FGF4-FSHB 19360 11815 11389 48533 47985 FSHB-LEF1-SFRP4 30991 41207 7433 5395 45348
FSHB-T-TLE1 20294 41198 2613 16496 22703 FRZB-FSHB-LEF1 54972 6560 43425 13496 36529
CSNK1G1-FSHB-TCF7L1 33263 14566 33778 3739 35177 CSNK2A1-FSHB-TCF7L1 28512 36022 53375 46 53291
CSNK1G1-FSHB-FZD1 30998 2577 47731 11545 47835 FSHB-JUN-SENP2 8504 11359 1933 11062 13593
CSNK2A1-FSHB-LEF1 57055 1237 51589 12220 31790 CTBP2-FOXN1-FSHB 23856 40293 23082 53127 1837
FSHB-LEF1-SLC9A3R1 34110 47269 1903 15643 45779 APC-FSHB-LEF1 56896 21413 52867 5217 28793
DIXDC1-FSHB-TCF7L1 13822 52661 54003 3177 5869 FRZB-FSHB-LRP5 34073 16424 34151 9778 55580
FSHB-NKD1-WIF1 24095 15522 18330 7234 28767 FSHB-GSK3A-WNT5A 3423 15383 30952 30779 42858
DIXDC1-FSHB-SFRP4 13901 35834 56885 9145 10625 CCND1-CTNNBIP1-FSHB 10903 57067 84 14969 9086
FSHB-MYC-WNT4 29072 10195 10094 2456 2568 DVL1-FSHB-FZD1 3557 48584 50929 1986 26905
FRZB-FSHB-PPP2CA 18947 29614 39286 5288 45027 EP300-FOXN1-FSHB 2802 1330 33722 46172 2708
FSHB-JUN-WNT4 52891 1241 4783 6486 15806 CSNK1G1-FSHB-LRP5 47118 5185 21879 18285 54603
CSNK1G1-FSHB-RHOU 47722 23571 41553 10002 20180 FSHB-JUN-TCF7 7804 30904 565 20582 2501
FSHB-FZD2-NKD1 17626 47853 2527 46929 13014 FSHB-PYGO1-WNT2 5673 7061 3497 1837 50147
CSNK1G1-FSHB-WNT2 50502 3033 48214 9696 46010 FSHB-LEF1-MYC 10894 16685 5708 19074 53792
FSHB-MYC-RHOU 13055 25852 10079 20516 29919 FSHB-NKD1-NLK 28359 8433 16880 40917 24813
FSHB-PYGO1-WNT3A 9583 23325 1231 22957 44849 DAAM1-FOXN1-FSHB 2820 39084 4512 44503 45
FBXW2-FSHB-FZD2 9636 32142 51450 29379 5217 CCND1-CTBP1-FSHB 36917 56532 211 43826 268
FSHB-JUN-LRP5 56899 1565 391 32348 8019 CSNK1D-CTNNBIP1-FSHB 15509 42611 40 44944 2520
Table 2: Rankings o FSHB-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
TCFDN a enua ed GnRH egula ion o CGA p omo e ac i i y, a known downs eam
a ge o JUN. Toge he , hei esul s indica ed ha GnRH egula ion o JUN an-
sc ip ion equi ed a unc ional in e ac ion be ween TCF/LEF and β-ca enin and ha
9
