o khead box N1 (FOXN1) : 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 : 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
FOXN1 belongs o he amily o FOX ( o khead box) p o eins ha ac as ansc ip ion
ac o s which play impo an oles in egula ing he exp ession o genes in ol ed in cell
g ow h, p oli e a ion, di e en ia ion, and longe i y. Guj al and MacBea h [1] p o ides
a quan i a i e, and dynamic s udy o WNT3A-media ed s imula ion o HEK 293 cells,
whe e hey eco d ime based exp ession p o iles o se e al esponse genes which co -
ela ed signi ican ly wi h p oli e a ion and mig a ion. By moni o ing he dynamics o
gene exp ession using sel -o ganizing maps, hey iden i ied clus e s o genes ha ex-
hibi simila exp ession dynamics and unco e ed p e iously un ecognized posi i e and
nega i e eedback loops. Howe e , hei s udy depic s/uses singula measu emen s o
indi idual gene exp ession a di e en ime snapsho s/poin s o in e he sys em wide
analysis o he pa hway. A any pa icula ime poin , i is o en he case ha genes
a e wo king syne gis ically in combina ions, e en hough hei exp ession measu e-
men s a e singula in na u e. He e, I •enume a e and ank all 2415 FOXN1 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 FOXN1-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 FOXN1 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1Aspec s o unpublished wo k we e p esen ed in a pos e session a Cell Symposia: Technology.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 20, 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 FOXN1 ela ed combina ions using indi idual gene exp essions measu ed in
ime, in WNT3A s imula ed HEK 293 cells.
2. In oduc ion
The de ails o he machine lea ning based sea ch engine has been ecen ly published in
Sinha [2] and deployed o explo e he 2nd o de combina ions o genes in he da a se
p o ided by Guj al and MacBea h [1]. Ne e heless, he e, I poin o he undamen als
o he published wo k o comple eness.
2.1. A combina o ial p oblem
Sensi i i y analysis plays a majo ole in compu ing he s eng h o he in luence o
in ol ed ac o s in any phenomena unde in es iga ion. When applied o exp ession
p o iles o a ious in a/ex acellula ac o s ha o m an in eg al pa o a signaling
pa hway, he a iance and densi y based analysis yields a ange o sensi i i y indices
o indi idual as well as a ious combina ions o ac o s. These combina ions deno e
he highe o de in e ac ions among he in ol ed ac o s. Compu a ion o highe o -
de in e ac ions is o en ime consuming bu i gi es a chance o explo e he a ious
combina ions ha migh be o in e es in he wo king mechanism o he pa hway. Fo
example, in a ange o ou h o de combina ions among he a ious ac o s o he Wn
pa hway, i would be easy o assess he in luence o he des uc ion complex o med by
APC, AXIN, CSKI and GSK3 in e ac ion. Bu he e ec o hese combina ions a y
o e ime as measu emen s o old changes and de ia ions in old changes a y. So
i is impe a i e o know how an in e ac ion o a combina ion o he in ol ed ac o s
beha e in ime and Sinha [2] de elops a p ocedu e o ack he beha iou by exploi ing
he in luences o hese in ol ed ac o s.
2.2. A possible solu ion
In his wo k, a e es ima ing he indi idual e ec s o ac o s o a highe o de combi-
na ion, he indi idual indices a e conside ed as disc imina i e ea u es. A combina ion,
2
hen, is a ea u e se in highe o de (≥2 ,i.e mul i a ia e). Wi h an excessi ely la ge
numbe o ac o s in ol ed in he pa hway, i is di icul o sea ch o impo an com-
bina ions in a wide sea ch space o e di e en o de s. Exploi ing he analogy wi h
he issues o p io i izing webpages using anking algo i hms, o a pa icula o de , a
ull se o combina ions o in e ac ions can hen be p io i ized based on hese ea u es
using a powe ul anking algo i hm ia suppo ec o s Joachims [3]. Reco ding he
changing ankings o he combina ions o e ime e eals how highe o de in e ac ions
beha e wi hin he pa hway and when an in e en ion migh be necessa y o in luence
he in e ac ion wi hin he pa hway.
2.3. o khead box N1 (FOXN1)
F anke e al. [4] calcula ed he nuclea po e low a e (NPFR) o ibosomal and ans e
RNA om he s eady-s a e alues (mean nuclea po e numbe and RNA syn hesis a es)
o he di e en ia ed a li e cells. They compa ed he e hepa ocy e alues wi h he
co esponding RNA anspo pe o mance o he nuclea po e complexes o o he cell
ypes.
The egion-speci ic homeo ic gene o k head ( kh) is known o p omo e e minal as
opposed o segmen al de elopmen in he D osophila emb yo. Weigel e al. [5] cloned
he kh egion by ch omosomal walking. P elemen -media ed ge m-line ans o ma-
ion and sequence compa ison o wild- ype and mu an alleles iden i ied he kh gene
wi hin he cloned egion. They obse ed ha kh was exp essed in he ea ly emb yo in
he wo e minal domains ha we e homeo ically ans o med in kh mu an emb yos.
The nuclea localiza ion o he kh p o ein sugges ed ha kh egula ed he ansc ip-
ion o o he , subo dina e, genes. The kh gene p oduc , howe e , did no con ain a
known p o ein mo i , such as he homeodomain o he zinc inge s, no was i sim-
ila in sequence o any o he known p o ein. Fo khead genes a e a subg oup o he
helix- u n-helix class o p o eins (B ennan and Ma hews [6]).
The hepa ocy e nuclea ac o 3 (HNF3) gene amily is composed o h ee p o eins
(α,β, and γ) ha a e ansc ip ion ac o s in ol ed in he coo dina e exp ession o
se e al li e genes. Pani e al. [7] ocused on he HNF3βp o ein, and epo ed he
localiza ion o wo ansc ip ional ac i a ion domains wi h a co ans ec ion assay wi h
HNF3β epo e and exp ession plasmids. Mo e speci ically, hey de eloped a co ans-
ec ion assay in Hep-G2 cells o de ine amino acid esidues esponsible o HNF3β
ansc ip ional ac i a ion. They de ined a posi ion-independen ac i a ion domain a
he HNF3βca boxyl e minus (361-458) which could po en ia e he exp ession o a
TATA box-CAT epo e cons uc con aining mul ime ic DNA ecogni ion si es o
he HNF3 p o ein. They ound ha his HNF3βac i a ion domain equi ed egion II
and III sequences which we e conse ed wi h he HNF3 amily and he D osophila o k
head p o ein.
Since hei disco e y, he conse ed amily o o k head/HNF3- ela ed ansc ip ion
ac o s gained inc easing impo ance o he analysis o gene egula o y mechanisms
du ing emb yonic de elopmen and in di e en ia ed cells. Di e en membe s o his
amily, which we e de ined by a conse ed 110 amino acid esidues encompassing
DNA binding domain o winged helix s uc u e ( ha has ou helices and a wo-s and
be a-shee ), se ed as egula o y keys in emb yogenesis, in umo igenesis o in he
3
main enance o di e en ia ed cell s a es. The e iew by Kau mann and Knochel [8]
summa ized he accumula ing amoun o da a on s uc u e, exp ession and unc ion o
o k head/HNF3- ela ed ansc ip ion ac o s.
FOXN1 belongs o he amily o FOX ( o khead box) p o eins. I is known ha
mu a ions a he nude locus o mice and a s dis up no mal hai g ow h and hymus
de elopmen , hus causing nude mice and a s o be immune-de icien . Nehls e al.
[9] showed ha one o he genes om he mouse nude locus which had been local-
ized on ch omosome 11 (wi hin a egion o <1 megabase), designa ed winged helix
nude (WHN / FOXN1), encoded a new membe o he winged-helix domain amily o
ansc ip ion ac o s and ha i was dis up ed on mouse nu and a nuN alleles. Fu -
he , mu an ansc ip s did no encode he cha ac e is ic DNA-binding domain, hus
poin ing o he ac ha WHN gene was he nude gene.
The di e en ia ion o p imi i e epi helial p ecu so cells in he hymic p imo dium
in o subcapsula , co ical, and medulla y epi helial cells o he ma u e hymus equi ed
he ac i i y o WHN. I was also equi ed o p ope ke a iniza ion o he hai sha .
Scho pp e al. [10] de e mined he nucleo ide sequence o a 58 kilobase egion on
mouse ch omosome 11 ha encompassed he mouse WHN and pa o he wo neigh-
bo ing genes. Using c oss-hyb idiza ion, hey isola ed he human o hologue o he
mouse WHN. They obse ed ha he human WHN p o ein also consis ed o 648 amino
acids, 85% o which was iden ical o he mouse WHN p o ein. They ound ha like he
mouse gene, he human gene consis ed o eigh coding exons and u ilized wo al e na-
i e i s exons in a issue-speci ic ashion.
I p esen 3 d o de combina ions o FOXN1 wi h o he genes, ha he machine
lea ning 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
4
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].
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 SV MRank
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.
5
No e ha he ollowing is he o de in which he iles should be execu ed in R, in
o de , o ob aining he desi ed esul s (No e ha he code will no be explained he e) - •
use sou ce(”mainSc ip -1-1.R”) wi h a gumen s o Dynamic da a •sou ce(”SVMRank-
Resul s-D.R”), o ank he in e ac ions (again his needs o be done sepa a ely o
di e en kinds o SA me hods), •use sou ce(”Combine-Time- iles.R”), i compu -
ing indices sepa a ely ia p e ious ile, •sou ce(”So -n-Plo -D.R”) o so he in e -
ac ions. No e ha he so ing is chages he in e ac ion anking in ime. Thus •use
sou ce(”In e ac ion-P io i y-In ime.R”) o ind he p io i ized anking o each and e -
e y in e ac ion o e he di e en ime poin s and inally •use sou ce(”P in -Ranking-
AND-In e ac ion-Rank.R”) o p in indi idual anking o he equi ed inpu ac o wi h
o he in e ac ion ac o s.
6. Resul s & Discussion
6.1. Time se ies da a by Guj al and MacBea h [1]
NOTE - Ranking was assigned on sco es ha we e so ed in DECREASING alues.
So, 1 was assigned o highes sco e and ice e sa.
Resul s o he 3 d o de in e ac ions a e p esen ed he e. The esul s i s discuss
he beha iou o in e ac ions ac oss he snapsho s o ime using he compu ed sensi-
i i ies on old change measu emen s pe ime snapsho . The analysis was done us-
ing 4 di e en sensi i i y indices. Ou o he 71C3combina ions, I conside /p esen
only hose combina ions ha show a anking wi hin i s 10,000 ou o 57,155. This
choice is libe al and biologis s/oncologis s can ha e a mo e s ic e choice as pe need.
Two obse a ions a e made, • he anking o a pa icula combina ion is conse ed (i.e
wi hin he 10,000 ange) in a pa icula ime poin o in he ea ly phase o la e phase
o WNT3A s imula ion, ac oss he majo i y o he ou sensi i i y me hods, which is a
s ic c i e ia o assessmen o • he anking o a pa icula combina ion is conse ed
ac oss ime poin s/phase (i.e hey a e wi hin he 10,000 ange) and he majo i y o he
ou sensi i i y me hods, which is elaxed c i e ia o assessmen . Applying his il e
helps e eal impo an combina ions o in e es ha migh be wo king syne gis ically
a a highe o de le el in he cell.
Rega ding echnical poin s o implemen a ion, he ankings we e gene a ed wi h-
ou scaling/no malizing he ime se ies da a p o ided by Guj al and MacBea h [1].
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
6.2. Enume a ion and anking o 2415 FOXN1-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 [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 FOXN1-X-X
combina ions
A o al o 2415, 3 d o de combina ions in ol ing FOXN1 we e ob ained om a ull
se o 71C3= 57155 combina ions. Fu he , om his selec ed se , using he abo e c i-
e ia o conse ed ankings, I epo / abula e he meaning ul combina ions ha migh
be wo king syne gis ically. Tables 2, 3 and 4 show he ankings o he same combi-
na ions as in able 1, bu using b ke nel o HSIC, 2002 implemen a ion o SOBOL
and ma inez implemen a ion o SOBOL, espec i ely. As one allies he ankings o
ac oss hese ables o a pa icula combina ion, one inds ha he ole o he combina-
ion o in e es is conse ed. This conse a ion poin s o he exis ence o he biological
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 KREMEN1-FOXN1-X combina ions
Fully ma u e and di e se epi helial mic oen i onmen o he hymus is equi ed o T
cell de elopmen and selec ion. The acquisi ion o hese cha ac e is ics is dependen
on exp ession o FOXN1, as a lack o unc ional FOXN1 esul s in abe an epi he-
lial mo phogenesis and an inabili y o a ac lymphoid p ecu so s o he hymus p i-
mo dium. Balciunai e e al. [16] epo ha sec e ed WNT glycop o eins, exp essed by
hymic epi helial cells and hymocy es, egula e epi helial FOXN1 exp ession in bo h
au oc ine and pa ac ine ashions. Thus, WNT signaling has been epo ed o egula e
hymocy e p oli e a ion and selec ion a se e al s ages du ing T cell on ogeny, as well
as he exp ession o FOXN1 in hymic epi helial cells (TECs). KREMEN1 (KRM1) is
a nega i e egula o o he canonical WNT signaling pa hway, and unc ions oge he
wi h he sec e ed WNT inhibi o Dickkop (DKK) by compe ing o he lipop o ein
ecep o - ela ed p o ein (LRP6) co- ecep o o WNTs. Osada e al. [17] used KRM1
knockou mice o examine KRM1 exp ession in he hymus and i s unc ion in hy-
mocy e and TEC de elopmen . They de ec ed KRM1 exp essionin bo h co ical and
medulla y TEC subse s, as well as in imma u e hymocy e subse s, beginning a he
CD25+CD44+ (DN2) s age and con inuing un il he, CD4+CD8+(DP) s age. They ob-
se ed ha neona al mice showed ele a ed exp ession o KRM1 in all TEC subse s,
while KRM1−/−mice exhibi ed a se e e de ec in hymic co ical a chi ec u e, includ-
ing la ge epi helial ee egions. Fu he , a TOPFlash assay e ealed a 2- old inc ease in
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
FOXN1-KREMEN1-WNT2B 170 28253 38493 15670 30910 FOXN1-KREMEN1-PPP2R1A 187 38390 45372 22596 7296
FOXN1-KREMEN1-WNT3 217 53012 56130 3088 18960 CSNK1G1-FOXN1-TLE2 226 3994 5316 18517 42187
FOXN1-KREMEN1-LRP5 260 52346 51858 34049 42979 FBXW11-FOXN1-KREMEN1 302 14589 4660 6330 19835
FZD5-FOXN1-LRP5 307 845 9386 28797 54200 DIXDC1-FOXN1-FBXW4 330 3748 17556 12016 30746
AES-FOXN1-FZD7 338 21947 6320 11489 39355 DKK1-FOXN1-SENP2 342 12351 29242 3585 34803
DKK1-FOXN1-FRZB 357 16995 47883 4088 8042 CSNK1G1-FOXN1-KREMEN1 390 10815 10745 12917 12924
FBXW11-FOXN1-SENP2 392 5743 6882 5378 5741 CSNK1G1-FOXN1-SLC9A3R1 395 1314 2366 9985 14166
AES-FOXN1-WNT2 402 20097 14407 14326 3071 AXIN1-FOXN1-WNT2 423 119 6856 11943 1929
FOSL1-FOXN1-FRZB 426 18495 3914 3925 50337 CSNK1D-FOXN1-LEF1 437 1005 10226 23334 20989
CTNNB1-FOXN1-FRZB 452 1997 7527 5341 50936 FZD5-FOXN1-SENP2 473 7569 6670 5785 7868
CTNNB1-FOXN1-RHOU 475 856 5984 14676 48295 DKK1-FOXN1-KREMEN1 480 13941 53110 3917 4043
CTNNB1-FOXN1-KREMEN1 484 1965 14838 7676 28339 AES-FOXN1-PPP2R1A 486 23646 9985 11397 37399
DAAM1-FOXN1-SFRP4 509 41264 31480 8686 35677 FOXN1-KREMEN1-SFRP4 510 51802 54641 8830 39747
FZD5-FOXN1-KREMEN1 511 1786 8577 7989 16170 AES-FOXN1-SENP2 518 22315 8527 7631 2724
FBXW11-FOXN1-SFRP4 561 2766 4849 11457 25387 FBXW11-FOXN1-FZD1 563 3393 3457 5989 33677
CTBP2-FOXN1-SENP2 566 19815 6401 2342 17983 DIXDC1-FOXN1-TLE2 581 740 1891 12323 28293
DIXDC1-FOXN1-FZD8 584 8409 1800 17070 5733 FZD5-FOXN1-SLC9A3R1 611 409 1127 6472 13971
DVL1-FOXN1-FRAT1 613 42124 2382 9631 35079 CSNK1D-FOXN1-SENP2 624 4830 5783 7469 36423
DVL1-FOXN1-FZD1 635 46799 7174 3229 38528 FOSL1-FOXN1-SFRP4 680 23049 4982 9425 19314
DAAM1-FOXN1-FZD6 681 45572 41066 6876 38373 CTNNB1-FOXN1-FZD6 691 3712 21540 10206 42092
FBXW11-FOXN1-FRZB 718 6154 5240 5636 29172 CSNK2A1-FOXN1-TCF7 736 11246 6655 11945 13187
FOSL1-FOXN1-KREMEN1 742 14009 6113 8523 21187 CSNK2A1-FOXN1-KREMEN1 775 7975 18477 6845 18012
AXIN1-FOXN1-FBXW4 790 11390 15320 10702 18907 DVL1-FOXN1-SFRP4 845 41274 6107 5709 48162
BTRC-FOXN1-FZD8 853 10860 9974 20227 10455 DKK1-FOXN1-WNT2B 864 31296 16351 4633 30734
DAAM1-FOXN1-FRZB 896 43246 33591 4294 17015 DVL1-FOXN1-SENP2 907 37314 6190 2465 34563
FBXW11-FOXN1-TCF7L1 916 13133 4109 10602 35818 CTNNB1-FOXN1-SLC9A3R1 917 2286 2153 7068 20569
FBXW11-FOXN1-LRP5 928 10686 10354 28842 25833 DIXDC1-FOXN1-KREMEN1 939 687 5376 7847 13303
CSNK1G1-FOXN1-PPP2R1A 960 4562 8931 10045 26824 FBXW11-FOXN1-FZD8 965 10160 4020 13865 51185
CTNNB1-FOXN1-PPP2R1A 966 1729 7371 11775 25639 DVL1-FOXN1-PPP2CA 977 25908 15392 4810 48741
AES-FOXN1-FZD8 981 7828 6668 16769 40810 FOSL1-FOXN1-GSK3B 983 14647 1776 17080 41173
FOXN1-KREMEN1-SFRP1 985 42401 54457 12806 414 CTNNB1-FOXN1-TLE2 988 5543 9432 15644 20859
FBXW11-FOXN1-WNT2B 989 38275 4532 5886 43707 AXIN1-FOXN1-FRAT1 990 1738 4384 15268 22220
FBXW11-FOXN1-TLE2 1004 10423 4208 12987 45489 DAAM1-FOXN1-LRP5 1009 45625 30080 34466 47125
DAAM1-FOXN1-PPP2CA 1014 50289 35190 8828 44977 FZD5-FOXN1-GSK3B 1022 3638 2875 20381 28710
CSNK2A1-FOXN1-WNT2B 1037 3175 7624 2538 6838 FOSL1-FOXN1-PPP2CA 1072 22257 16994 9632 25457
AES-FOXN1-GSK3A 1074 9734 18353 13099 38607 DKK1-FOXN1-TLE2 1080 18592 26323 11091 53163
CXXC4-FOXN1-SLC9A3R1 1089 2458 1605 6818 34700 CSNK1D-FOXN1-KREMEN1 1122 3656 11331 13933 38262
AXIN1-FOXN1-TCF7 1145 4915 2559 14562 18591 CSNK2A1-FOXN1-FZD8 1170 17750 11184 10347 9970
CCND3-FOXN1-FZD8 1183 34917 5116 11771 42093 CSNK1G1-FOXN1-FBXW4 1201 11491 20301 17590 26677
AES-FOXN1-SFRP4 1211 25834 8687 12229 21429 CSNK2A1-FOXN1-SENP2 1233 22059 16672 4525 3145
CTNNB1-FOXN1-TCF7L1 1237 6273 7647 12061 42159 AES-FOXN1-TLE1 1240 18162 8419 8417 9300
AES-FOXN1-FRZB 1247 23681 6537 2125 30062 CSNK1D-FOXN1-PPP2CA 1252 17427 16752 21509 34632
CXXC4-FOXN1-RHOU 1258 949 4951 19719 52102 DAAM1-FOXN1-FZD1 1262 38972 34040 4743 31791
FOSL1-FOXN1-SLC9A3R1 1275 28011 885 4881 51370 CSNK1G1-FOXN1-TCF7L1 1281 8663 5550 16139 11644
CSNK2A1-FOXN1-FZD6 1295 29245 25754 7254 28379 DAAM1-FOXN1-SLC9A3R1 1302 35893 19727 4671 23335
BTRC-FOXN1-WNT4 1304 7824 14183 18230 50189 FOSL1-FOXN1-FZD8 1314 19509 2343 16192 19984
DVL1-FOXN1-PPP2R1A 1316 40931 10069 3721 36477 FBXW11-FOXN1-FZD7 1326 9731 6528 14525 29594
CSNK1D-FOXN1-WNT2B 1328 17615 2986 7052 5219 EP300-FOXN1-TLE2 1334 6564 2241 419 8940
DKK1-FOXN1-WNT5A 1346 18310 46663 16625 23963 FBXW11-FOXN1-PPP2CA 1357 8568 16917 13742 17017
CSNK1D-FOXN1-FZD8 1359 10239 4066 27563 3347 DKK1-FOXN1-SFRP4 1382 19069 48285 11895 39000
DKK1-FOXN1-SLC9A3R1 1384 38790 31886 5342 8410 FOSL1-FOXN1-LRP5 1396 12463 8896 42948 43152
DVL1-FOXN1-SFRP1 1402 41345 3878 6051 10938 FBXW11-FOXN1-WNT4 1404 854 7156 11739 28714
CSNK2A1-FOXN1-PPP2CA 1415 18676 25465 10950 9597 APC-FOXN1-FRAT1 1418 596 2065 13524 33264
DVL1-FOXN1-RHOU 1438 38257 4549 11910 29038 AXIN1-FOXN1-TLE2 1439 321 2295 11236 23819
DIXDC1-FOXN1-FRZB 1442 1877 4968 4749 2791 AES-FOXN1-SLC9A3R1 1445 27866 3775 6327 34957
CTNNB1-FOXN1-FBXW4 1456 9113 25436 11398 551 AXIN1-FOXN1-FZD8 1457 7791 1656 16700 31078
CSNK2A1-FOXN1-SFRP4 1460 20356 19876 8892 40 FBXW11-FOXN1-PPP2R1A 1485 2012 6552 10843 25217
DIXDC1-FOXN1-SENP2 1491 2077 4322 6058 1110 CTNNB1-FOXN1-TCF7 1494 2724 3212 16430 18802
DVL1-FOXN1-FZD7 1495 41164 4874 7923 37918 CXXC4-FOXN1-FRZB 1502 99 9554 17642 46105
DAAM1-FOXN1-WNT2B 1503 50616 30398 2836 38247 FOSL1-FOXN1-TLE2 1506 19146 2341 7352 15489
CTNNB1-FOXN1-SFRP4 1509 2040 14820 13619 31097 AXIN1-FOXN1-RHOU 1511 738 4253 15602 21213
AXIN1-FOXN1-FZD7 1535 435 3620 13698 37972 AXIN1-FOXN1-SLC9A3R1 1537 5364 1010 5913 21906
FBXW11-FOXN1-FRAT1 1538 7360 6561 15328 24410 CSNK1G1-FOXN1-FRZB 1549 4678 4300 19697 7337
Table 1: Rankings o FOXN1-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
canonical WNT, signaling in TEC lines de i ed om KRM1−/−mice, when compa ed
wi h KRM+/+de i ed TEC lines. Fluo escence ac i a ed cell so ing (FACS) analysis
o dissocia ed hymus e ealed a educed equency o bo h co ical (BP1+EpCAM+)
and medulla y (UEA-1+EpCAMhi) epi helial subse s, wi hin he KRM1−/− hymus.
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
FOXN1-KREMEN1-WNT2B 20404 27973 36396 884 30778 FOXN1-KREMEN1-PPP2R1A 21547 15130 581 7154 55001
FOXN1-KREMEN1-WNT3 13156 54946 25548 2636 21279 CSNK1G1-FOXN1-TLE2 3512 4562 37294 11299 13219
FOXN1-KREMEN1-LRP5 11126 50667 3123 17750 44144 FBXW11-FOXN1-KREMEN1 332 11091 39492 5306 3719
FZD5-FOXN1-LRP5 6222 3631 43311 16286 12181 DIXDC1-FOXN1-FBXW4 3530 9085 27138 3878 80
AES-FOXN1-FZD7 1606 10949 51740 1419 32162 DKK1-FOXN1-SENP2 1097 45908 33686 6660 22662
DKK1-FOXN1-FRZB 2424 22948 17226 12105 45671 CSNK1G1-FOXN1-KREMEN1 7103 1710 43024 36832 31611
FBXW11-FOXN1-SENP2 128 6559 40566 11817 4361 CSNK1G1-FOXN1-SLC9A3R 4227 955 39936 15459 33468
AES-FOXN1-WNT2 1377 9293 42185 20047 814 AXIN1-FOXN1-WNT2 7365 9601 53234 18 663
FOSL1-FOXN1-FRZB 1736 15846 50466 9743 17620 CSNK1D-FOXN1-LEF1 6150 3755 45826 20298 28446
CTNNB1-FOXN1-FRZB 5276 5613 54024 3609 15356 FZD5-FOXN1-SENP2 755 12834 53767 11567 34539
CTNNB1-FOXN1-RHOU 3224 1823 49426 36371 35760 DKK1-FOXN1-KREMEN1 881 21837 24344 36049 9457
CTNNB1-FOXN1-KREMEN1 3133 5700 50426 39968 6394 AES-FOXN1-PPP2R1A 723 21691 22365 18788 29319
DAAM1-FOXN1-SFRP4 3365 36634 40858 22210 87 FOXN1-KREMEN1-SFRP4 21541 47835 13452 25040 38289
FZD5-FOXN1-KREMEN1 2473 6345 54537 47495 8093 AES-FOXN1-SENP2 1218 24082 52757 25404 14040
FBXW11-FOXN1-SFRP4 1186 2565 33626 28405 852 FBXW11-FOXN1-FZD1 808 5651 40991 17020 5225
CTBP2-FOXN1-SENP2 7635 19129 49786 125 42056 DIXDC1-FOXN1-TLE2 1845 16001 48828 19868 1211
DIXDC1-FOXN1-FZD8 2700 34651 54623 33036 17361 FZD5-FOXN1-SLC9A3R1 7530 2172 55354 30145 48570
DVL1-FOXN1-FRAT1 5430 52202 38249 34820 8736 CSNK1D-FOXN1-SENP2 1289 8520 39455 7070 25852
DVL1-FOXN1-FZD1 12403 42347 27218 6716 3146 FOSL1-FOXN1-SFRP4 3581 13413 50450 24051 14731
DAAM1-FOXN1-FZD6 1927 40856 30711 15954 7578 CTNNB1-FOXN1-FZD6 11515 4963 34761 21621 26869
FBXW11-FOXN1-FRZB 399 8848 44432 12716 405 CSNK2A1-FOXN1-TCF7 215 23416 42414 29565 28676
FOSL1-FOXN1-KREMEN1 2901 4767 41791 45598 13163 CSNK2A1-FOXN1-KREMEN1 393 11453 41930 49554 21564
AXIN1-FOXN1-FBXW4 10622 8925 10482 6312 6744 DVL1-FOXN1-SFRP4 7106 43765 40948 42922 1021
BTRC-FOXN1-FZD8 2072 28008 41249 39653 29423 DKK1-FOXN1-WNT2B 1531 23991 43537 22407 32006
DAAM1-FOXN1-FRZB 941 43695 29607 4270 188 DVL1-FOXN1-SENP2 3061 30960 37499 23295 15142
FBXW11-FOXN1-TCF7L1 214 35323 46275 16010 3890 CTNNB1-FOXN1-SLC9A3R1 5536 1236 55646 25818 55006
FBXW11-FOXN1-LRP5 3329 14741 47735 14948 367 DIXDC1-FOXN1-KREMEN1 3184 5794 52693 37317 689
CSNK1G1-FOXN1-PPP2R1A 3415 1125 22606 474 39586 FBXW11-FOXN1-FZD8 898 23493 49484 30381 27534
CTNNB1-FOXN1-PPP2R1A 6266 597 36708 568 27942 DVL1-FOXN1-PPP2CA 3346 22402 46063 15109 11834
AES-FOXN1-FZD8 952 20530 45343 38349 44784 FOSL1-FOXN1-GSK3B 1043 18255 47348 52130 53425
FOXN1-KREMEN1-SFRP1 27973 46263 7809 24618 51572 CTNNB1-FOXN1-TLE2 6280 17739 53356 13450 21250
FBXW11-FOXN1-WNT2B 3637 18134 11920 13018 6771 AXIN1-FOXN1-FRAT1 11810 7964 53217 15423 27549
FBXW11-FOXN1-TLE2 703 4758 34636 12462 1117 DAAM1-FOXN1-LRP5 3170 48457 41942 33504 19
DAAM1-FOXN1-PPP2CA 609 47634 41711 1552 16742 FZD5-FOXN1-GSK3B 2002 13824 55143 46713 44426
CSNK2A1-FOXN1-WNT2B 1203 7766 40805 5216 17738 FOSL1-FOXN1-PPP2CA 2050 20570 56654 7068 31115
AES-FOXN1-GSK3A 5614 14376 46340 29843 24805 DKK1-FOXN1-TLE2 2263 27657 20761 18942 14648
CXXC4-FOXN1-SLC9A3R1 5835 4242 55138 22461 50612 CSNK1D-FOXN1-KREMEN1 3060 1922 41865 30003 8137
AXIN1-FOXN1-TCF7 13244 8054 54509 20647 45016 CSNK2A1-FOXN1-FZD8 255 29912 45362 18779 48225
CCND3-FOXN1-FZD8 1397 31287 48789 38612 24901 CSNK1G1-FOXN1-FBXW4 6456 4971 6852 14306 22075
AES-FOXN1-SFRP4 2089 18334 55582 19404 4752 CSNK2A1-FOXN1-SENP2 142 13206 48212 17145 40808
CTNNB1-FOXN1-TCF7L1 2168 10793 56115 29127 14142 AES-FOXN1-TLE1 4311 39109 52117 30044 17018
AES-FOXN1-FRZB 1605 12489 50016 38983 5419 CSNK1D-FOXN1-PPP2CA 677 25144 54449 22625 44186
CXXC4-FOXN1-RHOU 4808 4398 51558 29746 40158 DAAM1-FOXN1-FZD1 988 42509 43886 1406 307
FOSL1-FOXN1-SLC9A3R1 3153 36795 52930 36241 48302 CSNK1G1-FOXN1-TCF7L1 2258 13561 45680 37954 10050
CSNK2A1-FOXN1-FZD6 138 14978 35128 24657 38128 DAAM1-FOXN1-SLC9A3R1 1344 32331 48311 28919 3119
BTRC-FOXN1-WNT4 5283 22892 41659 11506 604 FOSL1-FOXN1-FZD8 2757 28761 55000 20964 54956
DVL1-FOXN1-PPP2R1A 7337 28739 5315 11310 23091 FBXW11-FOXN1-FZD7 145 35268 39903 13575 28607
CSNK1D-FOXN1-WNT2B 2477 5135 39962 11954 23525 EP300-FOXN1-TLE2 1483 17209 53526 19752 11793
DKK1-FOXN1-WNT5A 6288 34859 41852 39340 54131 FBXW11-FOXN1-PPP2CA 551 13483 50027 13549 5121
CSNK1D-FOXN1-FZD8 484 13507 45944 36021 49258 DKK1-FOXN1-SFRP4 6549 20275 30679 15932 4932
DKK1-FOXN1-SLC9A3R1 4065 38156 35913 28567 35213 FOSL1-FOXN1-LRP5 6494 8694 40579 24357 5973
DVL1-FOXN1-SFRP1 4030 42573 44105 866 23391 FBXW11-FOXN1-WNT4 740 5464 41361 27962 1025
CSNK2A1-FOXN1-PPP2CA 278 35287 48412 13705 23060 APC-FOXN1-FRAT1 3161 1596 53917 10232 43563
DVL1-FOXN1-RHOU 5953 29822 43726 42040 18510 AXIN1-FOXN1-TLE2 5475 3810 51423 13930 10241
DIXDC1-FOXN1-FRZB 2525 19420 51785 5266 224 AES-FOXN1-SLC9A3R1 1732 22096 50779 28472 22844
CTNNB1-FOXN1-FBXW4 11108 9146 31589 18968 25721 AXIN1-FOXN1-FZD8 1586 21758 56745 32193 53352
CSNK2A1-FOXN1-SFRP4 363 18135 45540 43080 8388 FBXW11-FOXN1-PPP2R1A 848 15861 49514 22629 20720
DIXDC1-FOXN1-SENP2 1565 39282 50555 4197 2183 CTNNB1-FOXN1-TCF7 5722 8679 50274 17038 47411
DVL1-FOXN1-FZD7 3648 31690 36198 26788 18813 CXXC4-FOXN1-FRZB 2132 6567 49876 23824 32081
DAAM1-FOXN1-WNT2B 1209 51245 29982 6041 1467 FOSL1-FOXN1-TLE2 2685 18230 45559 25782 35344
CTNNB1-FOXN1-SFRP4 3275 1822 55437 22387 4399 AXIN1-FOXN1-RHOU 7032 3608 53224 33577 37035
AXIN1-FOXN1-FZD7 2665 2108 55426 10290 29770 AXIN1-FOXN1-SLC9A3R1 19889 17956 54483 21472 34039
FBXW11-FOXN1-FRAT1 1470 1272 34561 8385 2950 CSNK1G1-FOXN1-FRZB 4205 5593 40227 150 17600
Table 2: Rankings o FOXN1-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
Howe e , hei da a sugges ed ha a loss o KRM1 led o a se e e de ec in hymic
a chi ec u e.
Looking a he ables abo e, one inds he ollowing combina ions o KREMEN1
along wi h FOXN1, o be p ominen a 3 d o de le el - FOXN1-KREMEN1-WNT2B,
FOXN1-KREMEN1-WNT3, FOXN1-KREMEN1-LRP5, CTNNB1-FOXN1-KREMEN1,
9
