RESEARCH ARTICLE Open Access
In eg a ed RNA-seq and DNase-seq
analyses iden i y pheno ype-speci ic BMP4
signaling in b eas cance
M. Ampuja
1,2*†
, T. Ran ape o
1†
, A. Rod iguez-Ma inez
1,2†
, M. Palm o h
1
, E. L. Ala mo
1
, M. Nyk e
1
and A. Kallioniemi
1,2
Abs ac
Backg ound: Bone mo phogene ic p o ein 4 (BMP4) plays an impo an ole in cance pa hogenesis. In b eas
cance , i educes p oli e a ion and inc eases mig a ion in a cell line-dependen manne . To cha ac e ize he
ansc ip ional media o s o hese pheno ypes, we pe o med RNA-seq and DNase-seq analyses a e BMP4
ea men in MDA-MB-231 and T-47D b eas cance cells ha espond o BMP4 wi h enhanced mig a ion and
dec eased cell g ow h, espec i ely.
Resul s: The RNA-seq da a e ealed gene exp ession changes ha we e consis en wi h he in i o pheno ypes
o he cell lines, pa icula ly in MDA-MB-231, whe e mig a ion- ela ed p ocesses we e en iched. These esul s we e
con i med when en ichmen o BMP4-induced open ch oma in egions was analyzed. In e es ingly, he ch oma in
in ansc ip ion s a si es o di e en ially exp essed genes was al eady open in uns imula ed cells, hus enabling
apid ec ui men o ansc ip ion ac o s o he p omo e s as a esponse o s imula ion. Fu he analysis and
unc ional alida ion iden i ied MBD2, CBFB, and HIF1A as downs eam egula o s o BMP4 signaling. Silencing
o hese ansc ip ion ac o s e ealed ha MBD2 was a consis en ac i a o o a ge genes in bo h cell lines, CBFB
an ac i a o in cells wi h educed p oli e a ion pheno ype, and HIF1A a ep esso in cells wi h induced mig a ion
pheno ype.
Conclusions: In eg a ing RNA-seq and DNase-seq da a showed ha he pheno ypic esponses o BMP4 in b eas
cance cell lines a e e lec ed in ansc ip omic and ch oma in le els. We iden i ied and expe imen ally alida ed
downs eam egula o s o BMP4 signaling ha ela e o he di e en in i o pheno ypes and hus demons a e
ha he downs eam BMP4 esponse is egula ed in a cell ype-speci ic manne .
Keywo ds: Bone mo phogene ic p o ein, B eas cance , NGS, RNA-seq, DNase-seq, T ansc ip ion ac o
Backg ound
Despi e many ad ances in diagnos ics and he apeu ics,
b eas cance emains he leading cause o cance dea h
in women [1]. Bone mo phogene ic p o eins (BMPs) a e
a g oup o g ow h ac o s ha a e impo an playe s
du ing de elopmen [2, 3] bu also con ibu e o cance
o ma ion and p og ession [4–6]. As a sub amily o he
ans o ming g ow h ac o β(TGF-β) p o ein supe am-
ily, BMPs a e ex acellula ligands ha bind as dime s o
hei speci ic ansmemb ane ecep o s and ac i a e he
in acellula SMAD signaling pa hway leading o phos-
pho yla ion o ecep o - egula ed SMADs (SMAD1/5/9).
The ac i a ed SMADs bind o SMAD4 and he complex
ansloca es o he nucleus whe e i egula es he exp es-
sion o BMP a ge genes [7, 8]. Al e na i ely, BMP signals
a e also media ed h ough he ac i a ion o ERK, JNK and
p38 mi ogen-ac i a ed p o ein kinase pa hways [7, 8].
The unc ional consequences o BMP signaling depend
on he BMP ligand and issue ype. We and o he s ha e
shown ha BMP4 educes he p oli e a ion o b eas can-
ce cell lines, while simul aneously inducing mig a ion
and in asion in a subse o cell lines [9–11]. Simila
* Co espondence: [email p o ec ed]
†
Equal con ibu o s
1
BioMediTech, Uni e si y o Tampe e, Tampe e, Finland
2
Fimlab Labo a o ies, Tampe e, Finland
© The Au ho (s). 2017 Open Access This a icle is dis ibu ed unde he e ms o he C ea i e Commons A ibu ion 4.0
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ep oduc ion in any medium, p o ided you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o
he C ea i e Commons license, and indica e i changes we e made. The C ea i e Commons Public Domain Dedica ion wai e
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Ampuja e al. BMC Genomics (2017) 18:68
DOI 10.1186/s12864-016-3428-1
dualis ic e ec s upon BMP4 s imula ion ha e also been
epo ed in o he umo ypes [12]. Conco dan ly, da a
om b eas cance pa ien samples poin o a co ela ion
be ween ele a ed BMP4 le els and educed p oli e a ion
as well as an inc eased isk o ecu ence [13]. These
BMP4- ela ed e ec s ha seem ei he de imen al
( educed cell g ow h) o bene icial (inc eased mobili y)
o he cance cells a e likely o be media ed by speci ic
BMP4 a ge genes. The iden i ica ion o such a ge
genes is hus impo an since i may allow gene a ion
o e ec i e cance he apies a ge ing each pheno ype
independen ly.
We ha e p e iously sea ched o BMP4 a ge genes in
a se o b eas cance cell lines ha p edominan ly e-
spond o BMP4 ea men by educ ion o p oli e a ion
[14]. He e, we used nex -gene a ion sequencing (NGS)
echnologies (RNA-seq and DNase-seq) o unco e
BMP4-media ed ansc ip ional e en s wi h a speci ic
ocus on compa ing cells in which BMP4 has opposing e -
ec s, namely an ip oli e a i e and p omig a o y. Ou o
he nine b eas cance cell lines we ha e p e iously s ud-
ied, T-47D shows one o he mos p ominen g ow h
educ ions and MDA-MB-231 cells display he mos o e
induc ion o mig a ion [9, 10], and we e hus selec ed o
his s udy.
RNA-seqme hodquan i ies hele elo geneexp es-
sion ac oss he genome [15] while DNase-seq allows
iden i ica ion o open ch oma in egions ha a e sensi-
i e o diges ion by he DNase I endonuclease [16].
Open ch oma in egions a e conside ed as si es whe e
ansc ip ional egula ion can ake place since hey a e
accessible o egula o y molecules o bind and exe
hei unc ion. By combining da a om RNA-seq and
DNase-seq, and using addi ional da a analysis ools, i
was possible o iden i y candida e ansc ip ion ac o s
in ol ed in he obse ed ansc ip ional esponses. This
app oach hus p o ides he means o be e unde s and
he ansc ip ional e en s ha link BMP4 signaling and
i s esul ing pheno ypes.
Resul s
We pe o med RNA-seq and DNase-seq analyses in wo
b eas cance cell lines, T-47D and MDA-MB-231. The
cell lines we e ea ed wi h BMP4 and ehicle con ol
o 3 h, hus allowing us o speci ically ocus on ea ly e-
sponse e en s. Bo h ehicle- and BMP4- ea ed cell lines
we e sequenced (see me hods).
BMP4-elici ed ansc ip ional egula ion is highly
di e gen in he wo b eas cance cell lines wi h di e en
unc ional esponses o BMP4
Sequencing eads om RNA-seq and DNase-seq we e
aligned o he human genome and u he analyzed as de-
sc ibed in he me hods. To con i m ha he wo da ase s
we e consis en , we compa ed he ch oma in openness as
de e mined by DNase-seq signal a he ansc ip ion s a
si e (TSS) o he exp ession le el o he gene as de e -
mined by RNA-seq. As expec ed, we ound ha he in-
c eased openness o TSS globally co ela ed wi h
inc eased gene exp ession (Addi ional ile 1: Figu e S1,
Panels A and B). Howe e , he a iance is high, indica ing
ha he di e ences in he ch oma in s a e only pa ly ex-
plain gene exp ession pa e ns.
Nex we compa ed he exp ession le els om RNA-seq
be ween he ehicle- and BMP4- ea ed cells. This analysis
iden i ied 91 di e en ially exp essed genes (DEGs) in
MDA-MB-231, o which 58 we e up egula ed and 33
down egula ed (Addi ional ile 2: Table S1). In T-47D,
he e we e 203 DEGs, o which 160 we e up egula ed and
43 we e down egula ed (Addi ional ile 3: Table S2). In
o al, 10 DEGs (ATOH8,BDKRB2,BMF,GS1-124 K5.4,
ID1,ID2,ID3,SKIL,SMAD6,andSMAD9)we esha ed
by he wo cell lines and all o hem we e up egula ed ex-
cep GS1-124 K5.4 which was down egula ed in bo h cell
lines. To illus a e ha BMP4 induces ma kedly di e gen
ansc ip ional esponses in hese wo cell lines, we gene -
a ed a hea map o show he exp ession le els o he
p o ein-coding DEGs (Fig. 1a). Using he DNase-seq da a,
we examined he ch oma in s a us a he ansc ip ion
s a si es (TSSs) o hese p o ein-coding DEGs. Fo he
majo i y o he cases he ch oma in was open a he TSS
be o e BMP4 s imula ion (app oxima ely 86% o all DEGs
in bo h cell lines) (Addi ional ile 1: Figu e S1, Panels C
and D). Fo he emaining DEGs, we obse ed ei he
opening o closing o he TSS a e s imula ion o no
change in he closed ch oma in s a us (Fig. 1a). These da a
indica e ha , a his ea ly ime poin , he BMP4-induced
di e en ial exp ession mainly in ol es genes whose an-
sc ip ion does no equi e changes in he ch oma in s a us
a TSS.
The DEG lis s included a numbe o genes in ol ed
in he canonical BMP pa hway. As expec ed, ID1, ID2
and ID3, known BMP4 a ge genes, we e up egula ed
in bo h cell lines (Fig. 1b). Simila ly, he ecep o -
egula ed SMAD9 was up egula ed in bo h cell lines
whe eas no signi ican di e ence in he o he ecep o -
egula ed SMADs o SMAD4 exp ession was obse ed.
Among he inhibi o y SMADs, SMAD6wasup egu-
la ed in bo h cell lines and SMAD7 in T-47D. In
addi ion, he BMP ype I ecep o BMPR1A and nega-
i e egula o s o BMP signaling, NOG and BAMBI,
we e up egula ed in T-47D while in MDA-MB-231
hei exp ession was no signi ican ly changed (Fig. 1b).
Thus BMP4 s imula ion leads o exp ession changes
ha ing cha ac e is ics o bo h eedback and eed o wa d
loops.
We hen e alua ed whe he he di e en ially exp essed
genes pa icipa e in speci ic biological p ocesses and
Ampuja e al. BMC Genomics (2017) 18:68 Page 2 o 15
A
B
Fig. 1 The RNA-seq and DNAse-seq da a e eal cell line-speci ic esponses o BMP4. aGene exp ession le els o di e en ially exp essed
p o ein-coding genes con e ed o log2 scale a e shown o bo h cell lines and ea men s, up egula ed genes on he le and down egula ed
genes on he igh . The s a us column deno es he cell line in which he gene is di e en ially exp essed. The igh mos columns indica e he
s a us o he ch oma in a ansc ip ion s a si es (TSS) o he DEGs as measu ed by DNAse-seq. bIllus a ion o he di e en ially exp essed
componen s o he BMP signaling pa hway upon BMP4 ea men
Ampuja e al. BMC Genomics (2017) 18:68 Page 3 o 15
especially assessed whe he he non-common DEGs ha e
di e ing unc ions. To his end we used DAVID o
sea ch o GO e ms en iched in he se s o non-
common p o ein-coding DEGs. In MDA-MB-231, mos
o he en iched e ms we e ela ed o cell mig a ion
whe eas o gan de elopmen and mo phogenesis as well
as in acellula signaling we e he mos signi ican GO
e ms in T-47D (Table 1). These indings imply ha he
ansc ip ional changes a e indeed likely o explain he
dissimila i ies in he pheno ypic esponses o hese wo
cell lines o BMP4 ea men .
The ea e , we also wan ed o in es iga e whe he he
exp ession le els o DEGs could be linked wi h su i al
in b eas cance pa ien s. Fo his pu pose, we used he
da a publicly a ailable in he TCGA da abase. The e-
sul s showed ha 20 DEGs in he MDA-MB-231 and 46
DEGs in he T-47D cells associa ed wi h ei he good o
poo p ognosis (Addi ional ile 4: Tables S3 and S4). O
he nine sha ed p o ein-coding DEGs, ou (ATOH8,
ID3,SMAD6 and SMAD9) we e co ela ed wi h su i al,
all being associa ed wi h poo p ognosis.
To alida e he esul s o he RNA-seq analysis and o
ex end he scope o he s udy beyond he 3 h ime poin
in wo cell lines, qRT-PCR was used o s udy he
exp ession le els o 15 selec ed DEGs in MDA-MB-231
and T-47D cells as well as in i e addi ional b eas
cance cell lines (BT-474, HCC-1954, MCF-7, MDA-
MB-361, and MDA-MB-436) and one no mal b eas
epi helial cell line (MCF-10A) ea ed wi h BMP4 and
ehicle o 3, 6 and 24 h. The genes we e selec ed based
on hei exp ession le els and epo ed cance associ-
a ion in he li e a u e, and i e o hese we e up egu-
la ed acco ding o he RNA-seq in bo h MDA-MB-231
and T-47D. The exp ession pa e ns o he majo i y o
he genes showed simila i ies ac oss he cell line panel
and ime poin s wi h he clea excep ion o MDA-MB-
436, in which he exp ession changes we e e y limi ed
(Fig. 2). Pa icula ly he i e sha ed genes (ATOH8,ID2,
SKIL,SMAD6 and SMAD9)aswellasDLX3 we e con-
sis en ly up egula ed upon BMP4 ea men h oughou
he ime se ies hus con i ming ha hey ep esen
common BMP4 a ge genes. The emaining genes
showed mo e a iabili y wi h al e ed exp ession ypic-
ally in only wo o h ee cell lines, sugges ing ha hei
exp ession is likely o be in luenced by ac o s ha a e
cell line-speci ic.
Ch oma in landscape and dynamics ollowing BMP4
ea men
To gain mo e insigh in o he changes o ch oma in s uc-
u e du ing BMP4 ea men , we pe o med peak de ec-
ion in a genome-wide manne o iden i y he a eas o
open ch oma in. The peak de ec ion app oach was bench-
ma ked by compa ison o publicly a ailable DNase-seq
da a o uns imula ed T-47D cell line om ENCODE (see
me hods), showing ha mos o he peaks iden i ied in
ou da a a e p esen also in ENCODE samples (Addi ional
ile 5: Table S5).
A e il e ing p ocedu es (see me hods), he numbe s
o iden i ied DNase hype sensi i e si es (DHSs) in he
MDA-MB-231 cell line we e 89,830 and 97,349 in e-
hicle- and BMP4- ea ed samples, espec i ely. In T-
47D, he co esponding numbe s we e 68,000 and
73,881. To ob ain a uni ied se o peaks o bo h condi-
ions, he o e lapping DHSs we e me ged esul ing in a
o al o 106,154 DHSs in MDA-MB-231 and 110,028 in
T-47D. A e he me ging, he ac ion o sha ed DHSs
be ween BMP4 and ehicle con ol in MDA-MB-231 sam-
ples was 75% while he ac ion o unique DHSs in he e-
hicle was 9% and co espondingly in he BMP4 sample
16% (Addi ional ile 6: Figu e S2). In he T-47D cell line,
Table 1 Gene on ology analysis
Cell line GO accession GO e m Numbe o genes Adjus ed p- alue
MDA-MB-231 GO:0030334 egula ion o cell mig a ion 5 2.0 × 10
−2
GO:0030335 posi i e egula ion o cell mig a ion 4 2.3 × 10
−2
GO:2000145 egula ion o cell mo ili y 5 2.4 × 10
−2
GO:2000147 posi i e egula ion o cell mo ili y 4 2.5 × 10
−2
GO:0051272 posi i e egula ion o cellula componen mo emen 4 2.7 × 10
−2
T-47D GO:0048513 animal o gan de elopmen 45 2.6 × 10
−8
GO:0035556 in acellula signal ansduc ion 41 4.5 × 10
−8
GO:0009887 o gan mo phogenesis 22 4.0 × 10
−7
GO:0009966 egula ion o signal ansduc ion 36 4.5 × 10
−6
GO:0007166 cell su ace ecep o signaling pa hway 34 9.5 × 10
−5
The DAVID Func ional Anno a ion Tools was used o e eal signi ican ly en iched GO ca ego ies among he di e en ially exp essed p o ein-coding genes. The analysis
was done independen ly o each cell line and sha ed di e en ially exp essed genes we e omi ed. The op i e biological unc ion GO e ms a e shown
Ampuja e al. BMC Genomics (2017) 18:68 Page 4 o 15
he ac ion o sha ed DHSs be ween he wo condi-
ionswas27%whe eas he ac iono uniqueDHSsin
he ehicle was 34% and in he BMP4 sample 39%
(Addi ional ile 6: Figu e S2).
Anno a ion o he me ged DHSs o genomic ea u es
e ealed a simila dis ibu ion in he wo cell lines in he
ehicle- ea ed condi ion, wi h he la ges ac ion (>30%)
o DHSs loca ing in in ons (Fig. 3a). When compa ing
he dis ibu ions o he BMP4-induced DHSs be ween he
cell lines appa en esemblances we e also obse ed. In
bo h cell lines, he p opo ion o DHSs associa ed wi h
in onic and in e genic egions inc eased a e BMP4
s imula ion wi h a co esponding dec ease a o he gen-
omic loca ions, including he p omo e egions (Fig. 3b).
Fig. 2 Exp ession le els o selec ed BMP4 a ge genes by qRT-PCR in a b eas cance cell line panel. The exp ession le els o 15 DEGs we e measu ed
a e 3, 6 and 24 h o BMP4 ea men in he indica ed cell lines. The colo code illus a es he ela i e exp ession le els in he BMP4- ea ed sample as
compa ed o he co esponding ehicle con ol. FC = Fold change, n.a. = mRNA le el oo low o allow eliable measu emen
A
B
Fig. 3 Dis ibu ion o open ch oma in egions. Anno a ion o open ch oma in egions in MDA-MB-231 and T-47D a e (a) ehicle ea men (basal
openness) and (b) BMP4 ea men (consis ing only o he ch oma in ha opened a e BMP4 ea men )
Ampuja e al. BMC Genomics (2017) 18:68 Page 5 o 15

To assess he unc ional impac o he BMP4-induced
global changes in he ch oma in s uc u e we conduc ed
an en ichmen analysis using GREAT [17] which maps
he DHSs o pu a i e egula o y egions o genes and
conduc s a gene on ology en ichmen analysis. The e-
sul s highligh ed e.g. cell mo ili y and o gan mo phogen-
esis as en iched biological unc ions o MDA-MB-231
and T-47D, espec i ely (Addi ional ile 7: Tables S6 and
S7). These esul s a e consis en wi h hose ob ained by
en ichmen analysis o he di e en ially exp essed genes
om RNA-seq (Table 1) and he eby sugges ha , o-
ge he wi h speci ic a ge genes, BMP4-induced changes
a ch oma in le el may con ibu e o he eme gence o
he di e en BMP4-media ed pheno ypes.
T ansc ip ion ac o binding si e en ichmen analysis in
open ch oma in egions o p omo e s e eals
ansc ip ion ac o s in ol ed in BMP4 signaling
egula ion
Based on ou TSS openness analysis (Fig. 1a), a domin-
an ea u e o ou da a is ha he ch oma in o he pu a-
i e BMP4 a ge genes (iden i ied by RNA-seq) is open
al eady in ehicle- ea ed cells. This is u he suppo ed
by ou genome-wide peak analysis, whe e he p omo e
egions we e no o e ep esen ed a e he ea men
(Fig. 3b). The e o e, he al e a ions in he ch oma in
s a e only pa ially explain gene exp ession di e ences
induced by he BMP4 ea men . Howe e , di e en ial
ansc ip ion ac o binding o open p omo e s may ex-
plain he di e en esponses in he cell lines. The e o e
we pe o med ansc ip ion ac o (TF) mo i binding
analysis. To assess which TFs migh be egula o s o he
BMP4 esponse, he sequences o open ch oma in si es in
he p oximal p omo e s o up egula ed genes we e ana-
lyzed wi h a o al o 426 posi ion weigh ma ixes (PWMs),
ep esen ing 401 indi idual TFs o TF-complexes (see
me hods). Fo each TF we calcula ed an en ichmen sco e
(see me hods) o he numbe o binding si es in ei he
MDA-MB-231 o T-47D cells.
This analysis led o he iden i ica ion o candida e
egula o TFs, including mul iple membe s o he SMAD
amily o TFs, as expec ed, as well as a numbe o sha ed
common egula o TFs. To ocus on biologically ele an
candida es, we il e ed ou hose TFs ha we e no
exp essed based on ou RNA-seq da a. In addi ion, we
included only hose TFs whose binding si es (TFBSs) in
open ch oma in egions o he p omo e s o DEGs we e
en iched in one and deple ed in he o he cell line. The
op 15 TFs ha a e exp essed in bo h cell lines bu ha e
a high en ichmen sco e only in one o he cell lines a e
lis ed in Tables 2 and 3. Examples o a ge gene p o-
mo e s wi h binding mo i s o p edic ed TFs a e shown
in Fig. 4a.
Fo mo e in-dep h unc ional analysis we selec ed
pa icula TFs om he op en iched candida es using
he ollowing c i e ia: 1) a binding mo i wi h a quali y
ca ego y o A-C in he HOCOMOCO da abase, 2) ele-
ance in he con ex o ou model based on li e a u e,
3) no a highly common egula o o pa o a la ge TF
amily, and 4) high exp ession le el o he TF (>1000
eads) in a leas one cell line and di e en ial exp es-
sion be ween cell lines acco ding o he RNA-seq. The
Table 2 Top 15 ansc ip ion ac o s en iched in MDA-MB-231 cells
TF name Mo i Selec ion by: TF binding si es Re . si es Expec ed si es in e . Ra io o en ichmen Mean ead coun
MYBL2 MYBB_ 1 2, 3, 4 12 2930 6.2 1.92 2197
BACH1 BACH1_si 1, 2, 3 15 3904 8.3 1.81 531
MYC MYC_ 1 1, 2, 4 10 2698 5.7 1.74 3044
MAFK MAFK_si 2, 3 16 4428 9.4 1.70 688
RELA TF65_ 2 1, 2, 4 19 5467 11.6 1.63 1398
PPARA PPARA_ 1 1, 2, 3 9 2747 5.8 1.54 185
NFIA/B/C/X
a
1, 2, 3 15 4669 9.9 1.51
b
NFIL3 NFIL3_si 1, 2, 3 11 3494 7.4 1.48 474
FOXA2 FOXA2_ 1 1, 2, 3 36 11477 24.4 1.47 434
REL REL_do 1, 2, 3 17 5422 11.5 1.47 69
ZFHX3 ZFHX3_ 1 2, 3 46 14683 31.2 1.47 66
RXRB RXRB_ 1 1, 2, 4 20 6414 13.6 1.47 1015
SMARCC1 SMRC1_ 1 1, 4 20 6443 13.7 1.46 1478
ETV5 ETV5_ 1 2, 3 16 5199 11.1 1.45 641
NR3C1 GCR_si 1, 2, 4 15 4910 10.4 1.44 1087
The a io o en ichmen is he esul o di iding he numbe o TF binding si es by he numbe o expec ed si es. Mo i s a e de i ed om he HOCOMOCO
da abase.
a
NFIA + NFIB + NFIC + NFIX_ 2,
b
Read coun ange (51, 148, 748, 444, espec i ely). Re . e e ence
Ampuja e al. BMC Genomics (2017) 18:68 Page 6 o 15
las c i e ia was used o ensu e me hodological success
in subsequen unc ional assays. Wi h he c i e ia de-
sc ibed abo e CBFB, HIF1A, and MBD2 we e selec ed
o u he s udy. O hese, MBD2 had a la ge numbe
o binding si es in he p omo e s o ou DEGs while
binding si es o he o he wo TFs we e less widesp ead.
Inaddi ion,SMAD4wasusedasaposi i econ ol.
As SMAD4 is a known egula o o BMP signaling,
we pe o med co-occu ence analysis o he binding
si es be ween ou h ee candida e TFs and he SMAD
mo i s. We ound ha he MBD2 mo i was signi i-
can ly co-localized wi h he GC- ich SMAD4 consensus
mo i s CGCC (P= 1.1e-9), GCCGnCGC (P=1.3e-14),
and GGCGCC (P= 2e-10). As binding si es o CBFB o
HIF1A we e less equen ac oss DEGs, s a is ical signi i-
cance o co-localiza ion wi h SMAD mo i s could no be
eliably e alua ed. Howe e , we did ind se e al p omo e s
whe e SMAD binding si es co-localized wi h hese ac o s.
Silencing o selec ed TFs (SMAD4, CBFB, HIF1A, and
MBD2) was hen used o u he e alua e hei impac
on BMP4 signaling. A e 48 h o silencing, he cells
we e ea ed wi h BMP4 o 24 h and he mRNA le els
o he alida ed DEGs we e measu ed o assess whe he
he silencing in luences BMP4 a ge gene exp ession
(Fig. 4b and Addi ional ile 8: Figu e S3). Down egula-
ion o SMAD4 was able o e e se he BMP4-media ed
change in he exp ession o all he es ed a ge genes in
bo h MDA-MB-231 and T-47D cells (Fig. 4c) indica ing
ha hese exp ession changes a e indeed ansmi ed ia
he canonical BMP pa hway. Fo mos o he a ge
genes, MBD2 silencing led o ab oga ion o he BMP4-
media ed induc ion in gene exp ession in bo h cell lines.
In T-47D cells, simila da a was also ob ained o mos
o he genes upon CBFB (9/10) and HIF1A deple ion
(6/10). Howe e in MDA-MB-231, silencing o HIF1A
esul ed exclusi ely in up egula ion o he a ge genes
and bo h enhanced and diminished exp ession was seen
a e CBFB down egula ion. O no e, silencing o all o
he TFs in T-47D cells led o he enhanced exp ession
o he DLL1 gene, which was consis en wi h i being
down egula ed upon BMP4 ea men . These da a imply
ha he TFs may unc ion as ei he ep esso s o enhance s
o BMP4 a ge gene exp ession in a con ex -dependen
manne .
Discussion
We ha e p e iously cha ac e ized ansc ip ional esponses
o b eas cance cell lines o BMP4 by using mic oa ay
echnology[14].Howe e ,in ha s udywe ocusedonly
on cells ha espond o BMP4 by educed p oli e a ion.
E o s by o he s o examine BMP signaling a ge genes
ha e concen a ed exclusi ely on non-cance ous cells
[18–20]. He e we se ou o unco e he ansc ip ional
esponses o b eas cance cell lines wi h di e en phe-
no ypes by using one cell line ha esponds o BMP4
by educed p oli e a ion (T-47D) and ano he ha e-
ac s wi h inc eased mig a ion (MDA-MB-231). Being
able o unco e he mechanisms o hese wo di e en
esponses is essen ial o he unde s anding o he ole
o BMP4 in b eas cance pa hogenesis. To his end, we
used a subs an ially new app oach o combining DNase-
seq, RNA-seq and unc ional expe imen s.
In o de o ind he ea ly media o s o BMP4 esponse,
we ea ed he cells wi h BMP4 o ehicle con ol o
Table 3 Top 15 ansc ip ion ac o s en iched in T-47D cells
TF name Mo i Selec ion by: TF binding si es Re . si es Expec ed si es in e . Ra io o en ichmen Mean ead coun
MBD2 MBD2_si 1, 2, 3, 4 101 6664 39.6 2.55 571
TFAP2A AP2A_ 2 1, 2, 3 115 10363 61.6 1.87 941
E4F1 E4F1_ 1 2, 3 18 1750 10.4 1.73 310
SP1 SP1_ 1 1, 2 392 41453 246.3 1.59 838
CUX1 CUX1_ 1 1, 2, 3 13 1462 8.7 1.50 141
E2F2 E2F2_ 1 1, 2 17 1941 11.5 1.47 215
AHR AHR_si 1, 2, 3 9 1030 6.1 1.47 791
SP2 SP2_si 1, 2 140 16512 98.1 1.43 672
CREB1 CREB1_ 1 1, 2, 3 23 2720 16.2 1.42 177
CBFB PEBB_ 1 1, 2, 3, 4 46 5461 32.4 1.42 457
ZIC2 ZIC2_ 1 1, 2, 3 46 5487 32.6 1.41 118
ZFX ZFX_ 1 1, 2, 3 127 15650 93.0 1.37 287
HIF1A HIF1A_si 1, 2, 3, 4 15 1890 11.2 1.34 1847
E2F3 E2F3_si 1, 2, 3 16 2019 12.0 1.33 322
XBP1 XBP1_ 1 1, 3, 4 12 1545 9.2 1.31 22744
The a io o en ichmen is he esul o di iding he numbe o TF binding si es by he numbe o expec ed si es. Mo i s a e de i ed om he HOCOMOCO
da abase. Re . e e ence
Ampuja e al. BMC Genomics (2017) 18:68 Page 7 o 15
3 h. A his ime poin , he canonical BMP pa hway
h ough SMAD1/5/9 is al eady ac i a ed [9]. The esul s
o RNA-seq e ealed ha he cell lines esponded o
BMP4 by up egula ing o down egula ing a se o genes
ha we e mos ly cell line-speci ic, wi h only en com-
mon DEGs iden i ied. Consis en wi h he sequencing
da a, alida ion wi h qRT-PCR ac oss mul iple ime poin s
(3, 6, and 24 h) and i e addi ional cell lines u he con-
i med in a wide con ex he exis ence o common BMP4
a ge genes as well as cell line-speci ic exp ession pa -
e ns. O he en sha ed DEGs, h ee we e known BMP4
a ge genes (ID1-3) and wo membe s o he BMP
A
BC
Fig. 4 Examples o p edic ed TFBSs and he impac o ansc ip ion ac o s on BMP4 a ge gene exp ession. aThe p edic ed binding si es o
ansc ip ion ac o s MBD2, HIF1A and CBFB a e depic ed a he p omo e s o NOG,SMAD7 and ID1 genes, espec i ely. In addi ion, known BMP- esponse
elemen s (BRE) loca ed nea he binding si es a e illus a ed. bThe TFs we e silenced and he cells we e ea ed wi h BMP4 o ehicle con ol ollowed by
measu emen o a ge gene exp ession by qRT-PCR. Examples o ela i e exp ession le els o SKIL a e HIF1A silencing in MDA-MB-231 cells ( op panel)
and NOG exp ession a e MBD2 silencing in T-47D cells (bo om panel) a e shown. cG aphical summa y o he TF silencing expe imen s. The o de o
he genes is iden ical o ha in Fig. 2. Blue colo (dec eased a ge gene exp ession) deno es TFs ha we e essen ial o a ge gene exp ession and ed
colo hose whose silencing led o enhanced a ge gene exp ession. No applicable indica es cases whe e BMP4 did no al e he baseline
gene exp ession. Da a on he DLL1 gene, which is down egula ed in T-47D upon BMP4 ea men , a e highligh ed wi h a bold line
Ampuja e al. BMC Genomics (2017) 18:68 Page 8 o 15
signaling pa hway (SMAD6,SMAD9) [3]. The ac i a-
ion o he inhibi o y SMAD6 indica es a nega i e
eedback loop, which in T-47D is ein o ced by he
up egula ion o BMP an agonis NOG and he pseudo -
ecep o BAMBI. On he o he hand, ac i a ion o he
ecep o - egula ed SMAD9 seems o poin o a posi i e
eedback loop, as alongside o he R-SMADs, SMAD9
has been ound o enhance BMP signaling [21, 22].
Howe e , one s udy indica ed ha SMAD9 may ha e
an inhibi o y ole in BMP signaling [23]. In any case,
up egula ion o SMAD9 due o BMP4 ea men has
also been ecen ly epo ed in a ious cell ypes, o
example in p ima y ib oblas s, hepa ocellula ca cin-
oma and melanoma cells [24].
To unde s and he unc ion o he cell line-speci ic
DEGs, we used GO analysis o seg ega e he DEGs in o
biological p ocess ca ego ies and disco e ed ha he e-
sul s e lec ed he esponse o he cell lines o BMP4.
P ocesses ela ed o mig a ion we e en iched in he
MDA-MB-231 cells, whe eas mo e di e se esponses
we e ound in T-47D, including ca ego ies comp ised o
signaling, de elopmen and mo phogenesis. These ind-
ings we e co obo a ed by he DNase-seq da a, whe e
we ound ha BMP4-induced global open ch oma in
si es we e en iched wi h he same biological ca ego ies
ha we e ound wi h RNA-seq da a. While ca ego ies
associa ed wi h signaling we e obse ed in bo h cell
lines, in MDA-MB-231 hose ela ed o mig a ion we e
en iched. These da a ex end ou p e ious esul s show-
ing en ichmen o di e en ially exp essed genes in GO
ca ego ies ha we e associa ed wi h he BMP4-induced
dec ease in p oli e a ion [14]. Taken oge he , he di e -
en esponses o he cell lines o BMP4 a e e lec ed
bo h a he ansc ip ional and ch oma in le els.
In he analysis o TSS ch oma in s a e we could
obse e changes in only a ew o he genes ha we e di -
e en ially egula ed by BMP4. This migh be due o he
ac ha he 3 h s imula ion o BMP4 is oo sho o
mos o he TSSs o change hei ch oma in s a us.
Mo eo e , we could obse e ha in many cases he
ch oma in was al eady open a he TSS, in which case
u he changes a e no needed o enhance he ansc ip-
ional ac i i y. Toge he wi h he obse a ion ha he e
is a la ge a ia ion be ween he ch oma in s a us and
gene exp ession when we ex end he analysis o he
whole se o p o ein-coding genes, i can be concluded
ha he ch oma in s a e o TSSs explains he obse ed
exp ession pa e ns only o a small ex en . This esul
was no unexpec ed, as gene exp ession is also com-
monly egula ed om egions loca ed a om he TSS,
such as enhance s [25, 26].
Wi h genome-wide de ec ion o open ch oma in a eas
we no iced ha BMP4 s imula ion induces opening o he
ch oma in mos ly in he in onic and in e genic egions.
This is consis en wi h he ac ha changes in he TSS
and p omo e egions we e obse ed wi h only a ew o
he di e en ially exp essed genes. Opening o he in-
onic sequences may indica e inc eased le el o RNA
polyme ase ac i i y a gene bodies. Ch oma in opening
a in e genic egions migh sugges ha addi ional
egula o y con ol is being a ained in la ge ex en
h ough dis al egula o y elemen s such as enhance s
and silence s. Thus, al eady a he ea ly 3-h ime poin
we a e able o obse e con o ma ional changes ha
cells may u ilize in mo e de ailed egula ion o he
BMP esponse. Un o una ely, based on his analysis we
we e no able o de ine a speci ic ansc ip ion ac o
ch oma in signa u e ha could be used o de ine BMP-
speci ic egula o y sequences. Hence de ailed analysis
o he pu a i e enhance egions would equi e mo e
speci ic measu emen da a abou he ch oma in in e -
ac ions in hese cells.
To u he cha ac e ize he egula ion o BMP4 a ge
genes, we analyzed ansc ip ion ac o binding si es
(TFBSs) in he open ch oma in egions loca ed on gene
p omo e s. Among he op 15 en iched TFs, he e we e a
ew which had p e iously been linked o BMP a ge gene
egula ion. Fo example, XBP1 and RELA ha e been
shown o be ep esso s o BMP a ge genes X en -2 and
Id1, espec i ely [27, 28]. Using en ichmen o he TFBSs
be ween cell lines as well as o he c i e ia, we selec ed
h ee TFs (CBFB, HIF1A, and MBD2) o unc ional
cha ac e iza ion and silenced hem in he wo cell lines. In
addi ion, we used SMAD4, a key componen o he ca-
nonical BMP pa hway, as ou posi i e con ol and indeed
SMAD4 was equi ed o ansc ip ional egula ion o all
he BMP4 a ge genes in he assay. Al hough BMPs can
signal h ough al e na i e pa hways [7, 8], his esul
poin s o egula ion h ough he canonical pa hway. In
con as , he esponse o o he ansc ip ion ac o s was
mo e a iable and cell line-speci ic.
MBD2 is a me hyl-CpG-binding ansc ip ion ac o
ha plays a ole in de elopmen [29, 30]. Se e al s udies
ha e shown ha MBD2 ac s as a ansc ip ional ep es-
so by ec ui ing co- ep esso complexes o p omo e s,
which in u n leads o o ma ion o ep essi e ch oma in
h ough ch oma in emodelling [31, 32]. Howe e , he e
is also e idence ha MBD2 can ac i a e ansc ip ion by
emo ing me hyla ion om CpG islands loca ed in p o-
mo e s [33]. In bo h cell lines, MBD2 seemed o ac
mainly as an ac i a o o ansc ip ion, al hough i s ole
was mo e p ominen in MDA-MB-231. In ou analysis,
MBD2 had a la ge numbe o binding si es ac oss DEGs
and i was highly exp essed in bo h cell lines, consis en
wi h he obse ed beha io in he silencing expe imen .
The key ole o MBD2 in con olling he BMP4 esponse
sugges s ha DNA me hyla ion may be in ol ed in BMP4
signaling.
Ampuja e al. BMC Genomics (2017) 18:68 Page 9 o 15