In e na ional Jou nal o
Molecula Sciences
A icle
The In luence o Quad uplex S uc u e in P oximi y o
P53 Ta ge Sequences on he T ansac i a ion Po en ial
o P53 Alpha Iso o ms
O ília Po ubiako á1,2,†, Na ália Bohálo á2,3,†, Albe o Inga 4, Na ália Vado iˇco á2,5,
Jan Cou al 2, Mi osla Foj a 2and Václa B ázda 1,2,*
1Facul y o Chemis y, B no Uni e si y o Technology, Pu kyˇno a 118, 61200 B no, Czech Republic;
[email p o ec ed]
2Ins i u e o Biophysics, Academy o Sciences o he Czech Republic, K álo opolská135, 61265 B no,
Czech Republic; [email p o ec ed] (N.B.); [email p o ec ed] (N.V.); [email p o ec ed] (J.C.);
[email p o ec ed] (M.F.)
3Depa men o Expe imen al Biology, Facul y o Science, Masa yk Uni e si y, Kamenice 5, 62500 B no,
Czech Republic
4Labo a o y o T ansc ip ional Ne wo ks, Depa men CIBIO, Uni e si y o T en o, ia Somma i e 9,
38123 T en o, I aly; [email p o ec ed]
5Depa men o Biology, Facul y o Medicine, Masa yk Uni e si y, Kamenice 5, 62500 B no, Czech Republic
*Co espondence: [email p o ec ed]; Tel.: +420541517231; Fax: +420541211293
†These au ho s con ibu ed equally o his wo k.
Recei ed: 30 Oc obe 2019; Accep ed: 23 Decembe 2019; Published: 24 Decembe 2019
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Abs ac :
p53 is one o he mos s udied umo supp esso p o eins ha plays an impo an ole in
basic biological p ocesses including cell cycle, DNA damage esponse, apop osis, and senescence.
The human TP53 gene con ains al e na i e p omo e s ha p oduce N- e minally unca ed p o eins
and can p oduce se e al iso o ms due o al e na i e splicing. p53 unc ion is ealized by binding
o a speci ic DNA esponse elemen (RE), esul ing in he ansac i a ion o a ge genes. He e, we
e alua ed hein luenceo quad uplexDNAs uc u eon he ansac i a ionpo en ialo ull-leng hand
N- e minal unca ed p53
α
iso o ms in a panel o S. ce e isiae luci e ase epo e s ains. Ou esul s
show ha a G-quad uplex p one sequence is no su icien o ansc ip ion ac i a ion by p53
α
iso o ms, bu he p esence o his ea u e in p oximi y o a p53 RE leads o a signi ican educ ion o
ansc ip ional ac i i y and changes he dynamics be ween co-exp essed p53αiso o ms.
Keywo ds: p53 p o ein; p o ein-DNA in e ac ion; ansac i a ion po en ial
1. In oduc ion
The umo supp esso p o ein, p53, is called he “gua dian o he genome” due o i s c ucial ole
in main aining gene ic s abili y and inhibi ing cance o ma ion [
1
,
2
]. To exe his ole, once ac i a ed
a e cell inju y, p53 induces a numbe o cellula p ocesses, esul ing in cell epai and su i al o in
p og ammed cell dea h [
3
–
5
]. The canonical p53 p o ein, also named p53
α
, FLp53
α
, o TAp53alpha
(he ea e e e ed o as FLp53
α
), was he i s iden i ied p53 o m [
6
]. Human FLp53
α
is 393-amino
acids long and has se en unc ional domains. The N- e minal domain con ains wo ansac i a ion
(TA) domains, which a e equi ed o induce a dis inc subse o p53- a ge genes. O he domains a e a
p oline- ich domain (PRD), a DNA-binding domain (DBD), a hinge domain (HD), and a C- e minal
domain composed o an oligome iza ion domain (OD) and a nega i e egula ion domain (
α
) [
7
].
The nega i e egula ion domain is ich in lysine and unde goes many pos ansla ional modi ica ions
ha egula e FLp53
α
ac i i y and s abili y [
8
]. The DBD con ains se e al conse ed cys eines
In . J. Mol. Sci. 2020,21, 127; doi:10.3390/ijms21010127 www.mdpi.com/jou nal/ijms
In . J. Mol. Sci. 2020,21, 127 2 o 12
and his idines ha coo dina e Zn
2+
o Mg
2+
ions, which a e essen ial o FLp53
α
con o ma ion and
DNA-binding ac i i y [
9
]. Di e en N- e minal iso o ms o p53
α
ha e been iden i ied due o al e na i e
ansla ion ini ia ion, splicing si es, o al e na i e p omo e usage:
∆
40p53
α
,
∆
133p53
α
, and
∆
160p53
α
lack he 39, 132, and 159 N- e minal amino acids, espec i ely, compa ed wi h FLp53
α
[
10
,
11
]. As a
consequence,
∆
40p53
α
lacks one o he wo TA domains while he o he wo iso o ms lack bo h TA and
he PR domains, plus pa o he conse ed cys eine box in he DBD [
12
]. Based on expe imen s o e he
pas en yea s, i has been shown ha p53 iso o ms a e physiologically ac i e p o eins. Mis egula ion
o p53 iso o m exp ession can lead o cance , p ema u e aging, neu odegene a i e diseases, o e en
emb yo mal o ma ions [13,14].
p53 is pa o an ex ensi e ansc ip ional ne wo k ha coo dina es he esponse o in acellula
and ex acellula s esses o damage [5]. The main unc ion o p53 is p o ided by i s in e ac ion wi h
DNA [
15
–
19
]. p53 egula es a ge gene exp ession mainly by ac i a ion o p53- esponsi e p omo e s.
The DNA esponse elemen (RE) o p53 binding comp ises wo copies o a 5
0
- RRRC(A/T)(T/A)GYYY-3
0
sequence [
15
,
20
,
21
] accommoda ing he binding o wo p53 dime s o o m a p53 e ame ha is
conside ed he unc ional uni o ansc ip ional modula ion [
16
]. The domain esponsible o
sequence-speci ic DNA binding is he co e DBD, e en hough he OD is c i ical o e ame o ma ion
and modi ica ions o he C- e minal domain in luence binding a ini y and speci ici y [
22
]. p53-DNA
in e ac ions wi h p53 REs a e sensi i e o DNA opology and his is a key pa ame e con ibu ing o
p53-DNA a ini y and speci ici y [
18
,
23
]. I was demons a ed ha p53 also binds o a ious local DNA
s uc u es s abilized by DNA opological s ess such as c uci o ms [
24
,
25
], quad uplex [
26
], iplex [
27
],
bulged [28], and hemica ena e [29] DNAs.
The unicellula yeas Saccha omyces ce e isiae has been p e iously employed o s udy he
ansc ip ional ac i i y o many human ansc ip ion ac o s including p53 and i s iso o ms [
30
–
32
].
He e, we ha e enginee ed yeas epo e s ains o s udy he impac o posi ioning a G-quad uplex (G4)
p one sequence alone o in p oximi y (ups eam o downs eam) o a p53 RE on he ansac i a ion
induced by FLp53
α
and he N- e minally unca ed iso o ms (
∆
40p53
α
,
∆
133p53
α
, and
∆
160p53
α
),
exp essed bo h indi idually and in combina ion.
In pa icula , we in es iga ed whe he G4 p one sequences a e capable o inducing p53-dependen
ansac i a ion pe se, and/o whe he hey modi y ansc ip ion when p esen in close p oximi y o an
es ablished p53 binding si e. We also in es iga ed whe he G4 p one sequences impac on he c oss alk
be ween co-exp essed p53 iso o ms and mapped he p esence o G4 o ming sequences nea by p53
PUMA RE in genomic con ex . Ou esul s u he emphasize he po en ial ole o s uc u al DNA
ea u es as modi ie s o p53 p o ein unc ions a a ge p omo e si es.
2. Resul s
2.1. Cons uc ion o Isogenic Yeas S ains
To elucida e he in luence o a G4 on p53
α
ansc ip ional ac i i y, we exploi ed yeas isogenic
epo e s. We used he ollowing G- ich DNA sequence GGGGCGGGGGACGGGGGAGGGG, which
is e y highly p one o o m a G4, based on he p opensi y sco e gi en by he G4Hun e ool [
33
,
34
]
(G4Hun e sco e 3.182), which is e en highe han he sequence om he c-Myc p omo e egion
(G4Hun e sco e 2.941) whe e he p esence o he G4 s uc u e has been e alua ed bo h
in i o
and
in i o
[
35
,
36
]. We con i med he p opensi y o his sequence o o m G4 by CD spec oscopy (Figu e 1).
The measu emen s showed ha he G- ich sequence o ms a hyb id ype o G4 wi h dominan pa allel
G4 ep esen ed by he peak a 264 nm and an an ipa allel G4 s uc u e esul ing in he seconda y peak
a 295 nm. The slow d op o o he cu e a e he ypical 264 nm peak is in keeping wi h he e idence
ha opologically di e en G4 in e media es may coexis [
37
,
38
]. Sequences wi h an addi ional PUMA
p53RE egion showed highe p e e ence o he an ipa allel G4 s uc u e wi h a mo e p ominen peak
a ound 295 nm.
In . J. Mol. Sci. 2020,21, 127 3 o 12
In . J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 3 o 12
Figu e 1. Ci cula Dich oism }CD] spec oscopy o used DNA sequences. CD spec a o he bu e
(ligh blue), and oligonucleo ides om he Table 1 (G4, blue, PUMA- ed, G4-PUMA-g een, PUMA-
G4 iole ).
Nex , we in eg a ed he p53 RE de i ed om he human PUMA/BBC3 p omo e and he G4
sequence alone o combined ups eam o a minimal p omo e d i ing he luci e ase epo e gene a
he ade2 locus in yeas . Two e sions o he combined elemen we e cons uc ed, di e ing in he
posi ion o he G4 sequence ei he ups eam o downs eam o he p53 RE (Figu e 2).
Figu e 2. Scheme o he es ed sequences in he luci e ase epo e p omo e egion.
2.2. T ansac i a ion Ac i i y o p53α
The epo e yeas s ains we e used o measu e he ansac i a ion po en ial o ou p53α
iso o ms. Fi s , exploi ing he galac ose inducible sys em o con ol p53 exp ession, we analyzed he
le el o ansc ip ion o he epo e in he p esence o he PUMA p53RE wi hou galac ose and wi h
0.2% o 2% galac ose. The esul s showed ha bo h FLp53α and ∆40p53α ansac i a e he epo e ,
al hough o di e en ex en s (Figu e 3). Inc easing he amoun o galac ose led o a p opo ional
inc ease in ansac i a ion o bo h iso o ms. The ∆133α and ∆160α iso o ms did no induce
ansac i a ion o he PUMA p53 RE.
Figu e 1.
Ci cula Dich oism }CD] spec oscopy o used DNA sequences. CD spec a o he
bu e (ligh blue), and oligonucleo ides om he Table 1(G4, blue, PUMA- ed, G4-PUMA-g een,
PUMA-G4 iole ).
Nex , we in eg a ed he p53 RE de i ed om he human PUMA/BBC3 p omo e and he G4
sequence alone o combined ups eam o a minimal p omo e d i ing he luci e ase epo e gene
a he ade2 locus in yeas . Two e sions o he combined elemen we e cons uc ed, di e ing in he
posi ion o he G4 sequence ei he ups eam o downs eam o he p53 RE (Figu e 2).
In . J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 3 o 12
Figu e 1. Ci cula Dich oism }CD] spec oscopy o used DNA sequences. CD spec a o he bu e
(ligh blue), and oligonucleo ides om he Table 1 (G4, blue, PUMA- ed, G4-PUMA-g een, PUMA-
G4 iole ).
Nex , we in eg a ed he p53 RE de i ed om he human PUMA/BBC3 p omo e and he G4
sequence alone o combined ups eam o a minimal p omo e d i ing he luci e ase epo e gene a
he ade2 locus in yeas . Two e sions o he combined elemen we e cons uc ed, di e ing in he
posi ion o he G4 sequence ei he ups eam o downs eam o he p53 RE (Figu e 2).
Figu e 2. Scheme o he es ed sequences in he luci e ase epo e p omo e egion.
2.2. T ansac i a ion Ac i i y o p53α
The epo e yeas s ains we e used o measu e he ansac i a ion po en ial o ou p53α
iso o ms. Fi s , exploi ing he galac ose inducible sys em o con ol p53 exp ession, we analyzed he
le el o ansc ip ion o he epo e in he p esence o he PUMA p53RE wi hou galac ose and wi h
0.2% o 2% galac ose. The esul s showed ha bo h FLp53α and ∆40p53α ansac i a e he epo e ,
al hough o di e en ex en s (Figu e 3). Inc easing he amoun o galac ose led o a p opo ional
inc ease in ansac i a ion o bo h iso o ms. The ∆133α and ∆160α iso o ms did no induce
ansac i a ion o he PUMA p53 RE.
Figu e 2. Scheme o he es ed sequences in he luci e ase epo e p omo e egion.
2.2. T ansac i a ion Ac i i y o p53α
The epo e yeas s ains we e used o measu e he ansac i a ion po en ial o ou p53
α
iso o ms.
Fi s , exploi ing he galac ose inducible sys em o con ol p53 exp ession, we analyzed he le el o
ansc ip ion o he epo e in he p esence o he PUMA p53RE wi hou galac ose and wi h 0.2% o
2% galac ose. The esul s showed ha bo h FLp53
α
and
∆
40p53
α
ansac i a e he epo e , al hough
o di e en ex en s (Figu e 3). Inc easing he amoun o galac ose led o a p opo ional inc ease in
ansac i a ion o bo h iso o ms. The
∆
133
α
and
∆
160
α
iso o ms did no induce ansac i a ion o he
PUMA p53 RE.
In . J. Mol. Sci. 2020,21, 127 4 o 12
In . J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 4 o 12
Figu e 3. p53-dependen ansac i a ion po en ial in yeas . All p53α iso o ms a e exp essed unde an
inducible GAL1 p omo e . His og ams show he a e age old induc ion o e emp y ec o in h ee
biological eplica es (mean ± S.D.). The esul s wi h h ee le els o p53 induc ion (no induc ion,
mode a e, high) ob ained a e 24 h in inducing media a e p esen ed. As e isks indica e a signi ican
induc ion o p53 dependen ansac i a ion (p < 0.05).
Simila ly, he ansac i a ion po en ial o cons i u i ely exp essed p53 (GPD p omo e ) was
signi ican ly highe o he FLp53α iso o m compa ed o he ∆40p53α iso o m, while ∆133 and ∆160
iso o ms we e no able o ansac i a e he epo e (Figu e 4).
Figu e 4. p53-dependen ansac i a ion po en ial in yeas . All p53α iso o ms a e exp essed unde a
cons i u i e glyce aldehyde-3-phospha e dehyd ogenase (GPD) p omo e . The esul s o he
indica ed p53α iso o ms ob ained a e 24 h in media wi hou induc ion a e p esen ed. As e isks
indica e a signi ican induc ion o p53 dependen ansac i a ion (p < 0.05).
To elucida e he ole o G4 s uc u e on he ansc ip ional ac i i y p53α iso o ms, we es ed
h ee addi ional yeas isogenic s ains con aining he G4 alone o combina ions o he p53 RE wi h
he G4 sequence ups eam o downs eam. All s ains we e co- ans o med so ha he ac i i y o
FLp53α exp essed alone o combined wi h he o he p53α iso o ms could be assessed in he a ious
Figu e 3.
p53-dependen ansac i a ion po en ial in yeas . All p53
α
iso o ms a e exp essed unde
an inducible GAL1 p omo e . His og ams show he a e age old induc ion o e emp y ec o in
h ee biological eplica es (mean
±
S.D.). The esul s wi h h ee le els o p53 induc ion (no induc ion,
mode a e, high) ob ained a e 24 h in inducing media a e p esen ed. As e isks indica e a signi ican
induc ion o p53 dependen ansac i a ion (p<0.05).
Simila ly, he ansac i a ion po en ial o cons i u i ely exp essed p53 (GPD p omo e ) was
signi ican ly highe o he FLp53
α
iso o m compa ed o he
∆
40p53
α
iso o m, while
∆
133 and
∆
160
iso o ms we e no able o ansac i a e he epo e (Figu e 4).
In . J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 4 o 12
Figu e 3. p53-dependen ansac i a ion po en ial in yeas . All p53α iso o ms a e exp essed unde an
inducible GAL1 p omo e . His og ams show he a e age old induc ion o e emp y ec o in h ee
biological eplica es (mean ± S.D.). The esul s wi h h ee le els o p53 induc ion (no induc ion,
mode a e, high) ob ained a e 24 h in inducing media a e p esen ed. As e isks indica e a signi ican
induc ion o p53 dependen ansac i a ion (p < 0.05).
Simila ly, he ansac i a ion po en ial o cons i u i ely exp essed p53 (GPD p omo e ) was
signi ican ly highe o he FLp53α iso o m compa ed o he ∆40p53α iso o m, while ∆133 and ∆160
iso o ms we e no able o ansac i a e he epo e (Figu e 4).
Figu e 4. p53-dependen ansac i a ion po en ial in yeas . All p53α iso o ms a e exp essed unde a
cons i u i e glyce aldehyde-3-phospha e dehyd ogenase (GPD) p omo e . The esul s o he
indica ed p53α iso o ms ob ained a e 24 h in media wi hou induc ion a e p esen ed. As e isks
indica e a signi ican induc ion o p53 dependen ansac i a ion (p < 0.05).
To elucida e he ole o G4 s uc u e on he ansc ip ional ac i i y p53α iso o ms, we es ed
h ee addi ional yeas isogenic s ains con aining he G4 alone o combina ions o he p53 RE wi h
he G4 sequence ups eam o downs eam. All s ains we e co- ans o med so ha he ac i i y o
FLp53α exp essed alone o combined wi h he o he p53α iso o ms could be assessed in he a ious
Figu e 4.
p53-dependen ansac i a ion po en ial in yeas . All p53
α
iso o ms a e exp essed unde a
cons i u i e glyce aldehyde-3-phospha e dehyd ogenase (GPD) p omo e . The esul s o he indica ed
p53
α
iso o ms ob ained a e 24 h in media wi hou induc ion a e p esen ed. As e isks indica e a
signi ican induc ion o p53 dependen ansac i a ion (p<0.05).
In . J. Mol. Sci. 2020,21, 127 5 o 12
To elucida e he ole o G4 s uc u e on he ansc ip ional ac i i y p53
α
iso o ms, we es ed
h ee addi ional yeas isogenic s ains con aining he G4 alone o combina ions o he p53 RE wi h
he G4 sequence ups eam o downs eam. All s ains we e co- ans o med so ha he ac i i y
o FLp53
α
exp essed alone o combined wi h he o he p53
α
iso o ms could be assessed in he
a ious epo e s ains. FLp53
α
was exp essed unde he cons i u i e GPD p omo e while
∆
p53
α
iso o ms we e unde he GAL1 p omo e and we e exp essed bo h a mode a e (Figu e 5A) and
high le els (Figu e 5B). Pe o ming wes e n blo o p53 iso o ms is challenging due o he lack o
comme cially a ailable iso o m-speci ic an ibodies, bu wes e n blo wi h he DO-1 an ibody ha
de ec s an N- e minal epi ope ( esidues 11–25) in FLp53
α
has shown ha exp ession o ull-leng h
p53 by he cons i u i e GPD p omo e in yeas was no d ama ically a ec ed by he co-selec ion o
exp ession plasmids o p53
α
iso o ms (Figu e S1). FLp53
α
induced ansac i a ion in he s ain wi h
jus he p53 RE ups eam o he luci e ase epo e , bu had no ansac i a ion ac i i y on G4 alone.
The ansac i a ional ac i i y o FLp53
α
was a ec ed by he G4 sequence placed ei he ups eam
o downs eam o he p53RE. In e es ingly, he p esence o he G4 in close p oximi y o he p53 RE
dec eased p53-dependen ansac i a ion (Figu e 5, ed ba s), bu he posi ion o he G4 sequence
in luenced his e ec . The inhibi o y e ec was g ea e wi h he G4 inse ed a e he p53 RE (i.e., close
o he TSS) han when he G4 was posi ioned ups eam o he p53 RE. None o he
∆
p53
α
iso o ms
impac ed he low ansc ip ion ac i i y o he epo e con aining he G4 sequence only. In he p53
PUMA RE epo e s ain,
∆
160p53
α
dec eased ansac i a ion by FLp53
α
, pa icula ly when exp essed
a high le els (Figu e 5B). Such a dec ease was no obse ed wi h
∆
40p53
α
(consis en wi h he esidual
ansac i a ion po en ial o his iso o m), bu i sligh ly po en ia ed FLp53
α
ansac i a ion ac i i y.
Howe e , placing he G4 sequence downs eam o he p53 RE led o changes in he appa en unc ional
in e ac ion be ween co-exp essed p53
α
iso o ms, and
∆
40p53
α
gained an inhibi o y e ec o e FLp53
α
,
while
∆
133p53
α
and
∆
160p53
α
los ha p ope y. Indeed, when exp essed alone,
∆
40p53
α
was
impac ed by he p esence o he G4 sequence in a manne simila o FLp53α(Figu e 6).
In . J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 5 o 12
epo e s ains. FLp53α was exp essed unde he cons i u i e GPD p omo e while ∆p53α iso o ms
we e unde he GAL1 p omo e and we e exp essed bo h a mode a e (Figu e 5A) and high le els
(Figu e 5B). Pe o ming wes e n blo o p53 iso o ms is challenging due o he lack o comme cially
a ailable iso o m-speci ic an ibodies, bu wes e n blo wi h he DO-1 an ibody ha de ec s an N-
e minal epi ope ( esidues 11–25) in FLp53α has shown ha exp ession o ull-leng h p53 by he
cons i u i e GPD p omo e in yeas was no d ama ically a ec ed by he co-selec ion o exp ession
plasmids o p53α iso o ms (Figu e S1). FLp53α induced ansac i a ion in he s ain wi h jus he
p53 RE ups eam o he luci e ase epo e , bu had no ansac i a ion ac i i y on G4 alone. The
ansac i a ional ac i i y o FLp53α was a ec ed by he G4 sequence placed ei he ups eam o
downs eam o he p53RE. In e es ingly, he p esence o he G4 in close p oximi y o he p53 RE
dec eased p53-dependen ansac i a ion (Figu e 5, ed ba s), bu he posi ion o he G4 sequence
in luenced his e ec . The inhibi o y e ec was g ea e wi h he G4 inse ed a e he p53 RE (i.e.,
close o he TSS) han when he G4 was posi ioned ups eam o he p53 RE. None o he ∆p53α
iso o ms impac ed he low ansc ip ion ac i i y o he epo e con aining he G4 sequence only. In
he p53 PUMA RE epo e s ain, ∆160p53α dec eased ansac i a ion by FLp53α, pa icula ly when
exp essed a high le els (Figu e 5B). Such a dec ease was no obse ed wi h ∆40p53α (consis en wi h
he esidual ansac i a ion po en ial o his iso o m), bu i sligh ly po en ia ed FLp53α
ansac i a ion ac i i y. Howe e , placing he G4 sequence downs eam o he p53 RE led o changes
in he appa en unc ional in e ac ion be ween co-exp essed p53α iso o ms, and ∆40p53α gained an
inhibi o y e ec o e FLp53α, while ∆133p53α and ∆160p53α los ha p ope y. Indeed, when
exp essed alone, ∆40p53α was impac ed by he p esence o he G4 sequence in a manne simila o
FLp53α (Figu e 6).
Figu e 5. Con .

In . J. Mol. Sci. 2020,21, 127 6 o 12
In . J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 6 o 12
Figu e 5. In luence o ∆p53α iso o ms wi h inducible exp ession on ansac i a ion ac i i y o
cons i u i ely exp essed FLp53α. (A) in media wi h 0.2 % galac ose; (B) in media wi h 2% galac ose.
Fou isogenic yeas s ains we e used, wi h he p53 a ge si e (PUMA), wi h he p53 a ge si e a e
G4 o ming sequence (G4-PUMA), wi h he p53 a ge si e be o e G-quad uplex o ming sequence
(PUMA-G4), and he G4 o ming sequence ups eam o he luci e ase gene. As e isks indica e a
signi ican induc ion o p53-dependen ansac i a ion (p < 0.05).
Figu e 6. Impac o he G4 p one sequence on Δ40p53α ansac i a ion ac i i y om he PUMA RE.
Δ40p53α was exp essed unde an inducible GAL1 p omo e . His og ams show a e age old induc ion
o e emp y ec o in h ee biological eplica es (mean ± S.D.). The esul s ob ained a e 24 h in 2%
galac ose inducing media a e p esen ed. As e isks indica e a signi ican induc ion o p53-dependen
ansac i a ion (p < 0.05).
Figu e 5.
In luence o
∆
p53
α
iso o ms wi h inducible exp ession on ansac i a ion ac i i y o
cons i u i ely exp essed FLp53
α
. (
A
) in media wi h 0.2 % galac ose; (
B
) in media wi h 2% galac ose.
Fou isogenic yeas s ains we e used, wi h he p53 a ge si e (PUMA), wi h he p53 a ge si e a e
G4 o ming sequence (G4-PUMA), wi h he p53 a ge si e be o e G-quad uplex o ming sequence
(PUMA-G4), and he G4 o ming sequence ups eam o he luci e ase gene. As e isks indica e a
signi ican induc ion o p53-dependen ansac i a ion (p<0.05).
In . J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 6 o 12
Figu e 5. In luence o ∆p53α iso o ms wi h inducible exp ession on ansac i a ion ac i i y o
cons i u i ely exp essed FLp53α. (A) in media wi h 0.2 % galac ose; (B) in media wi h 2% galac ose.
Fou isogenic yeas s ains we e used, wi h he p53 a ge si e (PUMA), wi h he p53 a ge si e a e
G4 o ming sequence (G4-PUMA), wi h he p53 a ge si e be o e G-quad uplex o ming sequence
(PUMA-G4), and he G4 o ming sequence ups eam o he luci e ase gene. As e isks indica e a
signi ican induc ion o p53-dependen ansac i a ion (p < 0.05).
Figu e 6. Impac o he G4 p one sequence on Δ40p53α ansac i a ion ac i i y om he PUMA RE.
Δ40p53α was exp essed unde an inducible GAL1 p omo e . His og ams show a e age old induc ion
o e emp y ec o in h ee biological eplica es (mean ± S.D.). The esul s ob ained a e 24 h in 2%
galac ose inducing media a e p esen ed. As e isks indica e a signi ican induc ion o p53-dependen
ansac i a ion (p < 0.05).
Figu e 6.
Impac o he G4 p one sequence on
∆
40p53
α
ansac i a ion ac i i y om he PUMA RE.
∆
40p53
α
was exp essed unde an inducible GAL1 p omo e . His og ams show a e age old induc ion
o e emp y ec o in h ee biological eplica es (mean
±
S.D.). The esul s ob ained a e 24 h in 2%
galac ose inducing media a e p esen ed. As e isks indica e a signi ican induc ion o p53-dependen
ansac i a ion (p<0.05).
In . J. Mol. Sci. 2020,21, 127 7 o 12
3. Discussion
p53 is a ansc ip ion ac o ha ecognizes a 20-bp long DNA mo i . Howe e , ch oma in
immunop ecipi a ion has shown ha many p53 a ge s do no con ain a classical ull-leng h p53 RE,
bu can be o med by hal -si e [
21
], o do no con ain classical a ge sequences [
39
]. Non-canonical DNA
mo i s a e ansc ip ionally ac i e o wild ype and mu an p53 p o eins [
40
] and local DNA s uc u es
a e impo an de e minan s o p o ein-DNA binding [
41
]. Recen ly, he in e ac ion o p53 wi h G4s
has been demons a ed [
26
]. E en i i was demons a ed ha G4 s uc u es a e o en loca ed in gene
egula o y sequences in he human genome [
42
] and he e a e many s udies o p53 a ge genes [
16
,
39
],
a combined s udy o bo h ea u es is missing. The e o e, we pe o med addi ional analyses o 100 bp
sequence su ounding he p53- a ge sequence in he PUMA gene p omo e . In e es ingly, he e a e
se e al po en ial G4-p one sequences in close p oximi y o he PUMA p53- a ge sequence (Figu e 7).
The G4-p one sequence is loca ed igh ly be o e p53 RE (
−
33 o
−
1 be o e p53 RE, max. G4Hun e sco e
in his a ea 1.84) and se e al G4-p one sequences a e loca ed a e he p53 RE including a G4Hun e
sco e o 1.32 immedia ely a e he p53 RE—loca ion 0–25—and ano he u he downs eam (s a ing
ei he 21, 45, and 58 nucleo ides a e he p53 RE; highes G4Hun e sco e o 3.2 o he sequence:
GGGGGCGGGG CGGGGCGGGG CGGGG, peak a 71 nucleo ides a e p53 RE).
In . J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 7 o 12
3. Discussion
p53 is a ansc ip ion ac o ha ecognizes a 20-bp long DNA mo i . Howe e , ch oma in
immunop ecipi a ion has shown ha many p53 a ge s do no con ain a classical ull-leng h p53 RE,
bu can be o med by hal -si e [21], o do no con ain classical a ge sequences [39]. Non-canonical
DNA mo i s a e ansc ip ionally ac i e o wild ype and mu an p53 p o eins [40] and local DNA
s uc u es a e impo an de e minan s o p o ein-DNA binding [41]. Recen ly, he in e ac ion o p53
wi h G4s has been demons a ed [26]. E en i i was demons a ed ha G4 s uc u es a e o en loca ed
in gene egula o y sequences in he human genome [42] and he e a e many s udies o p53 a ge genes
[16,39], a combined s udy o bo h ea u es is missing. The e o e, we pe o med addi ional analyses o
100 bp sequence su ounding he p53- a ge sequence in he PUMA gene p omo e . In e es ingly, he e
a e se e al po en ial G4-p one sequences in close p oximi y o he PUMA p53- a ge sequence (Figu e
7). The G4-p one sequence is loca ed igh ly be o e p53 RE (−33 o −1 be o e p53 RE, max. G4Hun e
sco e in his a ea 1.84) and se e al G4-p one sequences a e loca ed a e he p53 RE including a
G4Hun e sco e o 1.32 immedia ely a e he p53 RE—loca ion 0–25—and ano he u he downs eam
(s a ing ei he 21, 45, and 58 nucleo ides a e he p53 RE; highes G4Hun e sco e o 3.2 o he
sequence: GGGGGCGGGG CGGGGCGGGG CGGGG, peak a 71 nucleo ides a e p53 RE).
Figu e 7. Localiza ion o G4-p one sequences a ound p53 PUMA RE sequence (100 bp be o e and a e p53
PUMA RE). The analysis o G4-p one sequences has shown ha p53 PUMA RE ( ec angle) in he human
genome is su ounded by G4-p one sequences wi h peaks a 1.84 be o e p53 RE and long G4-p one
sequence wi h he peak a 1.32 jus a e p53 RE and wi h a maximum peak wi h G4Hun e sco e 3.2).
E en hough he localiza ion o bo h p53 RE and G4 sequences ha e been shown in he genome,
he oles o G-quad uplexes in egula ing ansc ip ion by p53 iso o ms ha e no been e alua ed.
The e o e, we p epa ed a model sys em and analyzed he impac o a sequence endowed wi h high
p opensi y o adop a G4 s uc u e posi ioned ei he ups eam o downs eam o a mode a ely ac i e
p53 RE using yeas epo e s ains. FLp53α p o ein and i s ∆-iso o ms ailed o ansac i a e a
minimal p omo e when only a G4-p one sequence was inse ed a he si e. I has been shown ecen ly
ha G4s ha e an inhibi o y e ec on ansla ion in i o in he yeas sys em [43]. Ou esul s showed
ha ∆160p53α exp essed oge he wi h FLp53α dec eased ansac i a ion a he p53 RE. These new
da a a e in ag eemen wi h p e iously published apop osis assays, whe e ∆160p53 inhibi s apop osis,
in con as o ∆133p53 [44]. On he o he hand, he ∆133p53α and ∆160p53α iso o ms ailed o
dec ease ansac i a ion o he p53 RE p esen ed oge he wi h a G4-p one sequence in on o he
RE; in ac , he e was a sligh inc ease in ansac i a ion (Figu e 5A). This esul sugges s ha he e o-
e ame iza ion o ∆133p53α o ∆160p53α wi h FLp53α (con a y o ∆40p53α) does no inhibi
Figu e 7.
Localiza ion o G4-p one sequences a ound p53 PUMA RE sequence (100 bp be o e and
a e p53 PUMA RE). The analysis o G4-p one sequences has shown ha p53 PUMA RE ( ec angle) in
he human genome is su ounded by G4-p one sequences wi h peaks a 1.84 be o e p53 RE and long
G4-p one sequence wi h he peak a 1.32 jus a e p53 RE and wi h a maximum peak wi h G4Hun e
sco e 3.2).
E en hough he localiza ion o bo h p53 RE and G4 sequences ha e been shown in he genome,
he oles o G-quad uplexes in egula ing ansc ip ion by p53 iso o ms ha e no been e alua ed.
The e o e, we p epa ed a model sys em and analyzed he impac o a sequence endowed wi h high
p opensi y o adop a G4 s uc u e posi ioned ei he ups eam o downs eam o a mode a ely ac i e
p53 RE using yeas epo e s ains. FLp53
α
p o ein and i s
∆
-iso o ms ailed o ansac i a e a
minimal p omo e when only a G4-p one sequence was inse ed a he si e. I has been shown
ecen ly ha G4s ha e an inhibi o y e ec on ansla ion
in i o
in he yeas sys em [
43
]. Ou esul s
showed ha
∆
160p53
α
exp essed oge he wi h FLp53
α
dec eased ansac i a ion a he p53 RE.
These new da a a e in ag eemen wi h p e iously published apop osis assays, whe e
∆
160p53 inhibi s
apop osis, in con as o
∆
133p53 [
44
]. On he o he hand, he
∆
133p53
α
and
∆
160p53
α
iso o ms ailed
In . J. Mol. Sci. 2020,21, 127 8 o 12
o dec ease ansac i a ion o he p53 RE p esen ed oge he wi h a G4-p one sequence in on o
he RE; in ac , he e was a sligh inc ease in ansac i a ion (Figu e 5A). This esul sugges s ha
he e o- e ame iza ion o
∆
133p53
α
o
∆
160p53
α
wi h FLp53
α
(con a y o
∆
40p53
α
) does no inhibi
ansac i a ion a p53 a ge s associa ed wi h a G4 s uc u e, while in he case o
∆
40p53
α
, compe i ion
be ween iso o m speci ic homo- e ame s o he o ma ion o he e o- e ame s can lead o he inhibi ion
o he ansac i a ion po en ial o FLp53αa hese si es (Figu e 8).
In . J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 8 o 12
ansac i a ion a p53 a ge s associa ed wi h a G4 s uc u e, while in he case o ∆40p53α,
compe i ion be ween iso o m speci ic homo- e ame s o he o ma ion o he e o- e ame s can lead
o he inhibi ion o he ansac i a ion po en ial o FLp53α a hese si es (Figu e 8).
Figu e 8. Schema ic model o p53 iso o ms binding o a RE associa ed wi h G4 sequence. (A) FLp53α
o ∆40p53α bind e ec i ely o he RE and he e is a high o mode a e le el o ansac i a ion. (B)
∆133p53α and ∆160p53α inhibi FLp53α ansac i a ion, (C) he p esence o a G4 close o he RE
dec eases accessibili y o he TA domains and FLp53α ansac i a ion, (D) which is no mo e inhibi ed
by ∆133p53α and ∆160p53α, al hough s e ic p o ein o ien a ion is impai ed due o he G4 s uc u e.
TA is he blue domain, es o he p o ein is in g een, column ep esen ansac i a ion induced by
p53 complex ( ed column FLp53α, blue FLp53α wi h ∆133p53α and ∆160p53α iso o ms).
The e o e, i appea s ha he composi ion o he p53 iso o ms could be a selec i e de e minan
in p53 ansac i a ion speci ici y, esul ing no only om he p53 RE sequence, bu also om
s uc u al DNA ea u es, pa icula ly a G4 ups eam o downs eam o he p53 RE. The G4-p one
sequences localized in close p oximi y o he PUMA p53 RE sugges s ha G4 o ma ion could be an
addi ional ea u e ha de e mines he e ec i eness o p53 ansc ip ional egula ion. The co-
exp ession o di e en p53 iso o ms may inc ease plas ici y h ough a comp omise be ween e ec i e
FLp53 homo e ame s a RE si es embedded in s uc u ally a o able con ex s and less e ec i e, bu
s e ically mo e bene icial he e o e ame s, a RE si es lanked by s uc u ed mo i s such as G4.
4. Me hods
4.1. P epa a ion o Plasmids o Exp ess p53α Iso o ms
Vec o s con aining he coding sequences o p53α iso o ms we e p epa ed by he Ga eway
cloning sys em (de ailed in [45]). As he des ina ion ec o , pAG414GALccdB-HA con aining he
inducible GAL p omo e and pAG415GPDccdB-HA wi h he cons i u i e GPD p omo e we e used.
Des ina ion ec o s con aining he cDNAs o p53α iso o ms we e isola ed om E. coli STBL3 s ain
using a comme cial plasmid ex ac ion ki (Omega-Bio ek, No c oss, USA).
4.2. P epa a ion o Yeas Isogenic S ains by Deli o Pe e o Homologous Recombina ion
S. ce e isiae haploid s ain yLFM-ICORE (MATα leu2–3nic s ains, 112 p1–1 his3–11,15 can1–
100; u a3–1; ade2:RE:pCyc1::LUC1) was used o de i ing a panel o isogenic epo e s ains, which
di e in he p esence o a p53 RE and a G4 p one sequence (Table 1). The double coun e selec able-
Figu e 8.
Schema ic model o p53 iso o ms binding o a RE associa ed wi h G4 sequence. (
A
)
FLp53
α
o
∆
40p53
α
bind e ec i ely o he RE and he e is a high o mode a e le el o ansac i a ion.
(
B
)
∆
133p53
α
and
∆
160p53
α
inhibi FLp53
α
ansac i a ion, (
C
) he p esence o a G4 close o he RE
dec eases accessibili y o he TA domains and FLp53
α
ansac i a ion, (
D
) which is no mo e inhibi ed
by
∆
133p53
α
and
∆
160p53
α
, al hough s e ic p o ein o ien a ion is impai ed due o he G4 s uc u e.
TA is he blue domain, es o he p o ein is in g een, column ep esen ansac i a ion induced by p53
complex ( ed column FLp53α, blue FLp53αwi h ∆133p53αand ∆160p53αiso o ms).
The e o e, i appea s ha he composi ion o he p53 iso o ms could be a selec i e de e minan in
p53 ansac i a ion speci ici y, esul ing no only om he p53 RE sequence, bu also om s uc u al
DNA ea u es, pa icula ly a G4 ups eam o downs eam o he p53 RE. The G4-p one sequences
localized in close p oximi y o he PUMA p53 RE sugges s ha G4 o ma ion could be an addi ional
ea u e ha de e mines he e ec i eness o p53 ansc ip ional egula ion. The co-exp ession o di e en
p53 iso o ms may inc ease plas ici y h ough a comp omise be ween e ec i e FLp53 homo e ame s a
RE si es embedded in s uc u ally a o able con ex s and less e ec i e, bu s e ically mo e bene icial
he e o e ame s, a RE si es lanked by s uc u ed mo i s such as G4.
4. Me hods
4.1. P epa a ion o Plasmids o Exp ess p53αIso o ms
Vec o s con aining he coding sequences o p53
α
iso o ms we e p epa ed by he Ga eway cloning
sys em (de ailed in [
45
]). As he des ina ion ec o , pAG414GALccdB-HA con aining he inducible
GAL p omo e and pAG415GPDccdB-HA wi h he cons i u i e GPD p omo e we e used. Des ina ion
ec o s con aining he cDNAs o p53
α
iso o ms we e isola ed om E. coli STBL3 s ain using a
comme cial plasmid ex ac ion ki (Omega-Bio ek, No c oss, USA).
In . J. Mol. Sci. 2020,21, 127 9 o 12
4.2. P epa a ion o Yeas Isogenic S ains by Deli o Pe e o Homologous Recombina ion
S. ce e isiae haploid s ain yLFM-ICORE (MAT
α
leu2–3nic s ains, 112 p1–1 his3–11,15 can1–100;
u a3–1; ade2:RE:pCyc1::LUC1) was used o de i ing a panel o isogenic epo e s ains, which di e
in he p esence o a p53 RE and a G4 p one sequence (Table 1). The double coun e selec able- REpo e
ICORE casse e was eplaced by a a ge ing oligonucleo ide, consis ing o 30 n lanking homology
and he RE +G4 as an in e ening sequence, ollowing he p o ocol desc ibed in [
46
]. Replacemen
was acili a ed by induc ion o a single si e-speci ic DNA double s and b eak a he ICORE si e by he
homing endonuclease I-SceI, selec ed by exploi ing esis ance o 5- luo o-o o ic acid caused by loss o
he ICORE casse e and con i med by colony PCR and Sange sequencing. The ob ained yeas epo e
s ains di e ing in he p53 a ge si e we e pu i ied and ans o med wi h a plasmid o he exp ession
o speci ic p53αiso o ms.
Table 1.
Sequences cloned in o luci e ase p omo e egions in o yeas isogenic epo e s ain (PUMA
sequence – highligh ed by g ey, G- epea s – bold).
Region Sequence 5’−3’
PUMA CTGCAAGTCCTGACTTGTCC
PUMA–G4 CTGCAAGTCCTGACTTGTCCGGGGCGGGGGACGGGGGAGGGG
G4–PUMA GGGGCGGGGGACGGGGGAGGGG CTGCAAGTCCTGACTTGTCC
G4 GGGGCGGGGGACGGGGGAGGGG
4.3. Ci cula Dich oism (CD) Spec oscopy
CD measu emen s we e ca ied ou in a Jasco 815 (Jasco In e na ional Co. L d., Tokyo, Japan)
dich og aph in 1 cm pa h-leng h qua z Hellma mic ocells placed in a he mos a ically egula ed cell
holde a 23
◦
C. A se o ou scans was a e aged o each sample wi h a da a pi ch o 0.5 nm and
100 nm/min scan speed. CD signal was exp essed as he di e ence in he mola abso p ion, ∆εo he
le - and igh -handed ci cula ly pola ized ligh , mola i y being ela ed o DNA s ands; bu e : 50 mM
KCl, 5 mM T is/HCl pH 8.
4.4. T ans o ma ion o Yeas S ains
Yeas we e ans o med by a me hod based on mixing cells and DNA in he p esence o li hium
ace a e, TE, PEG, DMSO and pe o ming hea shock, s a ing om sa u a ed o e nigh cul u es [
47
].
Double ans o man s we e selec ed by auxo ophic selec ion on pla es lacking bo h yp ophan
and leucine.
4.5. Luci e ase Assay
Pu i ied ans o man colonies we e inocula ed on 96-well pla es in 120
µ
L selec i e media
con aining 2% a inose as a ca bon sou ce and di e en concen a ions o galac ose o induce p53
α
iso o m exp ession om he GAL p omo e o he pAG414GAL ec o . Luci e ase was measu ed as
desc ibed [
40
]. To asce ain p53 p o ein exp ession, samples used o he ansc ip ion analysis we e
also used o p epa e p o ein ex ac s o immunode ec ion by wes e n blo ing.
4.6. Wes e n Blo
Yeas cell lysis was pe o med as desc ibed [
48
]. P o ein ex ac s we e quan i ied using he
B ad o d assay. P o eins (80
µ
g) we e elec opho esed using 12.5% ac ylamide sodium dodecyl sul a e
polyac ylamide gel elec opho esis (SDS-PAGE) and ans e ed o a ni ocellulose memb ane. Speci ic
an ibodies di ec ed agains p53 we e dona ed by D . Voj ˇešek and he memb anes we e incuba ed as
desc ibed [
49
–
51
]. The signal was de ec ed using he ECL Selec eagen (Pie ce Fas Wes e n Blo Ki ,
The mo Fishe , WA, USA) and esul s we e isualized as chemiluminiscence on LAS 3000. Resul s a e
shown in Figu e S1.