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www.impac jou nals.com/onco a ge / Onco a ge , Vol. 7, No. 15
Me hylomic p edic o s demons a e he ole o NF-κB in old-age
mo ali y and a e un ela ed o he aging-associa ed epigene ic
d i
Juulia Jylhä ä1,2, Lau a Kananen1,2, Jani Rai anen3,4, Saa a Ma ila1,2, Tapio
Ne alainen1,2, An i He onen2,3, Ma ja Jylhä2,3 and Mikko Hu me1,2,5
1 Depa men o Mic obiology and Immunology, School o Medicine, Uni e si y o Tampe e, Tampe e, Finland
2 Ge on ology Resea ch Cen e , Uni e si y o Tampe e, Tampe e, Finland
3 School o Heal h Sciences, Uni e si y o Tampe e, Tampe e, Finland
4 UKK Ins i u e o Heal h P omo ion Resea ch, Tampe e, Finland
5 Fimlab Labo a o ies, Tampe e, Finland
Co espondence o: Juulia Jylhä ä, email: [email p o ec ed]
Keywo ds: me hyla ion, mo ali y, aging, longe i y, Cox model, Ge o a ge
Recei ed: No embe 06, 2015 Accep ed: Ma ch 10, 2016 Published: Ma ch 22, 2016
ABSTRACT
Changes in he DNA me hyla ion (DNAm) landscape ha e been implica ed in aging
and cellula senescence. To un a el he ole o speci ic DNAm pa e ns in la e-li e
su i al, we pe o med genome-wide me hyla ion p o iling in nonagena ians (n=111)
and de e mined he pe o mance o he me hylomic p edic o s and con en ional isk
ma ke s in a longi udinal se ing. The su i al model con aining only he me hylomic
ma ke s was supe io in e ms o p edic i e accu acy compa ed wi h he model
con aining only he con en ional p edic o s o he model con aining con en ional
p edic o s combined wi h he me hylomic ma ke s. A he 2.55-yea ollow-up,
we iden i ied 19 mo ali y-associa ed ( alse-disco e y a e <0.5) CpG si es ha
mapped o genes unc ionally clus e ing a ound he nuclea ac o kappa B (NF-κB)
complex. In e es ingly, none o he mo ali y-associa ed CpG si es o e lapped wi h
he es ablished aging-associa ed DNAm si es. Ou esul s a e in line wi h p e ious
indings on he ole o NF-κB in con olling animal li e spans and demons a e he ole
o his complex in human longe i y.
INTRODUCTION
The in luen ial ole o genomic ac o s, such as
DNA me hyla ion (DNAm) in he cou se o de elopmen ,
aging and age- ela ed pa hologies is well es ablished.
Se e al s udies ha e also ep oducibly demons a ed ha
he le el o me hyla ion a speci ic CpG si es changes
as a unc ion o age [1-5], hence p o iding a ma ke
o ch onological and, po en ially, biological age. An
in iguing cha ac e is ic o age- ela ed DNAm signa u es
is ha many o he age-associa ed DNAm changes ha e
been obse ed o be common in se e al di e en issues,
such as whole blood, b ain, lung and ce ix [1, 3, 6].
These obse a ions sugges ha a global mechanism(s)
migh be esponsible o age-associa ed modi ica ions
in he epigene ic landscape. Ne e heless, s udies wi h
monozygo ic wins ha e demons a ed ha he a e o
di e gence in me hylomic pa e ns inc eases wi h age [7,
8], sugges ing ha he age- ela ed modi ica ions in DNAm
a e also subjec o a ious en i onmen al, s ochas ic and
li e s yle- ela ed e ec s.
Howe e , he consequences o he aging-
accompanied DNAm al e a ions o la e-li e heal h
and unc ional abili ies a e la gely unknown. A ecen
epigenome-wide associa ion s udy (EWAS) demons a ed
ha he associa ion be ween age- ela ed DNAm changes
and heal hy aging pheno ypes in indi iduals 32-80 yea s
o age is negligible [8]. The esul s o his s udy also
e eal ha he DNAm egions associa ed wi h aging
pheno ypes a e dis inc om hose associa ed wi h
ch onological age. These indings sugges ha he CpG
si es in ol ed in heal h- ela ed ou comes in la e li e a e
la gely egula ed by si es o he han he es ablished age-
ela ed DNAm egions [8]. In addi ion, using an EWAS
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app oach, we ha e ecen ly demons a ed ha he CpG
si es ha a e associa ed wi h aging- ela ed in lamma ion,
i.e., in lammaging [9] a e la gely di e en om he
si es associa ed wi h age [5]. This phenomenon is also
obse able in ega d o gene exp ession p o iles and old
age mo ali y. We ha e p e iously demons a ed ha he
genes exhibi ing aging- ela ed changes in exp ession
le els a e p edominan ly di e en om hose ha p edic
mo ali y in la e li e [10]. These indings unde sco e he
complexi y and unknown na u e o he genomic ac o s
ha con ol he human heal h span and la e-li e e en s.
Ne e heless, he mo ali y-p edic ing genes in ou
p e ious s udy we e ound o be unc ionally connec ed
o he nuclea ac o kappa B (NF-κB) complex, which
is a cen al media o in immunoin lamma o y esponses
and has been ad oca ed as he culp i in aging and cellula
senescence ( e iewed in [11]). Abe an ac i a ion o NF-
κB has been epo ed in a ious age-associa ed condi ions,
such as neu odegene a ion, immunosenescence,
in lammaging, sa copenia and os eopo osis ( e iewed in
[12-14]), whe eas s udies in ol ing mouse models ha e
obse ed ha NF-κB ac i a ion is a key de e minan o
accele a ed aging and longe i y [15, 16]. In he mouse
models, i was demons a ed ha he hypo halamic
ac i a ion o NF-κB is a d i ing o ce o sys emic aging
h ough immune-endoc ine connec ions [16].
Li e span egula ion in humans is a mul i ac o ial
p ocess, and e y li le is known abou he genomic
de e minan s ha con ol la e-li e mo ali y a e he
ages o he common kille s, i.e., ca dio ascula e en s
and cance , ha e passed. In his s udy, we sough o
explo e how he human genome-wide me hylome is
associa ed wi h old-age su i al wi hin a sho e (2.55
yea s) and a longe (4 yea s) ollow-up ime. A la ge
panel o adi ional (bio)ma ke s and mo ali y isk
ac o s was assessed alongside he me hylomic ma ke s
o elucida e he ela ionship be ween he aging- ela ed
biophysiological changes and epigene ics.
RESULTS
The cha ac e is ics o he s udy popula ion and
dis ibu ion o he a iables in he popula ion wi h
me hyla ion da a a ailable (n = 111) a e p esen ed in Table
1. The a iables (i.e., he con en ional ma ke s) exhibi ing
signi ican (p < 0.05) uni a ia e and mul i a ia e
associa ions a he 2.55 ollow-up a e p esen ed in
Supplemen a y Table 1. The p edic o s emaining in he
mul i a ia e model, body mass index (BMI) and Mini-
Men al S a e Examina ion (MMSE) es sco e, we e used
as he model ac o s in he assessmen o he p edic i e
accu acy o modeling (see Me hods). The measu e o
“epigene ic clock” [17], he DNA me hyla ion age was no
p edic i e o mo ali y in ou coho (p = 0.733).
In he Cox uni a ia e assessmen , 19,621 and 15,505
CpG si es we e associa ed wi h mo ali y (p < 0.05) in
he 2.55-yea and 4-yea ollow-up da a, espec i ely
(Supplemen a y Tables 2 and 3). A e B-H co ec ion
(FDR < 0.5), 19 CpG si es emained signi ican o he
2.55-yea ollow-up and 7 CpG si es o he 4-yea ollow-
up da a (Supplemen a y Tables 2 and 3). The Ingenui y
Pa hway Analysis (IPA) -gene a ed ne wo k om he 16
known genes ha bo ing he 19 signi ican CpG si es a
he 2.55-yea ollow-up is p esen ed in Figu e 1a. This
ne wo k displayed NF-κB as a cen al node and in ol ed
10 o 16 o he genes mapped o he 19 mo ali y-
associa ed CpG si es (FDR < 0.5). We also an he IPA
ne wo k and pa hway analyses om he genes ha bo ing
he 250 op- anking CpG si es acco ding o he 2.55-
yea ollow-up da a (si es p esen ed in Supplemen a y
Table 2). The highes - anking ne wo k in his analysis
also placed NF-κB as a cen al complex (Figu e 1b). The
signi ican B-H-co ec ed canonical pa hways om his
da a se a e p esen ed in Table 2. A he 4-yea ollow-up,
he unc ional implica ions o he me hylomic p edic o s
we e a enua ed as no signi ican B-H -co ec ed canonical
pa hways we e iden i ied in IPA om he genes ha bo ing
he 250 highes - anking CpG si es and no signi ican
ne wo k en ichmen was obse ed among he genes
ha bo ing he 7 CpG si es (FDR < 0.5).
Assessmen o he p edic i e accu acy o he es ed
models e ealed ha he Ridge eg ession con aining
only he me hylomic ma ke s (Ridge1) pe o med
be e han he o he models; i.e., a model con aining
only he con en ional p edic o s, a Ridge eg ession
model con aining bo h he con en ional p edic o s
(Ridge2) and he me hylomic ma ke s and a model
con aining only he me hylomic ma ke s selec ed on
he basis o hei signi icance le el in Cox uni a ia e
assessmen . Speci ically, he me hylomic ma ke s alone
exhibi ed he smalles median de iance om he null
model (Supplemen a y Figu e 1), and we e hus used in
assessing he inal mo ali y-p edic ing signa u e in he
Cox mul i a ia e model o 2.55-yea ollow-up da a. The
de iances o he con en ional ma ke s exhibi ed clea ly
he smalles a ia ion bu hei median was ne e heless
highe han ha o he me hylomic ma ke s in Ridge1.
The Ridge eg ession-o ganized 19 me hylomic
ma ke s en e ed o he Cox mul i a ia e model a e
p esen ed in Supplemen a y Table 4. Inclusion o he
me hylomic ma ke s in he inal model was based on
selec ion o he model wi h he bes goodness o i
(Akaike In o ma ion c i e ion, AIC), which o he
selec ed model was 239.0. The inal Cox mul i a ia e
model is p esen ed in Table 3 and he dis ibu ions o
he be a alues o he se en CpG si es (ba ch e ec
-co ec ed) included his mo ali y-p edic ing signa u e
a e p esen ed in Supplemen a y Figu e 2.
The disc imina i e powe (Ha ell’s C) o his
model was 89.9%. The p opo ionali y assump ion in he
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Table 1: Cha ac e is ics o he s udy popula ion (n = 111). Dis ibu ions o he a iables a e p esen ed acco ding o he da a
a he 2.55-yea s mo ali y ollow-up.
Non-su i o s Su i o s
Va iable Mean/Med SEM/IQR/% Mean/Med SEM/IQR/%
Women (n/%) 27 75.0 54 72.0
Age (mon hs) 1079.5 0.61 1080.2 0.37
Sys olic blood p essu e (mmHg) 145 4.6 149 3.4
Dias olic blood p essu e (mmHg)* 71.5 13.5 74.0 19.0
Weigh (kg) 61.9 2.2 70.6 1.6
BMI (kg/m2) 24.3 0.75 27.5 0.54
Wais ci cum e ence (cm) 89.6 2.1 95.5 1.4
Hip ci cum e ence (cm)* 98 10.0 102 12.0
MMSE* 23.5 8.0 26.0 4.0
Ba hel index* 95.0 20.0 95 5.0
Handg ip (kg)* 18.0 11.0 20.0 7.0
Able o pe o m chai - ise es (n = yes/%) 19 57.6 59 78.7
Able o pe o m chai -s and es (n = yes/%) 22 71.0 62 82.7
F ail y index (n/%)
Non- ail 3 8.3 26 34.7
P e- ail 22 61.1 37 49.3
F ail 11 30.6 12 16.0
CRP le el (ng/ml)* 1.8 3.3 1.9 3.5
IL-1β le el (pg/ml)* 14.2 27.6 19.0 34.0
IL-6 le el (pg/ml)* 4.5 3.3 3.8 3.8
IL-7 le el (pg/ml)* 7.8 5.3 6.4 5.2
IL-10 le el (pg/ml)* 1.8 1.5 1.5 2.6
c -DNA le el (μg/ml)* 0.93 0.19 0.87 0.16
Unme hyla ed c -DNA le el (μg/ml)* 0.75 0.20 0.67 0.15
Plasma m DNA (copy numbe )* 4.30E82.37E83.75E82.09E8
Alu epea c -DNA (GE)* 74.4 50.4 66.8 38.3
DHEAS (μg/ml)* 0.25 0.48 0.25 0.31
Co isol (ng/ml)* 133 54.3 117 68.0
IDO ac i i y (Kyn/T p)* 44.3 25.5 51.8 25.3
An i-CMV an ibody i e * 19.000 8.000 19.000 9000
An i-EBV an ibody i e * 405 315 410 410
DNAm age 76.1 1.04 76.1 0.64
CD3+ cells (%)*a62.0 15.8 57.9 12.0
CD4+ cells (%)b62.9 2.5 63.8 1.6
CD8+ cells (%)b30.6 2.3 28.9 1.5
CD4+/CD8+ cells ( a io)* 2.4 2.3 2.3 2.4
CD4+CD28- cells (%)* 9.2 16.2 9.2 13.0
CD8+CD28- cells (%) 63.3 2.8 63.3 2.1
CD14+ cells (%)*a8.3 5.9 8.1 6.3
*median alues and IQR p esen ed
ape cen age o li e-ga ed cells; bpe cen age o o al T lymphocy es (CD3+ cells);
cpe cen age o CD4+ cells; dpe cen age o CD8+ cells
Abb e ia ions: BMI, body mass index; CD, clus e o di e en ia ion; CMV, cy omegalo i us; CRP, C- eac i e p o ein; c -DNA, cell- ee DNA; DHEAS,
dehyd oepiand os e one sul a e; DNAm, DNA me hyla ion; EBV, Eps ein-Ba i us; GE, genomic equi alen ; IDO, indoleamine 2,3-dioxygenase; IL,
in e leukin; Kyn, kynu enine; MMSE, Mini-Men al S a e Examina ion; m DNA, mi ochond ial DNA; T p, yp ophan
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Figu e 1: The highes - anking ne wo ks om he 16 known genes ha bo ing he op 19 signi ican (FDR < 0.5) CpG
si es a. and om he genes ha bo ing he op 250 CpG si es b. in he 2.55-yea ollow-up (n = 111). Bo h ne wo ks displayed NF-κB
as a cen al node and we e en iched o he common e m Hema ological Sys em De elopmen and Func ion. The g een colo o he
molecule indica es ha hypome hyla ion o a CpG si e in he gene was associa ed wi h inc eased mo ali y, and he ed colo indica es
ha hype me hyla ion o a CpG si e in he gene was associa ed wi h inc eased mo ali y. The ne wo ks we e gene a ed h ough he use o
QIAGEN’s Ingenui y Pa hway Analysis (IPA®,QIAGEN Redwood Ci y, www.qiagen.com/ingenui y).
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Table 2: Canonical pa hways cons uc ed om he genes ha bo ing he op 250 CpG si es associa ed wi h mo ali y
a he 2.55-yea ollow-up.
Ingenui y Canonical Pa hways -log(p)* Ra io Molecules
Ch onic Myeloid Leukemia Signaling 1.91 7.61E-02
TGFBR2, GAB2, HDAC4, SMAD3, PIK3R2,
NFKB1, ATM
Ge m Cell-Se oli Cell Junc ion Signaling 1.58 5.13E-02
TGFBR2, MAP3K14, MAP3K10, ACTA2,
KEAP1, ITGA2, PIK3R2, ATM
Role o NFAT in Ca diac Hype ophy 1.58 4.55E-02
IL6ST, TGFBR2, HDAC4, ITPR3, IGF1R,
SLC8A3, PIK3R2, ATM
Cell Cycle: G1/S Checkpoin Regula ion 1.58 7,94E-02 CCND2, HDAC4, CCND3, SMAD3, ATM
Regula ion o he Epi helial-Mesenchymal
T ansi ion Pa hway 1.58 4.40E-02
MAML1, TGFBR2, FZD3, SMAD3, PIK3R2,
NFKB1, SMURF1, ATM
iCOS-iCOSL Signaling in T Helpe Cells 1.58 5.83E-02 GAB2, CD28, ITPR3, PIK3R2, NFKB1, ATM
Rac Signaling 1.58 5,83E-02 CYFIP2, ITGA2, PIK3R2, NFKB1, ATM, ANK1
NF-κB Ac i a ion by Vi uses 1.58 6.85E-02 MAP3K14, ITGA2, PIK3R2, NFKB1, ATM
Hepa ic Fib osis/Hepa ic S ella e Cell
Ac i a ion 1.58 4.08E-02
TGFBR2, TNFSF4, ACTA2, MYH14, SMAD3,
IGF1R, NFKB1, FAS
GADD45 Signaling 1.58 1.58E-01 CCND2, CCND3, ATM
PKCθ Signaling in T Lymphocy es 1.57 5.31E-02
MAP3K14, MAP3K10, CD28, PIK3R2, NFKB1,
ATM
Molecula Mechanisms o Cance 1.57 3.06E-02
TGFBR2, GAB2, CCND2, CCND3, FZD3,
SMAD3, ITGA2, PIK3R2, NFKB1, FAS, ATM
CNTF Signaling 1.46 8.16E-02 IL6ST, CNTF, PIK3R2, ATM
B Cell Recep o Signaling 1.44 4.09E-02
GAB2, MAP3K14, MAP3K10, PAG1, PIK3R2,
NFKB1, ATM
RANK Signaling in Os eoclas s 1.44 5.81E-02 MAP3K14, MAP3K10, PIK3R2, NFKB1, ATM
Vi us En y ia Endocy ic Pa hways 1.44 5.62E-02 ITSN1, ACTA2, ITGA2, PIK3R2, ATM
C oss alk be ween Dend i ic Cells and
Na u al Kille Cells 1.44 5.62E-02 CD28, ACTA2, KLRD1, NFKB1, FAS
Lympho oxin β Recep o Signaling 1.44 7.41E-02 MAP3K14, PIK3R2, NFKB1, ATM
Dea h Recep o Signaling 1.44 5.49E-02 MAP3K14, ACTA2, PARP12, NFKB1, FAS
Colo ec al Cance Me as asis Signaling 1.43 3.46E-02
IL6ST, TGFBR2, FZD3, SMAD3, PIK3R2,
NFKB1, PTGER4, ATM
Myc Media ed Apop osis Signaling 1.40 6.90E-02 IGF1R, PIK3R2, FAS, ATM
T Cell Recep o Signaling 1.40 5,21E-02 CD28, PAG1, PIK3R2, NFKB1, ATM
Es ogen-Dependen B eas Cance Signaling
1.30 6.45E-02 IGF1R, PIK3R2, NFKB1, ATM
CD40 Signaling 1.30 6.25E-02 MAP3K14, PIK3R2, NFKB1, ATM

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Cox Reg ession model was es ed using he global es by
calcula ing he scaled Schoen eld esiduals o each o he
independen p edic o s in he inal Cox model. S a is ically
signi ican dependence o mo ali y on ime was no
obse ed (p = 0.280) indica ing ha he p opo ionali y
assump ion was no iola ed.
Due o he small numbe o mo ali y-associa ed
CpG si es in he me hylomic da a a he 4-yea ollow-
up, no compa ison o he p edic ion accu acies o he
di e en modeling op ions o assessmen o he inal
mo ali y-p edic ing signa u e was pe o med o he
4-yea mo ali y da a.
Co ela ion analysis be ween he me hyla ion
le els in he mo ali y-associa ed CpG si es and he
co esponding gene p oduc (s) e ealed a signi ican
co ela ion be ween h ee CpG si e/ ansc ip pai s. In e se
co ela ions we e obse ed be ween he cg03348466
(CRTC3) and CRTC3 mRNA le el and be ween
cg04182483 (RGS10) and he RGS10 mRNA le el. A
di ec co ela ion was obse ed be ween cg22794214
(HIVEP3) and HIVEP3 mRNA le el. All he co ela ions
a e p esen ed in Supplemen a y Table 5.
Analysis o he genomic loca ions o he op 19
CpG si es (FDR < 0.5, in Supplemen a y Table 2) o
ansc ip ion ac o (TF) binding si es and o he genomic
egula o y elemen s e ealed ha a majo i y o he si es
we e loca ed on ac i e cis- egula o y egions; hey ei he
ha bo ed TF binding si es, DNAse I hype sensi i i y
egions, and/o we e iden i ied as “P edic ed p omo e
egion including ansc ip ion s a si e (TSS)”, “P edic ed
enhance (E)” o “P edic ed weak enhance o open
ch oma in (WE)”. In addi ion, six CpG si es demons a ed
unc ional signi icance as hey we e anno a ed o
“P edic ed ansc ibed egion (T)”. The mos abundan
TFs we e POLR2A and RELA which bo h had binding
si es on ou CpG si e loci. Full da a o his assessmen a e
p esen ed in Table 4.
DISCUSSION
We ha e p e iously demons a ed, using genome-
wide gene exp ession da a, ha he NF-κB complex is
cen ally in ol ed in con olling human old-age mo ali y
[10]. In he p esen s udy, we expanded he examina ion
o he genomic ac o s egula ing la e-li e su i al
by analyzing he p edic i e abili y o genome-wide
me hylomic da a a he 2.55-yea ollow-up. The esul s
o his s udy co obo a e he ole o NF-κB in all-cause
elde ly mo ali y; he molecula ne wo k cons uc ed
om he genes ha bo ing he mo ali y-associa ed CpG
si es displayed he NF-κB complex as a cen al media o
(Figu e 1). The genes nuclea ac o o kappa ligh
polypep ide gene enhance in B-cells 1 (NFKB1) and
a axia elangiec asia mu a ed (ATM) we e also iden i ied
in he ne wo k. In iguingly, bo h NFKB1 and ATM ha e
p e iously been linked wi h accele a ed aging and cellula
senescence in s udies wi h gene ically enginee ed mice
[15, 18, 19]. These s udies ad oca ed ha NFKB1 and
ATM- egula ed abe an NF-κB ac i a ion and he ensuing
ch onic sys emic in lamma o y s a e a e he ul ima e
HGF Signaling 1.30 4.81E-02 MAP3K14, MAP3K10, ITGA2, PIK3R2, ATM
Panc ea ic Adenoca cinoma Signaling 1.30 4.72E-02 TGFBR2, SMAD3, PIK3R2, NFKB1, ATM
NGF Signaling 1.30 4.72E-02 MAP3K14, MAP3K10, PIK3R2, NFKB1, ATM
T Helpe Cell Di e en ia ion 1.30 5.97E-02 IL6ST, TGFBR2, CD28, IL21R
IL-9 Signaling 1.30 8.82E-02 PIK3R2, NFKB1, ATM
*Benjamini-Hochbe g-co ec ed p- alue
Table 3: The inal mo ali y-p edic ing signa u e a he 2.55-yea ollow-up assessed om he Ridge
eg ession -o ganized me hylomic ma ke s.
HR (95% CI) S.E. Z p
cg08421934 (NA)0.41 (0.26-0.64) 0.10 -3.84 <0.001
cg15770702 (MAP3K14)0.40 (0.27-0.61) 0.08 -4.38 <0.001
cg08596308 (ATP6V1G2; NFKBIL1)0.50 (0.34-0.73) 0.10 -3.60 <0.001
cg23282964 (RIOK1)0.56 (0.37-0.84) 0.12 -2.82 0.005
cg16720947 (PLEC1)0.52 (0.34-0.80) 0.13 -2.94 0.003
cg27027151 (IL21R)2.09 (1.44-3.02) 0.39 3.90 <0.001
cg26843567 (NA)0.68 (0.46-0.99) 0.13 -2.01 0.045
Abb e ia ions: CI, con idence in e al; HR, haza d a io; NA, no a ailable; S.E., s anda d e o
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d i e s o senescence and aging-associa ed de e io a ion
[15, 18]. Al hough ou da a do no p o ide a mechanis ic
link be ween he hypome hyla ion o hese CpG si es and
he isk o mo ali y, we specula e ha he mechanism
in ol es an in lamma o y componen by which he
genomic ac o s con ol la e-li e mo ali y.
Analysis o he 19 mo ali y-associa ed CpG si es
(FDR < 0.5) o genomic egula o y elemen s e ealed ha
a majo i y o he si es we e loca ed on ac i e cis- egula o y
egions (Table 4). Tha is, hey ha bo ed TF binding
si es, loca ed on DNAse I hype sensi i i y a eas and/o
displayed one o he ollowing p edic ed genomic s a es:
Table 4: Assessmen o he 19 mo ali y associa ed CpG si es (FDR<0.5) in he 2.55-yea ollow-up (n = 111) o
ansc ip ion ac o binding si es and o he unc ional genomic elemen s using ENCODE da a in he UCSC genome
b owse .
CpG si e (gene) GRCh37/hg19
coo dina e T ansc ip ion Fac o s Genome
s a us
DNAse I
Hype sensi i i y
Clus e
cg24859528 (IQSEC1) ch 3:12941421 TNO
cg03348466 (CRTC3) ch 15:91104770 CEBPB TYES
cg02395768 (ATP5SL) ch 19:41945578
SIN3AK20, POLR2A, SP2, SP1,
CHD2, NFYB, PBX3, MAZ, NFIC,
GTF2F1, MTA3, TAF1, TBL1XR1,
JUND, KDM5B, STAT5A, HDAC1,
SAP30, FOS, YY1, PHF8, FOXM1,
TBP, CEBPB, REST, TCF12, IRF1,
TEAD4, ZBTB7A, GABPA, MEF2A,
PML, RELA
TSS YES
cg15770702
(MAP3K14)ch 17:43384845
PML, STAT5A, NFATC1, CEBPB,
BCL3, TCF12, EBF1, FOXM1,
EP300, RELA, STAT3, NFIC,
TBL1XR1, JUND, MEF2A, PAX5,
BHLHE40, MEF2C, ATF2, SP1,
BATF, RUNX3, IRF4, BCL11A
TSS/T YES
cg16720947 (PLEC1)ch 8:145048137 n.a. YES
cg22794214 (HIVEP3) ch 1:42123463 CTCF WE/R YES
cg08596308
(ATP6V1G2;
NFKBIL1)
ch 6:31516045
CHD1, RBBP5, ZNF274, POLR2A,
E2F6, E2F4, KDM5B, MYC, MAX,
MAZ
TSS YES
cg23282964 (RIOK1) ch 6:7417780 TNO
cg21200667 (NA) ch 2:30628085 R YES
cg08421934 (NA) ch 6:33942413 R NO
cg08486432 (ITPR3) ch 6:33598003 T/R YES
cg08352439 (VOPP1) ch 7:55637123 POLR2A, POU2F2 TSS YES
cg25356639 (FOXP1) ch 3:71349304 R NO
cg04395703 (METAP1)
ch 4:99982762 TYES
cg03171419 (GPR124)ch 8:37700802 POLR2A TYES
cg26843567 (NA) ch 12:104846281 R YES
cg00291478 (RGS10) ch 10:121301041 RELA, RUNX3, RBBP5 TSS YES
cg27027151 (IL21R) ch 16:27461638
POLR2A, MTA3, NFATC1, RELA,
BCLAF1, EBF1 E/R YES
cg04182483 (RGS10) ch 10:121259610 TNO
Abb e ia ions: TSS, P edic ed p omo e egion including ansc ip ion s a si e (TSS); T, P edic ed ansc ibed egion; WE,
P edic ed weak enhance o open ch oma in cis- egula o y elemen ; E, P edic ed enhance ; R, P edic ed Rep essed o Low
Ac i i y egion
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p omo e egion including ansc ip ion s a si e, enhance
o weak enhance /open ch oma in. I is possible ha he
associa ion be ween hese si es and longe i y is media ed
h ough al e ed binding o TFs o me hyl-binding domain
p o eins, o which he la e ec ui ch oma in-modi ying
p o eins o achie e a ep essi e ch oma in s a e. Howe e ,
ou da a do no allow us o de e mine whe he dis up ed
egula ion o ch oma in pe missi eness unde lies he
inc eased mo ali y isk. In e es ingly, RELA, which is a
subuni o he NF- κB complex, was iden i ied o ha e
a binding si e on ou o he analyzed 19 CpG si es.
This obse a ion u he suppo s he hypo hesis o he
unc ional ole o NF-κB in old-age mo ali y.
Region o a p edic ed ansc ip ion s a si e
was obse ed o cg02395768 (ATP5SL), cg15770702
(MAP3K14), cg08596308 (ATP6V1G2; NFKBIL1),
cg08352439 (VOPP1) and cg00291478 (RGS10).
Howe e , he me hyla ion le els in hese si es we e no
co ela ed wi h gene exp ession (Supplemen a y Table
5). Ins ead, me hyla ion le els o cg03348466 (CRTC3),
cg22794214 (HIVEP3) and cg04182483 (RGS10)
co ela ed wi h he co esponding ansc ip exp ession
le el. The obse a ion ha he co ela ions we e o e all
modes is, howe e , in line wi h p e ious indings on
minimal co ela ions be ween age-associa ed changes
me hyla ion and ansc ip ion [5, 20, 21]. Six si es,
including cg03348466 (CRTC3) and cg04182483 (RGS10)
esided in p edic ed ansc ibed a ea, and can hence also
be conside ed unc ionally signi ican . The po en ial
egula o y ole o hese si es (in he gene body egion)
may in ol e e.g. al e na i e splicing. Howe e , he exac
mechanism connec ing he mo ali y-associa ed changes
in me hyla ion o al e na i e splicing equi es u he
esea ch.
The canonical pa hways cons uc ed om he
genes ha bo ing he op 250 mo ali y-associa ed CpG
si es a he 2.55-yea ollow-up co e ed a wide a ie y
o cellula signaling unc ions among which se e al
in lamma ion and immuni y- ela ed p ocesses we e
ep esen ed. In e es ingly, pa hways e med NF-κB
Ac i a ion by Vi uses, GADD45 Signaling and Cell
Cycle: G1/S Checkpoin Regula ion we e also iden i ied.
The eme gence o hese pa hways sugges s ha NF-κB
migh also be in ol ed la e-li e con ol o cellula g ow h
and su i al, DNA epai and apop osis, as hese unc ions
a e asc ibed o he induc ion o he NF-κB- GADD45
cascade [22]. In e es ingly, in ou p e ious pape on he
ansc ip omic mo ali y p edic o s, we obse ed ha an
inc eased exp ession o GADD45B was p edic i e o an
inc eased isk o mo ali y in hese nonagena ians [10].
Howe e , as he numbe o mo ali y-associa ed
CpG si es was ma kedly educed om he 2.55-yea s
ollow-up o he 4-yea s ollow-up, we specula e ha
he me hylomic ma ke s migh exhibi a dynamic na u e
e en in he ex eme ages. Tha is, a subs an ial pa o
he genomic CpG si es migh be cons an ly emodeled,
and du ing 4 yea s, hei me hyla ion le els a e likely
o change o an ex en ha hei p edic i e abili y in ou
popula ion is educed. The longe ollow-up ime also
allows mo e ime o s ochas ic mo ali y de e minan s,
such as auma, o ope a e, which may hus weaken he
ole o he genomic p edic o s.
Al hough he me hylomic ma ke s did no exhibi
e y s ong s a is ical signi icances a e FDR-co ec ion
and we used a libe al h eshold o including hem in
he Ridge eg ession (FDR < 0.5), he me hylomic
da a demons a ed good pe o mance in e ms o
gene alizabili y and disc imina i e powe . Speci ically,
he me hylomic da a alone exhibi ed be e p edic i e
accu acy han he con en ional ma ke s alone o in
combina ion wi h he me hylomic ma ke s, and he se en
CpG si es in he inal Cox model had a disc imina i e
powe o 89.9%. In his espec , he me hylomic da a
also pe o med be e han he ansc ip omic mo ali y
p edic o s in ou p e ious s udy [10]. Ne e heless, we
acknowledge ha he majo weaknesses o ou s udy a e
a lack o a sepa a e e i ica ion coho and a a he small
s udy popula ion. Hence, he esul s mus be conside ed as
en a i e and hypo hesis-gene a ing. The s eng h o ou
s udy, howe e , is he ac ha all he s udy pa icipan s
we e 90 yea s o age a baseline. The e o e ou esul s
a e no con ounded by he e ec o ch onological age on
DNAm.
A ecen s udy by Moo e e al. analyzed genome-
wide me hylomic mo ali y p edic o s in indi iduals
wi h a wide age ange (30-100 yea s a 9-yea ollow-
up, mean mo ali y ollow-up ime 4.4 yea s) [23]. They
iden i ied 76 CpG si es whe e he a e o change in DNAm
was associa ed wi h mo ali y and 88 ma ke s whe e he
yea 9 le el o DNAm was associa ed wi h mo ali y.
In e es ingly, hei mo ali y-associa ed DNAm si es
also included genes wi h immunoin lamma o y unc ions
and a link o NF-κB egula ion. Howe e , no o e lap
be ween indi idual mo ali y-associa ed CpG si es we e
ound in ou da a se s. These di e ences may a ise due o
di e en popula ion cha ac e is ics, such as age ange and
he causes o dea h. Hence, u he s udies a e equi ed
o es ablish he po en ially age and popula ion-speci ic
ela ionships be ween DNAm and mo ali y.
When we examined he se en inal signa u e
mo ali y-p edic ing CpG si es and hei co esponding
genes (Table 3) o o e lap wi h he genes ha bo ing he
mos commonly aging-associa ed CpG si es - ELOVL2,
FHL2 [2, 21, 24-26], KLF14 [2, 21, 25, 26], SST [2,
25, 26], OTUD7A [2, 24, 26], PENK [2, 21, 24, 25] and
EDARADD [2, 6, 24, 26] - we ound no o e lap be ween
hese ma ke s. Mo eo e , none o ou op 250 mo ali y-
associa ed me hylomic si es (Supplemen a y Table 2) we e
among he 525 common age-associa ed CpG si es ha
ha e been obse ed in mo e han one s udy (summa ized
in [21]). Moo e e al. [23] ha e also in obse ed a simila
phenomenon in hei popula ion: e y ew ( < 0,05%)
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o he aging-associa ed CpG si s we e also mo ali y-
associa ed. These obse a ions sugges ha aging-
associa ed epigene ic d i and he epigene ic con ol o
he li e span in old age migh ope a e h ough di e en
genomic mechanisms. This hypo hesis is also in line wi h
ou p e ious indings on age-associa ed ansc ip s [27],
which displayed e y li le simila i y wi h mo ali y-
p edic ing ansc ip s [10].
Despi e he inc easing body o da a ha sugges s
ha se e al mani es a ions o o ganismal aging and
de elopmen a e o epigene ic o igin, he associa ions
epo ed hus a on DNAm and aging-pheno ypes a e
sca ce and/o he indings ha e been nega i e. Bell
e al. (2012) examined he genome-wide associa ions
be ween DNAm and 16 age- ela ed pheno ypes and
ound ha wo pheno ypes - lung unc ion and low-
densi y lipop o ein le els - exhibi ed an associa ion wi h
one CpG si e (cg16463460 in WT1 and cg03001305 in
STAT5A, espec i ely) and ma e nal longe i y exhibi ed an
associa ion wi h wo CpG si es (cg13870866 in TBX20 and
cg09259772 in ARL4A) [8]. In ano he EWAS, Ma ioni
e al. (2015) de ec ed no indi idual CpG si es associa ed
wi h physical o cogni i e i ness in an elde ly popula ion
[28]. Howe e , hey did ind a c oss-sec ional associa ion
be ween a measu e o DNAm age - he epigene ic clock
based on he Ho a h p edic o [17] -, and physical and
cogni i e i ness ye he DNAm age was no p edic i e
o a longi udinal chance in he i ness measu es [28]. The
DNAm age has also been ecen ly demons a ed o p edic
all-cause mo ali y in ou di e en coho s o elde ly
indi iduals [29] and in Danish wins [30]. Howe e ,
he DNAm age was no p edic i e o mo ali y in ou
s udy. One eason o he nega i e inding migh be ha
indi iduals in ou coho we e all e y old a baseline
(90 yea s), and dea h a his age likely has di e en
unde pinnings han a younge old ages and when assessed
in coho s wi h wide age spec a.
In conclusion, he esul s o his s udy suppo
he genomic-le el ole o NF-κB a he e y end o he
human li e span. We hypo hesize ha ou indings could
ela e o he ecen obse a ion o a p og amma ic ole o
hypo halamic NF-κB and IκB kinase-β ac i a ion in he
con ol o he li e span in expe imen al mouse models
[16]. Adhe ing o he conclusion o his mouse s udy
ha he decisi e ole o hypo halamic NF-κB is exe ed
sys emically le el h ough immune-neu oendoc ine
c oss alk [16], we sugges ha ou indings on immune
cells migh ep esen he pe iphe al co espondence o
hypo halamic NF-κB ac i a ion. Howe e , es ablishing
he sys emic-le el e en s ha connec NF-κB unc ion
o all cause-mo ali y in aged humans will equi e u he
esea ch.
MATERIALS AND METHODS
S udy popula ion
The s udy popula ion consis ed o nonagena ian
subjec s pa icipa ing o he Vi ali y 90+ s udy, which
is an ongoing, p ospec i e popula ion-based s udy on
indi iduals aged 90 yea s and abo e and who eside in
he ci y o Tampe e, Finland. The Vi ali y 90+ s udy was
ini ia ed in 1995, and since hen se e al nonagena ian
coho s ha e been ec ui ed and examined o biological,
clinical, demog aphic and social measu es. Mo ali y
a es ha e been analyzed longi udinally using comple e
ollow-ups. The ec ui men p o ocol and cha ac e iza ion
o he subjec s in he cu en s udy has been p e iously
desc ibed [10]. The da a in his s udy conce n indi iduals
bo n in 1920 and ec ui ed in 2010 o sample collec ion.
Genome-wide me hyla ion da a and he ull co a ia e da a
including cell ype p opo ions we e a ailable o 111
subjec s (n = 81 women and n = 30 men). The all-cause
mo ali y da a we e collec ed om he Popula ion Regis e
Cen e . As we wan ed o assess bo h sho e and longe -
e m su i al p edic o s o his coho , he mo ali y
da a was collec ed in wo di e en ime poin s. The i s
da a collec ion was pe o med on 31s o Janua y in 2013
co esponding o a 2.55-yea median ollow-up and he
second one was on 31
s
o May in 2014 co esponding o
a 4-yea ollow-up. The mo ali y a e a he 2.55-yea
ollow-up was 32.4% (36/111) and 47.7% (53/111) a he
4-yea ollow-up. All he pa icipan s ga e hei w i en
in o med consen . The s udy was conduc ed ollowing he
guidelines o he Decla a ion o Helsinki, and he s udy
p o ocol was app o ed by he e hics commi ee o he ci y
o Tampe e.
Sample collec ion and p ocessing
Venous blood samples we e collec ed in EDTA-
con aining ubes by a ained home- isi ing medical
s uden be ween 8 am and 12 am. Plasma was sepa a ed
and s o ed a -70°C. Genomic DNA and o al RNA we e
ex ac ed om PBMCs in which he blood samples we e
subjec ed o leucocy e sepa a ion using a Ficoll-Paque
densi y g adien (Ficoll-Paque™ P emium, ca . no. 17-
5442-03, GE Heal hca e Bio-Sciences AB, Uppsala,
Sweden). The PBMC laye was collec ed, and he cells
alloca ed o RNA ex ac ion we e suspended in 150 µl o
RNAla e solu ion (Ambion Inc., Aus in, TX, USA). Cells
ha we e alloca ed o FACS analysis and DNA ex ac ion
we e suspended in 1 ml o a eezing solu ion (5/8 FBS,
2/8 RPMI-160 medium, 1/8 DMSO; FBS ca . no. F7524,
Sigma-Ald ich, MO, USA; RPMI: ca . no. R0883, Sigma-
Ald ich, MO, USA; DMSO: ca . no. 1.02931.0500, VWR,
Espoo, Finland).