Seasonality Modifies Methylation Profiles in Healthy People
Citation: Ricceri F, Trevisan M, Fiano V, Grasso C, Fasanelli F, et al. (
Seasonality Modifies Methylation Profiles in Healthy People
Fulvio Ricceri 0
Morena Trevisan 0
Valentina Fiano 0
Chiara Grasso 0
Francesca Fasanelli 0
Chiara Scoccianti 0
Laura De Marco 0
Anna Gillio Tos 0
Paolo Vineis 0
Carlotta Sacerdote 0
Javier S. Castresana, University of Navarra, Spain
0 1 Unit of Cancer Epidemiology - CERMS, Department of Medical Sciences, University of Turin and Citta` della Salute e della Scienza Hospital , Turin , Italy , 2 Department of Mathematics, University of Turin , Turin, Italy, 3 IARC, Lyon, France, 4 Human Genetics Foundation (HUGEF), Turin , Italy , 5 Imperial College , London , United Kingdom
DNA methylation is a well-characterized epigenetic modification that plays an important role in the regulation of gene expression. There is growing evidence on the involvement of epigenetic mechanisms in disease onset, including cancer. Environmental factors seem to induce changes in DNA methylation affecting human health. However, little is known about basal methylation levels in healthy people and about the correlation between environmental factors and different methylation profiles. We investigated the effect of seasonality on basal methylation by testing methylation levels in the long interspersed nucleotide element-1 (LINE-1) and in two cancer-related genes (RASSF1A and MGMT) of 88 healthy male heavy smokers involved in an Italian randomized study; at enrolment the subjects donated a blood sample collected in different months. Methylation analyses were performed by pyrosequencing. Mean methylation percentage was higher in spring and summer for the LINE1, RASSF1A and MGMT genes (68.26%, 2.35%, and 9.52% respectively) compared with autumn and winter (67.43%, 2.17%, and 8.60% respectively). In particular, LINE-1 was significantly hypomethylated (p = 0.04 or 0.05 depending on the CpG island involved) in autumn and winter compared with spring and summer. Seasonality seems to be a modifier of methylation levels and this observation should be taken into account in future analyses.
DNA methylation is a well-characterized epigenetic modification
that involves the addition of a methyl group to cytosine when paired
to guanine (CpG). DNA methylation plays an important role in the
regulation of gene expression. Alterations in DNA methylation
pattern mostly lead to silencing of inducible genes (promoter
hypermethylation) or to re-expression of physiologically silenced
repetitive sequences and transposable elements (hypomethylation).
As a result, molecular pathway deregulation as well as chromosomal
instability and increased mutational events may occur, thus
favouring onset of diseases, including cancer . Environmental
factors seem to affect DNA methylation patterns.  Exposure to
carcinogens , air pollution [6,7], and nutritional factors 
were described to be associated with variation of DNA methylation
status in exposed subjects compared to unexposed ones.
Little is known about modulation in the basal methylation level
in healthy people and in particular about the correlation between
season-linked environmental factors and different methylation
profiles, although seasonality was described to sporadically
influence epigenetic events in different organisms, including plants
[9,10], animals , and humans .
To investigate in depth if seasonality may impact methylation
level in healthy people, we focused on selected genes frequently
tested in studies of association between DNA methylation and
cancer. We aimed at evaluating if variability in healthy subjects
basal methylation level has to be taken into account to avoid
biased results. Three genes were selected to be investigated in the
blood DNA of a series of healthy people enrolled in a previous
study of association between diet and DNA damage : LINE-1,
RASSF1A and MGMT. Transposable methylation of LINE-1
elements (long interspersed nucleotide element-1) is generalized to
reflect global DNA methylation [16,17]. RASSF1A
(Ras-association domain family 1 isoform A) is a tumour suppressor gene
regulated by promoter methylation, which leads to inhibition of its
expression. Promoter hypermethylation of this gene is related to
carcinogenesis.  MGMT (O6-methylguanine DNA
methyltransferase) is a gene involved in DNA repair mechanisms. The
promoter hypermethylation of MGMT inactivates different DNA
repair pathways and as suggested [21,22], this event occurs in
sporadic and hereditary cancer.
Subjects and Methods
In the frame of an Italian randomized trial (approved by the
local ethics committee) focused on the study of the association
between diet and DNA damage in heavy smokers (for further
details see ref [15,23,24]); hence 88 healthy heavy smokers of
aircured tobacco were recruited among Italian blood donors.
Participants were all males, aged 3570, residents in Turin
metropolitan area (Northern Italy). At the baseline, volunteers
filled in a validated food frequency questionnaire.  Twenty mL
of non-fasting blood were collected at the beginning of the study
and one year later; after processing, they were fractioned in plasma
and buffy coat and immediately stored in aliquots at 220. Herein,
only samples collected at time zero were used. Because the study
subjects were enrolled at different months of the year, we could
group the blood samples by season.
All participants signed an informed consent form. The study
was approved by the ethical committee of the Department of
Biomedical Sciences and Human Oncology, University of Torino,
and was monitored by the Association of General Practitioners of
the Province of Torino.
DNA extraction and sodium bisulfite treatment
Genomic DNA was extracted from 200 ml aliquots of buffy coat
through QIAamp DNA Blood Mini Kit (Qiagen, Hilden,
Germany) according to manufacturers instructions, with a final
elution in 70 ml of TE elution buffer.
All the genomic DNA samples, in addition to positive synthetic
controls for methylated and unmethylated status, underwent
bisulfite modification using Epitect Bisulfite Kit (Qiagen, Hilden,
Germany) according to manufacturers instructions.
Pyrosequencing assays were performed on a PyroMark Q24
using PyroMark Gold reagents (Qiagen). This updated system
incorporates a dedicate software for CpG analysis which allows a
more accurate quantization of methylation compared with AQ
software of PyroMark Q96 that is still being widely used for this
Primers were generated according to PyroMark Assay Design
software (Qiagen) for all the gene specific target sequences.
Preliminary PCR targeting the selected gene sequences to be
investigated were performed by using primers listed in Table 1 at
corresponding annealing temperature.
The preliminary PCR reactions were performed in a total
volume of 35 ml containing buffer (KCl) 1X, MgCl2 2 mM,
dNTPs 0.8 mM, 0.5 mM each primer Taq polymerase 1 U and
3 ml converted DNA with the following touchdown PCR profile
decreased of 3uC every 3 cycles until the specific annealing
temperature was reached: 94uC for 5 min followed by 3 cycles at
94uC for 30 sec, starting annealing temperature at 62uC for
30 sec, 72uC for 30 sec. When the gene specific annealing
temperature was reached, further 30 cycles were performed.
Amplicons were analyzed by gel electrophoresis on a 2% agarose
gel stained with ethidium bromide and visualized by ultraviolet
trans-illumination. Twenty ml of PCR product were added to 18 ml
of distilled water and incubated under shaking with 40 ml of
binding buffer pH 7.6 (10 mM Tris-HCl; 2 M NaCl; 1 mM
EDTA; 0.1% Tween 20) and 2 ml sepharose beads covered with
streptavidin. PCR products were washed with ethanol 70%,
denatured with NaOH 0.2 M and re-washed with Tris-Acetate
10 mM pH 7.6. Pyrosequencing reaction was performed in 25 ml
of annealing buffer [23.25 ml of 20 mM Tris-Acetate+5 mM
MgAc2, 0.15 ml of sequencing primer (0.3 mM) and 1.25 ml of
dimethyl sulfoxide (DMSO)]. Assays were created according to
manufacturers instruction and the nucleotide dispensation order
was suggested by the software used.
For consistency we tested the three genes by performing all the
methylation analyses on the PyroMark Q24, even though
RASSF1A and LINE-1 had been already analyzed in a previous
study.  Use of different pyrosequencing instruments
incorporating different analytical software may explain some discrepancies
in the absolute values of methylation percentage obtained.
We presented our results according to seasonality. Two groups,
one including samples collected in spring and summer and another
consisting of samples collected in autumn and winter, were
We presented summary data as means, medians and standard
deviations (SD) for continuous variables and as percentages for
categorical variables. Covariates/potential confounders were
tested using Wilcoxon rank sum test or Chi-square test, for
continuous variables or categorical variables, respectively. In order
to test differences in methylation levels we used Wilcoxon rank
sum test with continuity correction. We also presented the
differences in methylation levels for each tested CpG site. To
explain the seasonal differences, an analysis on the intake of
nutrients eaten the day before sampling was conducted.
Correlations between food groups and methylation were tested using
Pearsons correlation coefficient.
All tests were two-sided and we used a 5% significance
Analyses were performed using SAS V9.3.
The subjects mean age involved in the study was 52.42 (6.74
SD) and they were all males and heavy smokers at the time of the
analysis. The mean of Body Mass Index (BMI) was 26.40 kg/m2
(3.29 SD). Table 2 shows that no differences were found in the
study for covariates/potential confounders in different seasons.
AACTCCCTAACCCCTTAC Annealing T
* Target: 194 bp of MGMT (Gene ID 4255 at position 4452644719) promoter sequence including 6 CpGs at position: 44600, 44604, 44607, 44614, 44621, 44623.
1 Target: 136 bp of RASSF1A (Gene ID 11186 at position 16971833) promoter sequence including 6 CpGs at position: 1746, 1750, 1755, 1757, 1767, 1780.
# Target: 108 bp of LINE-1 (GeneBank X58075.1 at position 117224) promoter sequence including 6 CpGs at position: 156, 131, 165, 167, 172, 182.
Results of the overall methylation analysis are shown in Table 3
where methylation is reported as mean/median percentage of
methylation of all the CpG sites investigated for the specific gene
and in Figure 1, in which average methylation for the different
CpG islands is reported for each gene. In all the loci analyzed, the
methylation status was higher in spring and summer compared
with autumn and winter. In particular, LINE-1 was borderline
significantly hypermethylated (p = 0.05) in spring/summer
compared with autumn/winter and this difference is significant for 3
over 6 sites (CpG II, III, VI) in LINE-1 and for 1 site (CpG II) in
MGMT. The samples of the paired season group showed similar
methylation levels (data not shown).
The additional analysis on nutrients showed a slight and
nonsignificant difference for Vitamin B6 (1.45 mg in autumn/winter
vs 1.52 in spring/summer), B9 (195 mg vs 206 mg), and B12
(0.97 mg vs 1.00 mg), while a significant increase in autumn/
winter vs. spring/summer was found for flavonoids (107.79 mg vs
65.69 mg, p-value = 0.03).
Only low correlations (r,|0.30|) between methylation and
food groups were found, although some of correlation coefficients
were statistically significant (data not shown).
There is growing evidence on the involvement of epigenetic
mechanisms in disease onset, in particular cancer. However, little
is known about the determinants of basal methylation levels in
healthy people. Whereas studies in healthy people were mainly
focused on the identification of a correlation between methylation
and gene expression , few reports are available on intra- and
inter-individual differences in methylation among healthy people.
Commonly, healthy people are included in case-control studies
and sources of variability in methylation levels of the control group
subjects are rarely considered.
In our study we found differences in methylation levels of
selected genes in healthy adults according to seasonality. We
divided the methylation results into two groups showing overall
similar intra-group methylation levels: one including subjects
enrolled in spring and summer and the other including those
enrolled in autumn and winter. This seasonal grouping is in line
with previous reported data on seasonality  and allowed us to
compare groups with a larger sample size.
In the spring and summer group we observed a pattern of
higher methylation in all the promoter regions of genes we
analyzed, compared with the other. The differences, in spite of not
being statistically significant (likely due to the small sample size of
the study), but borderline for LINE-1, are suggestive of the
possible impact on basal DNA methylation levels of seasonal
determinants. The observed differences are not likely to reflect
exposure to inhaled tobacco carcinogens which did not vary
between seasons. Also, while the study subjects are slightly
overweight on average (Table 2), at present there is no evidence
of an association of DNA methylation with body mass index. 
Air pollution could be one of the likely determinants: Baccarelli et
al.  showed that variation in methylation levels of LINE-1 in
healthy subjects was not only associated with season, but also with
the day of the week, being higher on Wednesdays than Mondays.
Our data are in line with recent reports showing evidence of
seasonal variability in air composition, daytime light, temperature
and habits that may affect DNA methylation in all the living
organism, human, animals and plants.
In several studies  short-term effects of pollutants on
health were more evident in spring and summer than in the coldest
seasons, and more in southern countries than in northern
countries. One proposed interpretation of this finding has been
that higher exposure to outdoor air pollutants during the summer
period might be due to increased indoor ventilation and seasonal
differences in outdoor human activity. Other differences may be
due to the chemical mixture in outdoor pollution that varies with
season, source pattern and weather. 
Additionally, ozone levels have an opposite seasonality pattern
compared with other air pollutants; the highest levels of ozone in
the Northern Hemisphere were found in the spring and summer
and the lowest in the autumn and winter. Ozone has been shown
to have important detrimental health effects  and it is known
that some individuals are particularly susceptible to ozone adverse
effects. Genetic and epigenetic mechanisms have been invoked to
explain differential susceptibility and alterations in DNA
methylation could be involved.
Circadian rhythms were also described to affect basal DNA
methylation status.  Exposure to light at night through
long-term shift work can influence DNA methylation and may
result in epigenetic alterations of biological pathways and
Another possible explanation of the association between
variation in healthy peoples basal methylation levels and
seasonality is the change in outdoor temperature. Seasonal
environmental temperature changes regulate the life of plants
and aquatic species. Adaptation to cold and warm conditions
requires dramatic changes in gene expression. For example, in
eurythermal fish adaptation to cold conditions requires
considerable changes at cellular level in gene expression regulated by
epigenetic mechanisms, such as methylation.  According to
Baccarelli  LINE1 methylation showed an association with
mean outdoor temperature of the day when the blood was drawn;
despite not being significant, this factor was consistent with
changes observed by season.
We observed a similar relationship between biomarker and
seasonality in a pooled analysis on bulky DNA adducts. . DNA
adducts showed lower levels in spring than in winter. For this
result our explanation was more straightforward, i.e. a protective
effect of seasonal intakes of fresh fruit and vegetables and higher
levels of exposure to particulate outdoor pollution in winter.
Results from the European Prospective Investigation into
Cancer and Nutrition (EPIC study)  showed that dietary
folate intake is lower in summer, especially in southern European
countries, because summer heat causes bolting and foliage
Study subjects N = 88
Spring/Summer N = 29 mean% (SD)
Autumn/Winter N = 59 mean% (SD)
P values are based on Wilcoxon rank sum test or chi square test.
P-values are based on Wilcoxcon rank sum test.
Figure 1. Box plot for percentage of methylation of RASSF1A (Panel A), MGMT (Panel B), LINE1 (Panel C) divided by seasonality (0 =
autumn/winter; 1 = spring/summer). P-values are based on Wilcoxcon rank sum test.
deterioration in vegetables. Moreover, in a recent paper  the
authors observed that naturally occurring seasonal variations in
food consumption patterns have a profound effect on
methyldonor biomarker status. Therefore, despite expecting lower
methylation levels in summer, we found an opposite result,
possibly suggesting that dietary intake of folate may not be major
determinant of methylation levels. 
Similar evidence was highlighted for the increased level of
flavonoids in autumn/winter.  Although the debate is still
open , different in vitro studies showed that flavonoids (in
particular, genistein) act as a DNA demethylating agent, inducing
a dose-dependent inhibition of the DNA-methyltrasferase activity.
It is also to be taken into account that in different seasons can
occur a preferential selection of particular peripheral blood cell
types . Thus white blood cell may have different patterns of
DNA methylation.among seasons.
Finally, many seasonally related candidate gene pathways seem
to be regulated by epigenetic mechanisms, DNA methylation
included. This evidence is strengthened by the results described in
rodent species, where deeper laboratory investigations can be
performed.  For example, in blood of hibernating chipmunks,
the hibernation-specific protein-27 is known to be upregulated by
DNA methylation.  As well, in ground squirrels has been
shown that a protein linked to the regulation of the circadian
rhythms in the liver affected the de novo methyltransferase
probably resulting in the metabolic depression during hibernation.
 In mice, DNA methylation has been shown to play a role in
the suprachiasmatic nucleus of the hypothalamus, which regulates
circadian behaviour in mammals. 
Moreover, epigenetic changes may explain the seasonal
reproductive phenotypes in seasonally breeding vertebrates. In
hamsters, changes in photoperiod have been shown to induce
variation in reproductive phenotypes mediated by DNA
One limitation of our study is that the differences we found in
methylation levels by seasonality were small, but this is consistent
with variability of the basal methylation level, specifically in
cancer-related genes such as RASSF1A and MGMT which are a
priori expected to show small changes in promoter methylation in
healthy people. Otherwise, LINE-1 sequences are expected to be
highly methylated since they are physiologically silenced in healthy
people. Because these sequences are highly repetitive along the
genome, even hardly detectable differences could assume
relevance in terms of chromosomal instability or increased mutational
Another limitation is the study was performed only on male
heavy-smoker, hence further investigations are needed to
extrapolate our findings to more general population.
However the strength of this study is represented by
investigating for the first time in healthy subjects a seasonal pattern of DNA
methylation in genes usually studied in association with neoplastic
In conclusion, seasonality seems to be a modifier of basal DNA
methylation level in healthy people. These findings suggest that
variability in the basal methylation level of healthy controls should
be taken into account in future methylation data analyses.
Adjustment for season of sample collection is advisable to avoid
biased results, specifically when the difference in methylation levels
of the study groups is expected to be moderate.
This work has been made possible by grants from the World Cancer
Research Fund and Compagnia di San Paolo (Torino) to Paolo Vineis, and
partially supported by Piedmont Region. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of
the manuscript. We would like to thank dr. Andrea Serra for the language
Conceived and designed the experiments: FR LDM AGT PV CaS.
Performed the experiments: MT VF CG ChS LDM AGT. Analyzed the
data: FR FF CaS. Contributed to the writing of the manuscript: FR MT
VF CG FF ChS LDM AGT PV CaS.
1. Baylin SB , Jones PA ( 2011 ) A decade of exploring the cancer epigenome - biological and translational implications . Nat Rev Cancer 11 : 726 - 734 .
2. Jirtle RL , Skinner MK ( 2007 ) Environmental epigenomics and disease susceptibility . Nat Rev Genet 8 : 253 - 262 .
3. DeMarini DM ( 2013 ) Genotoxicity biomarkers associated with exposure to traffic and near-road atmospheres: a review . Mutagenesis 28 : 485 - 505 .
4. Pogribny IP , Beland FA ( 2012 ) DNA methylome alterations in chemical carcinogenesis . Cancer Lett.
5. Arita A , Shamy MY , Chervona Y , Clancy HA , Sun H , et al. ( 2012 ) The effect of exposure to carcinogenic metals on histone tail modifications and gene expression in human subjects . J Trace Elem Med Biol 26 : 174 - 178 .
6. Vineis P , Husgafvel-Pursiainen K ( 2005 ) Air pollution and cancer: biomarker studies in human populations . Carcinogenesis 26 : 1846 - 1855 .
7. Baccarelli A , Wright RO , Bollati V , Tarantini L , Litonjua AA , et al. ( 2009 ) Rapid DNA methylation changes after exposure to traffic particles . Am J Respir Crit Care Med 179 : 572 - 578 .
8. Lim U , Song MA ( 2012 ) Dietary and lifestyle factors of DNA methylation . Methods Mol Biol 863 : 359 - 376 .
9. Coustham V , Li P , Strange A , Lister C , Song J , et al. ( 2012 ) Quantitative modulation of polycomb silencing underlies natural variation in vernalization . Science 337 : 584 - 587 .
10. Sherman JD , Talbert LE ( 2002 ) Vernalization-induced changes of the DNA methylation pattern in winter wheat . Genome 45 : 253 - 260 .
11. Pinto R , Ivaldi C , Reyes M , Doyen C , Mietton F , et al. ( 2005 ) Seasonal environmental changes regulate the expression of the histone variant macroH2A in an eurythermal fish . FEBS Lett 579 : 5553 - 5558 .
12. Waterland RA , Kellermayer R , Laritsky E , Rayco-Solon P , Harris RA , et al. ( 2010 ) Season of conception in rural gambia affects DNA methylation at putative human metastable epialleles . PLoS Genet 6 : e1001252 .
13. Kurian JR , Terasawa E ( 2013 ) Epigenetic control of gonadotropin releasing hormone neurons . Front Endocrinol (Lausanne) 4: 61.
14. De Prins S , Koppen G , Jacobs G , Dons E , Van de Mieroop E , et al. ( 2013 ) Influence of ambient air pollution on global DNA methylation in healthy adults: a seasonal follow-up . Environ Int 59 : 418 - 424 .
15. Talaska G , Al-Zoughool M , Malaveille C , Fiorini L , Schumann B , et al. ( 2006 ) Randomized controlled trial: effects of diet on DNA damage in heavy smokers . Mutagenesis 21 : 179 - 183 .
16. Schulz WA ( 2006 ) L1 retrotransposons in human cancers . J Biomed Biotechnol 2006 : 83672 .
17. Heyn H , Esteller M ( 2012 ) DNA methylation profiling in the clinic: applications and challenges . Nat Rev Genet 13 : 679 - 692 .
18. Richter AM , Pfeifer GP , Dammann RH ( 2009 ) The RASSF proteins in cancer; from epigenetic silencing to functional characterization . Biochim Biophys Acta 1796 : 114 - 128 .
19. Gsur A , Haidinger G , Hinteregger S , Bernhofer G , Schatzl G , et al. ( 2001 ) Polymorphisms of glutathione-S-transferase genes (GSTP1, GSTM1 and GSTT1) and prostate-cancer risk . Int J Cancer 95 : 152 - 155 .
20. Campaner S , Doni M , Verrecchia A , Faga G , Bianchi L , et al. ( 2010 ) Myc, Cdk2 and cellular senescence: Old players, new game . Cell Cycle 9 : 3655 - 3661 .
21. Fahrer J , Kaina B ( 2013 ) O6-methylguanine-DNA methyltransferase in the defense against N-nitroso compounds and colorectal cancer . Carcinogenesis 34 : 2435 - 2442 .
22. Bujko M , Kowalewska M , Danska-Bidzinska A , Bakula-Zalewska E , Siedecki JA , et al. ( 2012 ) The promoter methylation and expression of the O6-methylguanine-DNA methyltransferase gene in uterine sarcoma and carcinosarcoma . Oncol Lett 4 : 551 - 555 .
23. Malaveille C , Hautefeuille A , Pignatelli B , Talaska G , Vineis P , et al. ( 1998 ) Antimutagenic dietary phenolics as antigenotoxic substances in urothelium of smokers . Mutat Res 402 : 219 - 224 .
24. Guarrera S , Sacerdote C , Fiorini L , Marsala R , Polidoro S , et al. ( 2007 ) Expression of DNA repair and metabolic genes in response to a flavonoid-rich diet . Br J Nutr 98 : 525 - 533 .
25. Pisani P , Faggiano F , Krogh V , Palli D , Vineis P , et al. ( 1997 ) Relative validity and reproducibility of a food frequency dietary questionnaire for use in the Italian EPIC centres . Int J Epidemiol 26 Suppl 1 : S152 - 160 .
26. Scoccianti C , Ricceri F , Ferrari P , Cuenin C , Sacerdote C , et al. ( 2011 ) Methylation patterns in sentinel genes in peripheral blood cells of heavy smokers: Influence of cruciferous vegetables in an intervention study . Epigenetics 6 : 1114 - 1119 .
27. van Eijk KR , de Jong S , Boks MP , Langeveld T , Colas F , et al. ( 2012 ) Genetic analysis of DNA methylation and gene expression levels in whole blood of healthy human subjects . BMC Genomics 13 : 636 .
28. Zhang FF , Santella RM , Wolff M , Kappil MA , Markowitz SB , et al. ( 2012 ) White blood cell global methylation and IL-6 promoter methylation in association with diet and lifestyle risk factors in a cancer-free population . Epigenetics 7 : 606 - 614 .
29. Samoli E , Aga E , Touloumi G , Nisiotis K , Forsberg B , et al. ( 2006 ) Short-term effects of nitrogen dioxide on mortality: an analysis within the APHEA project . Eur Respir J 27 : 1129 - 1138 .
30. Biggeri A , Bellini P , Terracini B ( 2004 ) [Meta-analysis of the Italian studies on short-term effects of air pollution-MISA 1996-2002] . Epidemiol Prev 28 : 4 - 100 .
31. Bell ML , Dominici F ( 2008 ) Effect modification by community characteristics on the short-term effects of ozone exposure and mortality in 98 US communities . Am J Epidemiol 167 : 986 - 997 .
32. Bauer AK , Kleeberger SR ( 2010 ) Genetic mechanisms of susceptibility to ozoneinduced lung disease . Ann N Y Acad Sci 1203 : 113 - 119 .
33. Shi F , Chen X , Fu A , Hansen J , Stevens R , et al. ( 2013 ) Aberrant DNA methylation of miR-219 promoter in long-term night shiftworkers . Environ Mol Mutagen 54 : 406 - 413 .
34. Zhu Y , Stevens RG , Hoffman AE , Tjonneland A , Vogel UB , et al. ( 2011 ) Epigenetic impact of long-term shiftwork: pilot evidence from circadian genes and whole-genome methylation analysis . Chronobiol Int 28 : 852 - 861 .
35. Vollmers C , Schmitz RJ , Nathanson J , Yeo G , Ecker JR , et al. ( 2012 ) Circadian oscillations of protein-coding and regulatory RNAs in a highly dynamic mammalian liver epigenome . Cell Metab 16 : 833 - 845 .
36. Ricceri F , Godschalk RW , Peluso M , Phillips D , Agudo A , et al. ( 2010 ) Bulky DNA adducts in white blood cells: a pooled analysis of 3600 subjects . Cancer Epidemiol Biomarkers Prev.
37. Park JY , Nicolas G , Freisling H , Biessy C , Scalbert A , et al. ( 2012 ) Comparison of standardised dietary folate intake across ten countries participating in the European Prospective Investigation into Cancer and Nutrition . Br J Nutr 108 : 552 - 569 .
38. Dominguez-Salas P , Moore SE , Cole D , da Costa KA , Cox SE , et al. ( 2013 ) DNA methylation potential: dietary intake and blood concentrations of onecarbon metabolites and cofactors in rural African women . Am J Clin Nutr 97 : 1217 - 1227 .
39. Rietjens IM , Sotoca AM , Vervoort J , Louisse J ( 2013 ) Mechanisms underlying the dualistic mode of action of major soy isoflavones in relation to cell proliferation and cancer risks . Mol Nutr Food Res 57 : 100 - 113 .
40. Attwood JT , Yung RL , Richardson BC ( 2002 ) DNA methylation and the regulation of gene transcription . Cell Mol Life Sci 59 : 241 - 257 .
41. Fang MZ , Chen D , Sun Y , Jin Z , Christman JK , et al. ( 2005 ) Reversal of hypermethylation and reactivation of p16INK4a, RARbeta, and MGMT genes by genistein and other isoflavones from soy . Clin Cancer Res 11 : 7033 - 7041 .
42. Li Y , Liu L , Andrews LG , Tollefsbol TO ( 2009 ) Genistein depletes telomerase activity through cross-talk between genetic and epigenetic mechanisms . Int J Cancer 125 : 286 - 296 .
43. Wu HC , Delgado-Cruzata L , Flom JD , Kappil M , Ferris JS , et al. ( 2011 ) Global methylation profiles in DNA from different blood cell types . Epigenetics 6 : 76 - 85 .
44. Alvarado S , Fernald RD , Storey KB , Szyf M ( 2014 ) The Dynamic Nature of DNA Methylation: A Role in Response to Social and Seasonal Variation . Integr Comp Biol . 54 : 68 - 76 .
45. Fujii G , Nakamura Y , Tsukamoto D , Ito M , Shiba T , Takamatsu N ( 2006 ) CpG methylation at the USF-bindingsite is important for the liver-specific transcription of thechipmunk HP-27 gene . Biochem J 395 : 203 - 9 .
46. Maekawa F , Shimba S , Takumi S , Sano T , Suzuki T , et al. ( 2012 ) Diurnal expression of Dnmt3b mRNA in mouse liver is regulated by feeding and hepatic clockwork . Epigenetics 7 : 1046 - 56 .
47. Azzi A , Dallmann R , Casserly A , Rehrauer H , Patrignani A , et al. ( 2014 ) Circadian behavior is light-reprogrammed by plastic DNA methylation . Nat Neurosci 17 : 377 - 82 .
48. Stevenson TJ1 , Prendergast BJ ( 2013 ) Reversible DNA methylation regulates seasonal photoperiodic time measurement . Proc Natl Acad Sci USA 110 : 16651 - 6 .