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THE EPIGENETIC DISTURBANCES IN
HUMAN BLOOD LEUKOCYTES IN REMOTE
PERIOD AFTER RADIATION EXPOSURE
Nina Kuzmina, Nellya Lapteva,
Aleksandr Rubanovich
N.I. Vavilov Institute of General Genetics,
Russian Academy of Sciences,
Moscow, Russia
[email protected]
DNA methylation - a main epigenetic
genomic modification
CpG islands (CGIs)

short genomic regions (500 bp to a few kb)

C+G ≥ 0.5, CpG obs ≥ 0.6
CpG exp

located in the proximal promoter region of
approximately 75% of human genes

unmethylated CpG dinucleotides of CGIs are
associated with active promoters

hypermethylation of CGIs leads to transcriptional
repression and gene inactivation
Radiation-induced changes in DNA methylation patterns
The investigations in vivo using
animal models (rodents)
The investigations in vitro
using cells
Main results
Authors
1. DNA hypomethylation LINE-1 and Alu repetitive elements in
Aupar et al., 2011
various cell lines (16-20 population doublings).
Goetz et al., 2011
DNA hypermethylation at SAT2 and MLT1A repetitive elements in
Kaup et al., 2006
HPV-G cells (20 population doublings,), but hypomethylation, which is
not identified with respect to the sequence context
Association with genomic instability.
2. Promoter hypermethylation
Antwih et al., 2013
Association of promoter methylation with radiosensitivity of malignant Zhou et al., 2007; Leung et al.,
cells
2008; Xu et al., 2008
1. Reduction of the levels of various epigenetic indicators :
a) global genomic hypomethylation, tri-methylation of
histone H4-Lys20, methyltransferases Dnmt1 and
Dnmt3a/b, methyl-binding proteins MeCP2 and MBD2)
b) loss of imprinting
с) relationship between methylation disturbances, genomic
instability, and bystander effect
2. promoter hypermethylation and its locus-specific and
tissue-specific character
Koturbash et al., 2006; Pogribny et
al., 2005; Loree et al., 2006;
Raiche et al., 2004
Zhu et al., 2006
Bernal et al., 2013; Nandakumar et
al., 2011; Kovalchuk et al., 2004;
Wang et al., 2014; Lima et al., 2014
The results of the study of methylation pattern changes in blood
cells of BALB/c
mice exposed
chronic
irradiation
The most pronounced
effects to
were
observed
in blood cells.(Х-rays,
0,05
Gy/d
x 10d)
These changes
were
sustained
after 1 month.
Jingzi Wang, Youwei Zhang, Kai Xu et al. Genome-wide screen of DNA methylation
changes induced by low dose X-ray radiation in mice // PLoS One. 2014; 9(3)
Most
pronounced
effect
Most
pronounced
revealed
effect in
1. Significant loss of global DNA methylation was
blood cells 2 h
postirradiation. However, these changes had been repaired after 1 month
2h
(HPLC-method).
2. Hypermethylation of 811 promoter regions, which covered almost all key
biological processes, was revealed
1 month 2 h postirradiation (MeDIP-on-chip).
Eight hypermethylated genes (Rad23b, Tdg, Ccnd1, Ddit3, Llgl1, RasI11a,
Tbx2, Scl6a15) were verified by MeDIP-qPCR.
3. The hypermethylation of Rad23b and Ddit3 genes was revealed in cells
of blood and other tissues 2 hours and 1 month postirradiation (bisulfate
sequencing)
Actuality of problem
In the several wide-genome investigations the association between hypermethylation
and chronological age (r = 0.80 - 0.95 for different tissues) was established (Day K. et al.,
Genome Biology, 2013; Johansson Asa et al., PLoS One, 2013; Horvath S., Genome Biology, 2013; Hannum G,
et al., Mol Cell, 2013).
The accuracy of the age forecast based on hypermethylation estimation of 353 CpG
sites is +/- 3.6 years (Horvath S., Genome Biology, 2013)
Hypermethylation of some genes observed
in malignant cells in patients with
oncological diseases, was revealed in
leukocytes peripheral blood of these subjects
also (Al-Moundhri M. S. et al., PLoS One, 2010;
Flanagan J. M., et al., Human Molecular
Genetics, 2009; Tahara T. et al., Cancer Prev. Res.,
2013)
Ionizing radiation may make
additional contribution in
the hypermethylation
processes
Hypermethylation of some genes in leukocytes
blood DNA samples was revealed in patients
with nononcological age-related disease,
above all with cardiovascular
(Lakshmi Sana V. et al., Molecular and Cellular
Biochemistry, 2013; Kim G. H. et al., Antioxid
Redox Signal, 2013
Radiation-induced
premature aging and
age – related disease
«epigenetic clock»
of irradiated organism
The aim of investigation: to evaluate
long-term epigenetic consequence
associated with hypermethylation
promoter of genes of basic protective
functions of cells in human body
exposed to radiation
Examined subjects
I. Irradiated subjects: from 24 to 78 years, n=124
1. Liquidators of the ChNPP accident in 1986-1987: n=83
The individual doses ranged from 50 to 460 mSv (average dose 221 mSv).
2. Nuclear specialists (All-Russian Research Institute of Experimental
Physics, Sarov, Russia): n=21
The summarized accumulated doses over a period of work with tritium and
tritium oxide ranged from 37 to 994 mSv
3. Residents of territories with radioactive contamination Novozybkov town
and Novozybkov district, Bryansk region, 135-688 kBq/m2 , 137Cs): n=20
II. Offsprings born from irradiated parents: from 2,5 to
49 years, n=74
III. Unirradiated subjects (control group): from 19 до 76
years, n=208
The collection of peripheral blood
and DNA samples (2003 – 2007)
1. Federal State Institution Russian Scientific Center of
Roentgenology and Radiolology Rosmedtechnology
(Dr. Galina P. Snigiryova)
2. Federal Unitary State Enterprise “Russian Federal Nuclear
Center All-Russian Scientific Research Institute of
Experimental Physics” (Dr. Tatyana I. Khaimovich)
3. Federal Children’s Scientific and Practical Centre of
Radiation Safety (Dr. Larisa S. Baleva)
DNA methylation analysis
Genomic DNA was isolated from blood leukocytes
Restriction
Methylated DNA
Unmethylated DNA
AciI (5'…C↓C GC…3')
Restriction sites are cleaved
Restriction sites are not cleaved
PCR
no product of amplification
Amplification of DNA fragment
electrophoresis in 2% agarose gel
Nucleotide sequence of analysed fragment of promoters
of p16/CDKN2A gene (as example)
F
GGATTTCTTTTTAACAGAGTGAACGCACTCAAACACGCCTTTGCTGGCAGGCGG
GGGAGCGCGGCTGGGAGCAGGGAGGCCGGAGGGCGGTGTGGGGGGCAGGTGG
GGAGGAGCCCAGTCCTCCTTCCTTGCCAACGCTGGCTCTGGCGAGGGCTGCTTC
CGGCTGGTGCCCCCGGGGGAGACCCAACCTGGGGCGACTTCAGGGGTGCCACA
TTCGCTAAGTGCTCGGAGTTAATAGCACCTCCTCCGAGCACTCGCTCACGGCGT
CCCCTTGCCTGGAAAGATACCGCGGTCCCTCCAGAGGATTTGAGGGACAGGGTC
GGAGGGGGCTCTTCCGCCAGCACCGGAGGAAGAAAGAGGAGGGGCTGGCTGGT
R
CACCAGAGGGTGGGGCGGACCGCGTGCGCTCGGCGGCTGCGGAGAGGGGGAG
AGCAGGCAGCGGGCGGCGGGGAGCAGCATG
The analysed CpG - dinucleotides in gene promoters
The total
number of АсіІ
sites in the
studied
fragments of
promoters
The total
number of CpG
- dinucleotides
in analyzed
fragments of
promoters
The analysed CpG dinucleotides (%)
Gene
Function
р16/CDKN2A
Cell cycle
2
23
8,7 %
р14/ARF
Cell cycle
3
35
8,6 %
RASSF1A
Cell cycle
7
32
21,9 %
GSTP1
Xenobiotic
4
31
12,9%
detoxification
The analysis of promoter methylation of p14/ARF and
RASSF1A genes in five liquidators (as an example)
1 - 5: undigested DNA samples; 1a – 5a: digested DNA samples. A - RASSF1A gene, B p14/ARF gene, C - RAR-β2 gene (control of DNA preservation after restriction), D - ING1
gene (positive control methylation ), M - molecular ladder (step - 50 bp), K – water (no DNA).
The arrows indicate the detected cases of methylation
Frequency, %
No significant differences were
of the total number of
foundThe
in the distribution
frequency of children
born
to irradiated and unirradiated
hypermethylation
cases in examined groups
(control group) parents with the
revealed promoter hypermethylation
The frequency of individuals with
of studied genes
promoter methylation of at least one
(power100
of the test is about 90%).
of the analyzed genes in exposed
90
group is significantly higher
80
compared to the control group
70
(p = 3.9 • 10-7).
60
50
40
30
20
10
0
0
1
2
number of methylation genes
control group
offsprings of irradiated parents
irradiated subjects
Revealed cases of hypermethylation of studied gene
promoters (%) in examined subjects (two age categories)
12
10
8
6
4
2
0
p14/ARF
frequency, %
frequency, %
RASSF1A
yes
> 45
≤ 45
no
o
exp
age, years
e
sur
7
6
5
4
3
2
1
0
yes
> 45
frequency, %
frequency, %
ye s
≤ 45
age , ye ars
no
s ur
GSTP1
12
10
8
6
4
2
> 45
o
exp
age, years
p16/CDKN2A
0
≤ 45
no
e
sur
o
exp
12
10
8
6
4
2
0
ye s
> 45
≤ 45
age , ye ars
no
re
su
o
p
ex
e
General and private correlations* of hypermethylation
with age and exposure status
RASSF1A
p14/ARF
0,102
(0,041)
p16/CDKN2A
Status
0,117
(0,018)
Status (age is fixed)
0,011
(0,825)
0,032
(0,523)
0,186
(1,7 х 10-4)
Age
0,216
(1,1 х 10-5)
0,149
(2,5 х 10-3)
0,171
(5,4 х 10-4)
Age (status is fixed)
0,183
(2,0 х 10-4)
0,114
(0,021)
0,244
(6,5 х 10-7)
0,059
(0,238)
* - Coefficient of correlation and two-tailed p-value are demonstrated
GSTP1
0,214
(1,4 х 10-5)
0,182
(2,4 х 10-4)
0,115
(0,021)
0,010
(0,844)
The multiple regression analysis of dependence of number of
methylation genes on age and satus of subject
(control/exposed)
B*
β**
р-value
1. Methylation (RASSF1A + p14/ARF) ~ status +age
-0,066±0,029
Constant
Exposure
0,018±0,032
0,031
Age
0,003±0,001
0,242
Coefficient of determination R2 = 0,067
0,024
0,576
1,7 х 10-5
8,6×10-7
2. Methylation (p16/CDKN2A + GSTP1) ~ status + age
Constant
Exposure
0,655
-0,012±0,028
0,160±0,030
0,290
Age
0,001±0,001
0,053
Coefficient of determination R2 = 0,102
1,7 х 10-7
0,330
3,7×10-10
•
*- coefficient of linear regression;
•
** - standardized coefficient of linear regression (in units of standard
deviations)
Age-associated changes of DNA methylome.
DNA containing three
Schematic representation of DNA methylation
patterns
genes
and three classes of
in young (A) vs elderly (B) people interspersed repeats
Besides extensive genome-wide hypomethylation,
aging involves a progressive gain of DNA methylation,
which is associated with CGI-promoters
Zampieri, M., et al. Reconfiguration of DNA methylation in aging. Mech. Ageing Dev. (2015)
http://dx.doi.org/10.1016/j.mad.2015.02.002
CONCLUSION
1. The reality of hypermethylation of CpG islands in promoters of genes of
basic protective functions of cells is demonstrated in blood leukocytes in
remote periods after irradiation of human body.
2. Differential role of age and radiation exposure factor in the hypermethylation
of various genes was identified.
3. In present work in blood leukocytes of healthy subjects we observed
methylation changes which reproduce gene hypermethylation found in
malignant cells.
4. Additional studies with involvement in the analysis of a larger number of genes
are required to identify epigenetic markers of radiation-induced premature
aging of the organism and the development of age-associated oncological and
nononcological disease.
Thank you for attention!