METHYLMETABOLOMIC APPROACH OF GENOMIC INSTABILITY LINKED WITH DNA HYPOMETHYLATION BIOMARKERS
I Turcu2, M Matei3, G Anton4, N Belc5, D Duta5, LG Radu6, C Albu6, A Botezatu4,
C Posea7 and N Cucu1
1. University of Bucharest, Faculty of Biology, Dept of Genetics-MICROGEN-Epigenetics laboratory, Romania; 2. Ana Aslan International Academy of Anti_Aging, Bucharest, Romania; 3. CF2 Hospital Bucharest, Department of Assisted Reproduction, Genetics Laboratory, Romania; 4. “Stefan Nicolau” National Institute of Virology, Bucharest, Romania;
5. National Institute of Bioresources, Bucharest, Romania; 6. National Institute of Biology, Bucharest, Romania, 7. Dept of Hemathology, Universitary Emergency Hospital Bucharest, Romania
Abstract
Introduction
One carbon (methyl group) metabolism is actually considered one very important cell pathway linking the environment and the genome stability with a dramatic
impact on our health status. Epigenetic biomarkers such as the DNA and histone methylation and their associated chromatin covalent modifications (histone
acetylation/deacetylation) are proposed to be integrated with the already wellknown metabolites S-adenosylmethionine (SAM) and its product, S-adenosylhomocysteine
(SAH) when an investigation of the genome stability is performed. An experimental model is proposed to study the relation between the DNA hypomethylation induced
by aging process and certain pathogenetic states (cancer and diabetes) as well as by certain disease events and treatments for the young individuals.
In our study a cohort of six - elderly (60 plus years), adult (30-45 years), and young (18-30 years) groups, has been approached, as follows: one young group
with controlled sportive life, one young group with uncontrolled lifestyle, two adult groups with and without health problems, and two elderly groups, with vs. without
significant pathology. DNA methylation dynamics is similar both during aging and cancerogenesis process showing concomitant global DNA hypo- and local DNA
hypermethylation. Both biomarkers have been proved in tumors collected from breast cancer patients through methylation specific PCR method in local ER alpha and
RAR beta promoters and HPLC method, respectively, for the general DNA methylation level. As the median age of this group of individuals was 50 year, we approached
the biomarker study also using lymphocyte culture comparatively with the young and aged groups with and without health problems, proving similar methylation
profiles.
The study comprises a genetic (karyotyping) approach in lymphocyte cultures, which showed again similarities regarding the genome instability in terms of
frequent premature chromatides dissociations (PCDs) involving chromosome 1 and 16. Also, another epigenetic marker, the ratio of methyl donor SAM and its
methylation reaction product, SAH was used to prove the similarity between the aging and cancerogenesis process. The mthfr (methylenetetrahydrofolate) gene
plymorphism is proposed for proving no alteration in diet folate assimilation.
Using specific epigenetic drugs, such as Trichostatin A and 5Aza cyditidine, as well as the Gerovital, a product containing procaine, were approached in order to
demonstrate the involvement of the heterochromatin remodeling through DNA methylation in satellite 2 DNA and HDAC in pericentromeric region during both
developmental processes.
The study envisaged the importance of the epigenetic biomarkers when new therapeutical or nutrition personalized strategies have to be approached.
Specific biochemical modifications of chromatin, such as global DNA demethylation and HDAC activity alteration, are
considered hallmarks of the aging as well as cancerogenesis process, which affect primarily the chromosome architecture and
hence its function during cell division. Pericentromeric region of the chromosomes contains repetitive, satellite 2 DNA, in
interaction with specific centromeric proteins (CENP) and nonhistone proteins, HP1. The DNA hypomethylation processes are
frequently targeted towards this DNA region. Consequently, the chromatin conformation in this region may be dramatically
changed, carrying on altered DNA-protein and protein-protein interactions, due to specific conditions: aging process,
pathological condition, chemotherapy, methyl depleted diet and DNMT3b mutation (lake in the model ICF syndrome).
The effects on cell division may include premature chromatides divisions (PCD) and frequent aneuploidies as determinants
of genome instability. Such interesting phenomena are also encountered when karyotyping healthy young/adult people which
received specific treatments, or karyotyping women lymphocytes prelevated during menopause or hormonal treatment, and
very importantly, when karyotyping cancer patients. The experimentally 5azaC or TSA induced PCDs in lymphocyte culture
from young people plede for the role of epigenetic factors such as DNA demethylation and HDAC inhibition in the genome
instability characteristic to aging and pathological conditions.
Also, the newly emerged metabolomic domain is focusing on a major biochemical pathway concerning the one carbon (CH3), methyl group addition and depletion from DNA and histones, with interesting effects upon the genome stability and
chromosomal morphology. The SAM regeneration from its product, SAH, represents one of the central reaction involving
folates and B (12 and 6) vitamins and specific cathions (Mg and Zn). Therefore, the SAM/SAH ratio and further the Hcys level
may be good candidate markers for the general health status linked with chromatin methylation processes. In this concern, the
polymorphism of mthfr gene was considered as one major genetic factor involved in methyl metabolome linked with the SAH to
SAM regeneration.
Our goal was the study of the DNA hypomethylation effects on chromosomal stability during cell division in aging and
cancerogenesis process, considering also the methyl group metabolism factors. This is the first step in our research approach of
the epigenetic control of the genome stability during aging and cancer processes and the pathological conditions linked with this
critical human developmental stage. The further step envisages the focus on specific heterochromatin features which define the
centromere and pericentromere stability through specific histone covalent modifications, DNA-histone and histone-nonhistone
interactions.
Materials and methods
Genomic DNA methylation level, cell division patterns and methyl metabolomic aspects have been studied in six elderly (60 plus years), adult (30-45
years) and young (18-30 years) groups, as follows: one young group with controlled sportive life, one young group with uncontrolled lifestyle, two
adult groups with (cancer) and without health problems and two elderly groups, with (diabates, CVD, cancer) vs. without significant pathology.
Usual lymphocyte culture induction, chromosomal spreads obtaining and karyotyping methods have been performed. For the treatment of
lymphocyte cultures 6x10(-6)M 5azaC, and 1x10(7)M TSA were used. For DNA extraction a Promega/wizard DNA extraction kit was used. The
isoschizomer pair MspI and HpaII restriction enzymes has been used for DNA methylation sensible restriction in order to estimate the global DNA
methylation level. Another approach for this estimation was HPLC analysis of the relative 5-methyldeoxycytidine (5mdC) content: it was done after
hydrolysis of DNA with nucleases P1 and alkaline phosphatase. Also, the methyl group metabolites, SAM (methyl donor) and its product SAH have
been estimated by HPLC in individual blood samples. Genetic analyses by PCR with specific primers and restriction enzyme have been performed
for mthfr polymorphism estimation.
Results and discussions
Global DNA hypomethylation and local (ER alpha and RAR beta) DNA hypermethylation have been studied in tumor tissues of cancer patients. The same marks (ERalpha promoter hypermethylation and
global DNA hypomethylation) have been also approached in blood lymphocytes as the mean age of the cancer patients group was 58 and presented the same epigenetic features.
Our approach of the lymphocyte cultures obtained from the two groups of blood donators (a total cohort of 200 persons): (i) aged people (without major healthy problems) and (ii) young healthy people,
comprised the karyotype and epigenetic (HPLC and MspI / HpaII restriction) analyses. These analyses have been performed also with a third group of samples comprising treated cell cultures (from young persons)
with DNA inhibitor (5azacytidine, 5azaC) and HDAC inhibitor (trichostatin acid, TSA) as epigenetic agents.
In our study the untreated cell cultures obtained from the elderly group presented a specific karyotype, characterized by frequent premature chromatides dissociations(PCDs) and aneuploidy, as compared with
the cultures obtained from the young, untreated group (Fig.1 and 2). Further treatment of lymphocyte culture obtained from young group with epigenetic agents revealed the same frequent PCDs as in untreated
elderly lymphocytes (Fig.3). A link between the DNA demethylation processes in the pericentromeric region stability contributing to PCDs is suggested. Moreover, similar PCD phenomena have been observed in the
chromosome preparations obtained from adult-young groups having severe healthy (cancer) problems or in specific treatment conditions (antibiotics, hormonal treatments in young women) (Fig.4,6) ,
demonstrating the same genomic instability.
The cytogenetic results have been confirmed by the molecular and biochemical measurement of the global DNA methylation level (Fig.7,8, Table 1) and also by the SAM/SAH ratio estimations (Table 2). Not only
the DNA hypomethylation has been detected in elderly blood but also in treated lymphocyte culture from the young individuals. The ratio value presented a decreased level with age and pathological conditions. The
estimated mthfr polymorphism proved no implication of mutant MTHFR in DNA hypomethylation and SAH accumulation, for the studied cohort.
Global genomic DNA hypomethylation linked with genomic instability is recognized as a hallmark of aging processes and of certain pathological conditions, such as cancer. The extensive research of the
molecular basis of the centromere instability linked with PCD and aneuploidy and chromosomal rearrangements pointed towards an interesting syndrome (ICF, immunodeficiency, centromeric region instability,
facial anomalies) as a model to investigate the DNA hypomethylation effects. This is a pathological result of the DNMT3b mutation, linked with severe DNA 2 satellite demethylation that determined the
pericentromere instability linked chromosomal rearrangements between certain chromosomes (1-16) containing such repetitive DNA sequences (Fig.5). Intriguingly,however, this syndrome is not linked with cancer.
Heterochromatin is the major chromatin structure of centromere/pericentromere regions whose stability is of crucial importance for the cell division processes, and hence the proper cohesion and segregation of
the chromatides in chromosomes . Pericentric heterochromatin is found juxtaposed to centromere one and remains condensed throughout the cell cycle, but has to be flexible to external signals and endogenous
ones, linked especially with replication. Not only DNA methylation marks through satellite DNA 5-methylcytosine residues but also other methylation process at the level of pericentric heterochromatin have to be
seriously considered when studying the genome architecture protective factors which assure the proper chromosome functions (such as methylation of histones in specific lysine residues).
Certain researcher groups (T.Jenuwein, L.Almouzni) points to a crucial role of histone underacetylation within pericentric heterochromatin region and for their association with heterochromatin protein 1
(HP1) in order that centromere function to be properly performed. They point also towards a “magic” mark of the heterochromatin domains, encoded by histone methylation at H3lys9 (H3K9), more specifically its
SET domain. This is a heterochromatin subdomains (comprising minor and major satellite DNA) marks for specific interaction with centromeric proteins (as CENP A) and, respectively, HP1.
Moreover, the recently discovered miRNA implications in the heterochromatin structure and gene expression control oriented us towards further new exploration directions for the molecular details of the
centromere ctructures in order to explain their instability induced by the epigenetic factors during our experimental model of aging process and its related pathologies.
The study is aiming at finding molecular aging hallmarks an demonstrating the need for a personalized approach of the aged people when a correct diagnosis and therapeutic strategy have to be applied.
Karyotype analysis
Global DNA hypomethylation
Fig 8a,b. Msp/Hpa restriction diagrams showing high, respectively, low DNA
methylation level obtained from genomic DNA extracted from lymphocyte cultures
of young (a) people and aged group people (b).
Fig.5. Hypothetical model of the mechanism involved in radial
chromosome formation
(after Reik et al,1999 )
a
Methyl group metabolome
Scheme of one carbon (-CH3 group)
metabolome
(after L Sharp and J Little,
Polymorphisms in Genes Involved in
Folate Metabolism and Colorectal
Neoplasia: in Am J Epidemiol
2004;159:423–443)
b
Table 1. Variation of the global DNA methylation level estimated with
the two major groups- the young and aged groups
AGE
Fig. 6. Diagram of PCS percent estimated with the three
experimental models: treated and untreated young people,
untreated aged group
70
YOUNG
6,9
5,3
4,9
5,1
6,2
6,0
5,6
5,2
6,1
5,9
ELDERLY
4,5
4,1
4,2
3,9
4,1
4,3
4,5
4,6
4,9
4,7
DNA hypermethylation
65
60
Breast cancer patients-tumor tissue
55
50
40
Control young
40
Elderly
30
Young/TSA
20
Young/5-aza
10
GLOBAL GENOMIC DNA METHYLATION LEVEL
%5mC
[5mC] X 100
[C]+[5mC]
DIONA PROJECT- Romanian National Research Program
The main purpose of DIONA is to address nutritional issues that affect the ageing process in the hope of improving
the health and quality of life of the ageing population.
A target study group of around 1200 elderly persons, will be selected through socio-demographical screening on
different elderly population groups and the care needs of the elderly under study will be assessed in accordance with their
dependency degree as institutionalized or at home and their actually nutritional status and needs. Metabolomic approach of
nutrigenomics in elderly under study will be applied with focus on the impact of the methyl donor bioactive compounds.
Based on the results about nutritional needs of the elderly, scientific and technical improvements and more efficient
methods will be developed in order to produce the best food products. Three new foods will be designed, adapting them to
the traditional recipes of the different European countries.
Bioresources containing proper bioactive compounds for a personalized diet specific for healthy ageing and age-related
pathology as preventive/curative diets will be selected and will be included in new food recipes. Formulation and
development of food in easy opening containers, mainly plastic containers, with different grades of grinding for old people
with mastication problems will be considered.
The food products will be elaborated at pilot plant scale and will be
clinically tested and, then the proper ones will be transferred to the industry for production.
In addition, the results will be efficiently disseminated to the industry, researchers, consumers by creating tailored
training and detailed instructions as well as to policy’ makers for an establishment
and alignment of food strategy related to health/nutrition European claims.
2
0
RAR beta
Elderly-ER alpha-blood
ER alpha
Breast cancer patients -ER alpha- blood
References
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Conclusions
The results pointed out to a pronounced global DNA hypomethylation concomitant with local DNA hypermethylation (ER alpha) in the healthy elderly as well as in cancer
affected adult persons; similar epigenetic aspects have been observed by methyl metabolome analyses, which indicated a decrease in methyl donor level in these individuals. Such
results explained the altered cell division pattern characterized by frequent PCDs and even a marked genome instability proved by the radial chromosomal morphology. These
features have been identified also in the young subjects with cancer or antiviral or hormonal therapy as well as in adult cancer bearing patients, when compared with the young
sportive group. However, the involvement of the epigenetic factors associated with the pericentromeric heterochromatin structure has been proved by obtaining similar genome
instability features when treatment of the young group lymphocyte culture with DNA methylase and HDAC inhibitors have been performed. Epigenetic factors such as DNA
hypomethylation, accompanied by HDAC inhibition, Lsh gene alteration and histone H3K9 methylation in the pericentromeric region containing repetitive satellite DNA may
result in alterations of the heterochromatin structure which is of crucial importance for the correct cohesin, separase and ciclins regulation activity of the chromatides cohesion.
Karyotyping lymphocytes from different individuals grouped as function of age, health conditions and specific treatments revealed interesting preliminary results which
correlated the global DNA hypomethylation and premature chromatide separation processes.Treatment of lymphocyte cultures from the young healthy donors with the DNA
demethylating agent and the HDAC inhibitor resulted in similar PCDs frequency, as compared with the percentage observed with normal elderly karyotype, demonstrating the
relevance of the two epigenetic factors in maintaining the genome stability through the centromere-pericentromere heterochromatin structure.
The molecular Msp/Hpa restriction profiles and HPLC determinations proved the decrease in global DNA methylation level with aging.
PCR screening, performed in order to ensure that the mthfr mutations had no implications in this study, proved that the methyl group metabolism was not influenced by this
factor. By the other hand, this specific feature of elderly methyl group metabolome may be monitored by diets and supplements which may provide proper elements (folates,
vitamins B12 and B6, Mg and Zn cathions and other secondary methyl donors such as betaine and choline) for maintenance of a normal genomic stability in aging conditions and
perhaps improving the genomic structure in cancer.
This study is curently continued by targeting other molecular actors such as H3K9 and K27 di and trimethylated, LSH demethylase, and also the study of certain HDAC
inhibitors (SAHA, butyrate etc) upon the demethylation in promoter ER alpha and RAR beta and their gene expression reactivation. These studies are concomitant with other
epigenetic therapies alternatives, such as known procaine DNA demethylating agent.