Paolo VineisImperial College London and HuGeF Foundation Torino

EPIGENETICS

Firenze 20 june 2013

“Inheritance” in images, from Darwin’s “tree of life” to DNA’s iconic

crystallography to the epigenetic dynamics

http://course1.winona.edu/kbates/Bio241/dna.htm

http://www.lifesciencesfoundation.org/printer_events-Xray_photographs_of_DNA.html

The iconic power of the double helix is related to its heuristic simplicity, particularly as a mechanism to

explain inheritance.

However, the script needs to be interpreted and receives meaning only from the interplay with the

environment (interpretation of the script).

The emphasis has been originally put on “variation” (inherited mutations, common variants …),

i.e. an error in the script is at the basis of disease

Things become more complex with the discovery of DNA’s “metabolism”, with transposable elements,

repair, methylation, miRNA, histone acetylation, i.e. INTERPLAY WITH THE (internal and external)

ENVIRONMENT

What is Epigenetics?

• Epigenetics is the study of inherited changes in phenotype (appearance) or gene expression caused by mechanisms other than changes in the underlying DNA sequence.

• These changes may remain through cell divisions for the remainder of the cell's life and may also last for multiple generations.

• Changes in gene expression that do not involve alterations in DNA base sequence

MRC-HPA Centre for Environment and Health

Imperial CollegeLondon

Epigenetic Modifications

•DNA Methylation

•Histone Modification (e.g. Acetylation, methylation)

•Non-coding RNAs (e.g. microRNA)

•All Regulate Gene Expression

Epigenetic Modifications•DNA Methylation

–C-5 position of cytosine in CpG dinucleotides (Islands)

- When histones are tagged, or acetylated, chromatin is open and genes are potentially active;

- When histones are not chemically tagged, deacetylated, the chromatin condenses and genes silenced.

Epigenetic Modification: Histone Modifications

Epigenetic Modification: Non-coding RNAs

A new mechanism for gene regulation • RNA which is not used for making proteins (non-coding RNA) can be cleaved and used to inhibit protein-coding RNAs

•siRNAs, microRNAs (~22Nucleotides; fine tune geneExpression)

Epigenetic-Regulated Phenomena

•Cellular Differentiation–Totipotent cells become pluripotent cells of the embyro

which differentiate into specific lineages•X-chromosome Inactivation

–Gene expression on one of the female X-chromosomes is downregulated

–DNA methylation and histone modifications

•Imprinting–Epigenetic marking of a locus on the basis of parental origin–Results in monoallelic gene expression

H19

IGF2*

H19*

IGF2

Paternal Imprinting

Maternal Imprinting

Parental Imprinting

Epigenetics and Cancer

1. DNA methylation •Gene specific hypermethylation (eg RASSF1, MLH1) •Genome-wide hypomethylation (4% down to 2-3% of all cytosines)

2.Histone Modifications •Active vs Inactive histone marks •Polycomb group gene silencing (H3-K27-me3)

•Growing data on the importance of epigenetics in the aetiology and pathogenesis of cancer

Cancer Epigenetics Paradox: Global Loss of DNA methylation in addition to locus-specific gain in methylation are causally linked to human cancer

Many cancer risk factors cause epigenetic modifications

DNA Methylation Influences Cancer Processes

DNA Repair

Hormonal Regulation

Carcinogen Metabolism

Apoptosis

DifferentiationCell Cycle

DNA

Methylation

Epigenetics and The Environment

•Epigenetic changes can be inherited mitotically in somatic cells

•Pre-natal and early post-natal exposures can result in changes in risk of developing disease

–Nutrition–Xenobiotic chemicals–Behavioural Factors–Reproductive Factors, Hormonal Exposures

•Epigenetic alterations may also be inherited transgenerationally (developmental origins of adult-onset disease)

Epigenetics and The Environment

• Prenatal environment • Famine exposure, Folic acid use (Tobi et al HMG 2009,Steegers-Theunissen et al Plos One 2009)

• Adult methylome • Smoking, Diet (Breitling AMHG 2011, Zhang Journal of Nutrition

2010) • Cancer methylome • Alcohol and folate (Christensen et al Plos genetics 2010)• Methylation variability between monozygotic twins increases with age (Fraga et al PNAS 2005)

Mapping sequences with differential DNA methylation between MZ twins.

Fraga M F et al. PNAS 2005;102:10604-10609

1- 3 H

-Met

hyl A

ccep

tanc

e (%

)

100

50

0

Day of Depletion6 69

Jacob et al J. Nutr. 128:1204, 1998

Dietary Folate Deficiency Causes Hypomethylation in Human Lymphocytes

In Utero Nutritional Exposure and Changes in Offspring Phenotype

Odds ratio for the metabolic syndrome according to birth weight among 407 men born in Hertfordshire (adjusted for adult body mass index).

Hales C N , Barker D J P Br Med Bull 2001;60:5-20

Some examples from our research

Smoking and epigenetics

AHRR

1x10-5

1x10-7

Smoking intensity is directly correlated with hypomethylation at AHRR

and 2q37.1.

Methylation β-values are presented for non-smokers (0), former smokers (1)

and increasing intensities in current smokers as 2 (1-3 cigarettes per day), 3 (4-

8), 4 (9-13), 5 (14-18), 6 (19-23), 7 (24-28), 8 (29-33) and 9 (>34). The y-axis

represents methylation β-values with box and whisker plots showing the

median line, 25th-75th percentiles and whiskers showing the 95th percentile

range for each probe.

P=0.007P=0.006

Smoking intensity is directly correlated with hypomethylation at AHRR and 2q37.1. Shenker et al, Methylation and smoking in EPIC-Torino (submitted).

The relevance of the findings or environmental epidemiology is due to the key role played by AHR in th metabolism of dioxins, PAHs and other environmental toxicants.

P=0.004

Rischio cumulativo di cancro del polmone nei non-fumatori e in fumatori che hanno cessato di fumare a diverse età o non hanno cessato.

Vineis P et al. JNCI J Natl Cancer Inst 2004;96:99-106

© Oxford University Press

SES and epigenetics

Social inequalities in health

Gallo et al. PLoS One, 2012

Results: Total mortality among men with the highest education level is reduced by 43% compared to men with the lowest

(HR 0.57, 95% C.I. 0.52–0.61); among women by 29% (HR 0.71, 95% C.I. 0.64–0.78).

The risk reduction was attenuated by 7%in men and 3% in women by the introduction of smoking and

to a lesser extent (2% in men and 3% in women) byintroducing body mass index and additional explanatory

variables (alcohol consumption, leisure physical activity, fruit and vegetable intake) (3% in men and 5% in women).

In the last years, research on SES has expanded with the aim of identifying the biological mechanisms through which

socioeconomic status is embedded and eventually “gets under the skin”

In humans, low socioeconomic status across the lifecourse has been associated with greater diurnal cortisol production,

increased inflammatory activity, higher circulating antibodies for several pathogens (suggesting dampened cell-mediated

immune response), reduction in prefrontal cortical grey matter and greater amygdale reactivity to threat, among others.

Human and animal studies have shown that socioeconomic status influences DNA Methylation and gene expression, in

particular across genome regions regulating the immune function.

Social and financial adversities over the entire lifespan (and more critically in early life) would program a “defensive”

phenotype that, through glucocorticoid receptor resistance, would lead to exaggerated glucocorticoid levels and

uncontrolled inflammatory responses to challenges in adult life, both increasing the risk of developing chronic diseases.

Dominance rank and expression level of pro-inflammatory genes (macaques)

Tung et al. Social environment is associated with gene regulatory variation in the rhesus macaque immune system.Proc Natl Acad Sci U S A. 2012 Apr 24;109(17):6490-5.

Unpublished data from the EPIC-Torino cohort (n=830)(analysis by Silvia Stringhini)

Illumina 450K results under investigation

16 candidate genes have been selected by S.S. as being implicated in psycho-social stress

Results after Bonferroni correction:

-1.5

-1

-0.5

0

0.5

1

Met

hyla

tion

diff

eren

ce b

etw

een

SES

extr

emes

(%

)

NFATC1 NFATC1 is one of the three genes whose expression was more strongly associated with social rank in macaques (more expressed in macaques with low social rank)

NFATC1 gene is involved in the expression of cytokine genes in T-cells, especially in the induction of the IL-2 or IL-4 gene transcription

NFATC1 regulates not only the activation and proliferation but also the differentiation and programmed death of T-lymphocytes as well as lymphoid and non-lymphoid cells

SES and DNA methylation – EPIC Turin

Diet and epigenetics

Dutch Hunger Winter 1944-1945

•Famine in The Netherlands towards the end of WWII-caused by Nazi occupation (blockade) and severe winter; led to severe malnutrition in Dutch population

•Dutch Famine Birth Cohort Study-prospectively studies offspring of mothers who were exposed to the famine

•Offspring shown to be increased risk of diabetes, obesity, CVD

•Offspring were smaller than those born to non-exposed mothers and then the children of these offspring were also, on average, smaller

–Suggestive of transgenerational inheritance-epigenetics

Difference in DNA methylation of CpG dinucleotides in siblings discordant for periconceptional exposure to famine.

Tobi E W et al. Hum. Mol. Genet. 2009;18:4046-4053

•60 individuals pre-natally exposed to famine compared with matched, unexposed siblings

•Investigated several genes involved in metabolism

•Positive difference indicates higher methylation level among exposed individuals

Evidence for the effects of maternal malnutrition on offspring comes from a historical cohort of Dutch individuals whose

mothers were exposed during the wartime famine of 1944–1945. Offspring of women exposed during early pregnancy were

more likely to develop the metabolic syndrome in adulthood compared to offspring of women pregnant before or after the

famine.

Epigenetic analyses in these individuals nearly 60 years later show differential methylation in several genes involved in

growth and metabolic control, which are dependent on sex and time of exposure during gestation. Hypomethylation at the

promoter of IGF2, a maternally imprinted gene implicated in growth and development, has also been observed in those

exposed during the peri-conceptional period relative to unexposed siblings.

•1-carbon metabolism is involved in DNA synthesis and methylation key for epigenetics

•1-C metabolism is highly related to dietary intakes (e.g. folate, Methionine…)

study of the association between 1-C metabolism and epigenetics through dietary exposure

Lung cancer and 1-carbon metabolism in EPIC (Johansson, Vineis, Brennan) – JAMA 2010

Analysis by quartiles

Vitamin B6 1·00 (reference)0·76 (0·57 - 1·00)0·54 (0·40 - 0·73)0·30 (0·32 - 0·59)ptrend

5 = 5x10-7

Methionine 1.00 (reference)0·90 (0·69 - 1·18)0·51 (0·38 - 0·69)0·53 (0·40 - 0·72)ptrend

5 = 2x10-6

<= 8 years > 8 years

Controls Cases OR 95% CI Controls Cases OR 95% CI

CDKN2A/P16 (Tumor Suppressor)

0 6

41 1.00 48 8 1.00

>0 9 28 0.42 (0.08-2.19) 35 15 2.02 (0.71-5.77)

RASSF1A (Tumor Suppressor)

<1.82 5 31 1.00 48 7 1.00

>=1.82 10 38 0.51 (0.11-2.39) 35 16 2.91 (0.98-8.61)

Odds ratios (OR) and 95% confidence intervals (CI) for methylation levels (below/above median in controls) and lung cancer risk by time since blood drawing (Vineis et al, Epigenetics 2010)

Methylation patterns in sentinel genes in peripheral blood cells of heavy smokers: influence of cruciferous vegetables in an

intervention study

Scoccianti C, Vineis P et al, Epigenetics 2011

Intervention trial with 3 different diets in heavy smokers: methylation of repeat sequences, tumour suppressor genes,

MTHFR and other genes.

These data suggest that a healthy diet may stabilize global epigenetic (LINE-1 DNA methylation) patterns in peripheral white

blood cells, but do not provide specific evidence for potential prevention of methylation changes in specific genes.

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

Normal Enriched Supplemented

Dietary Groups

Me

an

% m

eth

yla

tio

n

LINE T0

LINE T4

MLH1 T0

MLH1 T4

RASSF1A T0

RASSF1A T4

CDKN2A T0

CDKN2A T4

MTHFR T0

MTHFR T4

ARF T0

ARF T4

Epigenetics and disease prediction

Flanagan J, Vineis P et al. (Cancer Res, 2012).

Hypermethylation of ATM and other DNA repair genes in breast cancer in four independent studies with pre-

clinical samples

Methylation of ATM and breast cancer. Flanagan J, Vineis P et al. (Cancer Res,2012).

Chemicals and epigenetics

There is much more than genotoxicity - need to explore several steps/segments of carcinogenic process

Simplified models of carcinogenesis (Vineis, Schatzkin, Potter, Carcinogenesis 2010)

Model 1 Model 2 Model 3 Model 4“mutational” “DNA instability” “non-genotoxic” “Darwinian”

Chemical carcinogens Familiarity Clonal expansion/ Clonal expansion/Viruses Genome instability Epigenetics Cell selection

Tobacco and lung Colon cancer Diet, hormones Beta-carotene,HPV Retinoblastoma folate,

chemotherapy

DNA adducts CI, MI, MMR, Rb, Methylation Selective advantagemutations BRCA1, TSG histone acetylationoncogenes

Mathematical models: Armitage-Doll Knudson Moolgavkar Nowak

Thank you