Epigenetics - appunti di lezione

EPIGENETICS AND ENVIRONMENT: Nature vs nurture debate

It is a hot topic in genetics and epigenetics studies about the gene-environment interaction: what we are, healthy/unhealthy state, depends more on what we inherited, our genome (nature), or on what we are exposed to during life, the environment (for example what we nourish with)?

We know that the phenotype can be explained by genetics: each individual is different from the other: the amount of genetic variation between individuals is 0.1%.

Genome-wide association studies demonstrated that many diseases are explained by genetics: for example, variations in susceptibility and severity of mendelian diseases can be easily explained by genetic variations as substitutions, insertions, deletions, inversions, translocations, SNPs. Anyway, the phenotype associated to complex diseases as obesity or cardiovascular diseases can be just partially explained by genetic variants, showing a missing heritability (for instance, 1000 variants found by GWAS for a

certain disease can explain only 60% of the phenotype). So, something else affects phenotype: the environment.

Ex. monozygotic twins: even if they are genetically identical, for some disease the concordance in the onset is very high, like autism or hypertension, but for others the concordance is very low. Epigenetics can contribute to the onset: DNA methylation and histone modifications are identical at the beginning of life, after 50 years they are different. Despite our genome remains the same during life, our epigenome changes.

Epigenome is affected by internal genetic factors and external environmental factors*; among these latter, toxins, diet, stress, exercise, smoking, alcohol, drugs, pathogens: some of them are also genotoxic, but not all of them. Epigenome modification induces changes in phenotype, affecting longevity and behaviour, for example, and changes in gene expression.

So, the studies about twins demonstrated that the phenotype is indeed given by the genetics, by the epigenetics.

Exposome => the totality of our exposure to environmental factors from the conception onward and their effect on the epigenome: air pollution, tobacco smoke, oxidative stress, organic chemicals, and, recently discovered, also nutrients.

How does the environment alter epigenetics?

Ex. sex determination in turtles => sex determination is not a genetic process, but it is due to the temperature of eggs incubation with a probable epigenetic contribution:

• cold and wet => male specific protein expression and differentiation of testis
• hot and dry => female specific proteins expression and differentiation of the ovary

Ex. Vernalization => some plants can flourish only if exposed to a certain period of cold. The environmental signal is perceived at one stage and then remembered later. This occurs thanks to a molecular mechanism which involves the epigenetic silencing of the flowering locus C (FLC), which is a

floral repressor. If this floral repressor is expressed, the plant cannot flower, but during the exposure to a cold period, it's downregulated and with a quantitative increase with the time of cold exposure: the plant can flourish. The downregulation is stably maintained even when the T goes up through an epigenetics mechanism.

Before cold exposure, FLC is highly expressed. Soon after cold exposure, FLC is downregulated concomitantly with the transcription of COOLAIR nc RNA. Longer exposure to cold triggers COLDAIR ncRNA expression that recruits a PCR2 at FLC locus with enrichment of H3K27me3 that stabilises FLC repression even after the return to warm conditions. The expression of COOLAIR and antisense COLDAIR is limited in time but the silencing of FLC is permanent thanks to the histone modification.

Ex. Worker bees and queen bees => even if they are genetically identical, queen bees and worker bees are phenotypically very different. The queen has ovaries and large abdomen, longer

Life-span and it is able to fight, while the worker bees are sterile and don't fight. The different phenotype is due to their diet: larvae fed with royal jelly become queens, if they are not, instead, constitutively they become workers.

The royal jelly induces different phenotypes because it regulates an epigenetic mechanism: it downregulates DNMT3 expression, which constitutively methylates and suppresses gene promoters associated with the development of the queen phenotype, determining a constitutive worker bees' phenotype. So, constitutively, worker bees are generated when DNMT3 is active, queen bees are generated thanks to royal jelly that inhibits the methylation of queen genes determining a different phenotype.

Demonstration: DNMT3 inhibition through siRNA induced the development of the queen.

Ex. Mammalian animal models exposed to food contaminants => exposure to endocrine disruptors induces an epigenetic effect and this can be transmitted also through generation.

- Vinclozolin

is a fungicide used in wine making that can induce sterility or sub-fertility when the F0 (mother or father) is exposed to vinclozolin during mid-gestation pregnancy. The period of exposure is essential: if during life, in particular during mid-gestation pregnancy, these animals are exposed to vinclozolin, their male offspring has a sub-fertility in 90% of the male. This sub-fertility remains, there's a sort of memory also in F2 and F3 generation!

- Methyoxychlor is a pesticide that has replaced DDT, that can be an endocrine disruptor in case of exposure of F0 in mid gestation pregnancy: it can induce the sub-fertility in males but also in the females, even in F3 generation.

It's quite difficult to distinguish between genetic and epigenetics effect. We can do that in animal models but quite difficult in humans, because these molecules might also induce genetic variations.

Ex. Maternal care affects epigenetics => maternal care toward the offspring can induce a less stressed

Phenotype during adulthood: early life is a sensitive period for epigenetic changes. This is mediated by an epigenetic regulation of the glucocorticoid receptor. If the mother licks the pups, there's a demethylation of the glucocorticoid receptor promoter. So it's open and can be expressed.

Given the abundance of this receptor, once they produce glucocorticoid (cortisol, stress H), the receptor can mediate the effect of this H and so they react in a normal way. In pups not having this demethylation, the promoter remains methylated and the genes not expressed, so there's fewer amount of the receptor, meaning that they're not going to respond well to the stress.

This was demonstrated by cross-fostering experiments in which the offspring of a mother was put in the cage of another mother, they exchanged the offspring.

Ex. Agouti mouse

It's a particular mouse that can have three different phenotypes based on the coat colour:

• Yellow
• Mottled (mixed phenotype)
• Grey

(pseudo-agouti)But identical genotype at every locus.

Agouti gene => regulates the colour of the coat. It’s constitutively expressed only in the first part of the life of these animals, then it’s suppressed. Its expression is regulated also by methylation state of IAP, a long terminal repeat retrotransposon element which is upstream the promoter of this agouti gene:

• If methylated, after a transient expression, the gene is suppressed, and the mouse is grey.
• If not methylated, agouti gene is expressed during the whole life of the mouse, and the coat is yellow.

The methylation is established during gastrulation, and it is stochastic: some cells have it, some others not, and it is mitotically heritable. This mosaicism explains the mottled phenotype, similar to calico cat. So, we can have a variable spectrum of phenotype.

The colour of the coat is not the only phenotype associated to the methylation of IAP: yellow mice are also obese, prone to cancer and to metabolic disorders.

offspring. What is the reason why we can observe this phenomenon: intrauterine environment or gametes?

By transferring the zygote obtained by crossing between non-agouti father and agouti mother to a non-agouti mother that carries on the pregnancy, the ratio of the offspring was similar to the original one: it's not the intrauterine environment, but it's about gametes.

Environmental exposure can alter the ratio:

• Altered maternal diet with methyl donors as folate, B12, choline in a specific period, from the fertilization time (preimplantation/post implantation period), can generate offspring with more pseudo-agouti. Considering the crossing between grey mother with yellow agouti father, control offspring is 40% agouti, 40% mottled, 20% pseudo-agouti, while altered diet offspring shows 30% mottled and 70% pseudo-agouti grey mice. If the diet is given after the specific period, the phenotype doesn't change.
• Altered maternal diet with genistein, a soy isoflavone with potential

hyper-methylating effect, affects the ratio in the offspring increasing the pseudo-agouti in the same way*.- Even xenobiotics and food contaminants can affect the offspring ratio: exposure to bisphenol A (BPA),present in plastics, increases the ratio of the agouti mouse, so we have about 60% of yellow mice andonly 10% of pseudo agouti. BPA seems to reduce the methylation of the agouti gene. However, whenmothers are fed with BPA and methyl-rich foods, these latter are able to counteract the effects of BPA,coming back to the normal condition (see pictures). Maternal diet is very important!

The epigenetic state is established in early development and is set for the life of the organism due tomitotic heritability (but switch through generations). Agouti gene represent an example of metastableepialleles = identical genes that differ in the extent of DNA methylation, showing not genetic butepigenetic differences. They are stable during life of the organism (agouti mouse will be agouti

for therest of his life) due to mitotic heritability and ca