DNA modifications

Early life environment- adult phenotype

Influence on DNA methylation patterns and ageing?

Genetic clocks

Age estimates in years resulting from a mathematical algorithm based on the methylation state of specific CpGs in the genome

Cytosines of CG(CpG) Dinucleotides:

Sites of DNA methylation

The human genome has about 28 million CpG sites (potential for cytosine methylation)

Microarrays for cytosines methylation analysis

2-3% of the CpG sites can currently be analysed by microarrays

Ideal for surveying thousands of DNA samples

Widespread global adoption

Consistent data sets/ huge data sets

Such data sets can be used to generate epigenetic clocks

Some epigenetic clocks are tissue specific

Cytosine methylation

A common form of post- replicative DNA modification

Adds information without changing of the actual DNA sequence

Donor of methyl group: S- adenosyl- L- methionine (SAM)

Mammalian DNA methylation

CpG dinuclotides are the predominant sites of cytosine methylation (there are exceptions)

*DNA has a polar direction

*Palindromic sequence- reads the same both ways

How does DNA become methylated?

Replication fork

Semiconservative replication

5’ to 3’

De novo- add methyl to DNA where there previously wase’t any

3 known mammalian DNA Methyltransferases: DNMT1, DNMT3A, DNMT3B

DNA methyltransferases are essential for normal mammalian development

Deletion of these genes in mice results in embryonic or postnatal lethality

What triggers DNA methylation?

• repetitive DNA

• histone modifications

• certain RNAs

• metabolites4

Cytosine methylation: gene silencing and passive demethylation

*X chromosomes in women 1 active and 1 inactive

Cytosine methylation: active versus passive demethylation

Uneven distribution of CpG dinucleotides in mammalian genomes- and their modification states

Gene bodies are often methylated with higher DNA methylation at exons than introns

5hmC is present at expressed gene bodies (white square at body of gene)

key:

• white circles: nonmethylated CpGs

• black circles: methylated CpGs (5mC)

• white squares: hydromethylated CpGs (5hmC)

• red boxes: active and transcribes exons

• grey boxes: inactive and silenced exons

• red arrow: active transcriptional state

• black cross: inactive transcriptional state

CpG islands

CpG- rich areas in the genome

Defined as >0.5 kb stretches of DNA with a G + C content >/ 55%

CpG islands are typically located in the promoter region, 5’ to the TSS

CpG islands

CpG islands are regions of high CpG density that often lack methylation

These “islands” are found at promoters of the most human genes

Long- term silencing of the gene can be established by methylation of the CpG island region. E.g. genes on the inactive X chromosome and certain imprinted genes are silenced in this way. In cancer cells, certain genes are aberrantly silenced by CpG island methylation. Methylation of gene promoters generally correlates with gene inactivity

Genome- wide DNA methylation: dynamic during development

Developmental epigenetic reprogramming during mouse pre- implantation and germ cell development

More cytosine modifications

5hmC is present in the male pronucleus of mouse zygotes

5- hydroxymethly- cytosine (5hmC) known biological roles

5hmC can be a stable modification enriched with gene bodies, concentrated at transcriptional regulatory elements and positively associated with transcriptional activity

5hmC recruits transcriptional regulators, splicing factors, DNA repair proteins and chromatin regulators that are distinct from those recruited by 5mC

5hmC plays an essential role in embryonic development, cellular differentiation and stem cell reprogramming

5hmC is a strong prognostic indicator in cancer and other diseases

5hmC is implicated in the regulation of neurological plasticity, immunology and other dynamic systems by the TET enzyme family

5hmC is an intermediate during the process of demethylation

DNA demethylation: passive and active mechanisms

Detection of DNA methylation

• Methylation- sensitive restriction enzymes

• Antibodies (dotblot/ immune precipitation)

• Nanopore sequening

• Bisulfite sequencing (targeted, whole genome, microarrays)

Bisulfite sequencing: detection of cytosine methylation in DNA

Environmental influences: metal programming

Poor nutrition (the environment) in early life leads to “epigenetic programming” of certain organs and tissues:

• a factor in the aetiology of type 2 diabetes, cardiovascular disease and related health problems

Example of how environmental factors may influence the epigenome

The dutch hunger winter 1944- 1945

daily rations: 400-80- calories

~20 years after dutch hunger winter children started to experience obesity

~54 years after DNA methylation differences after exposure to prenatal famine are common and timing- and sex- specific

A type of cellular memory system

Biological memory can be defined as a sustained cellular response to a transient stimulus

The ability of a daughter cell to retain a memory of the gene expression patterns that were present in the parent cell is an example of epigenetic inheritance

Migrant model (changing enviornment) at different points in the life course

Bangladesh/ UK

Childhood environment influences levels of reproductive steroid hormones

Comparing mouse and human data

Question: what is early life stress?

Hypothesis: early life immunological stress (gastrointestinal infection)

Experimental work: compare DNA methylation patterns between mouse and human samples, gene expression in mouse

Differential gene expression in mouse ovaries

13 genes in mice ←→ differential DNA methylation in human orthologs

Correlation with altered DNA methylation levels in woman?

1 of the down- regulated genes in mouse ovaries: steroid 5 alpha- reductase 1 (SRD5A1)

SRD5A1 processes steroid hormones

e.g. progesterone → dihydroprogesterone

reduced SRD5A1 expression reported:

• in adult rodents

• humans after early- life stress

• survivors of the Holocaust

Altered SRD5A1 methylation in a putative enhancer in mouse and human

Summary

Early life immune challenge alters reproductive phenotype

SRD5A1 is a strong candidate mediating long- term effects of reproduction

2 classic cellular memory (epigenetic) systems

Polycomb- trithorax system

DNA modification