2. Epigenetics - lecture notes for this module, written down in cardiff university lectures. PDF

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Epigenetics  Epigenetics is the study of how your behaviours and environments can cause changes that affect the way your genes work.  Epigenetics is anything other than DNA sequence that influences the development of an organism  Epigenetics is the the study of mitotically and/or meiotically heritable changes in gene function that do not entail a change in DNA sequence

Cell Differentiation    

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Each cell genetically identical (except germ cells, white blood cells) Epigenetic modifications control how and when genes are transcribed. Epigenetic modifications are inherited when cells divide. Inherited through mitotic cell division Each cell type has a unique epigenetic signature Inherited through meiotic cell division

Epigenetics and human disease Genetic disorders influenced by:

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X-inactivation Fragile X syndrome Imprinting disorders Friedreich's ataxia Cancer Aging Schizophrenia Obesity

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Epigenetic Mechanisms Two key types of epigenetic marks DNA methylation

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DNA can be tagged with tiny molecules called methyl groups that are added to some of its C residues

Histone modification - Tags can be added to histones that are closely associated with DNA

DNA methylation  DNA methylation is the chemical modification of DNA by the addition of a methyl group (CH3) to the number 5 carbon of the cytosine pyrimidine ring.  The occurs only* with cytosine is next to guanidine (CG)  Non-CG methylation can be found in stem cells, and can be detected in brain tissue 5-methylcytosine is produced by the action of the DNA methyltransferases: - DNMT 1 - DNMT3a - DNMT3b These catalyse the transfer of a methyl group (CH3) from a donor (ex. S-adenosylmethionine = SAM; folate) to the carbon-5 position of cytosine In vertebrates, all* methylated cytosines are found in CG pairs:

- 5’ CG 3’ (CH3 on C)

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- 3’ GC 5’ (CH3 on C)  Methylation is symmetric ie both DNA strands  Most CGs in the mammalian genome are methylated CG dinucleotides are 5 X less frequent than expected because mC has a tendency to mutate to T

CG occurs at a normal frequency in CpG islands • 0.5-2 kb regions • Commonly located at 5ʼ end of many genes • Often overlap with promoter and 1st exon • 56% human genes and 47% mouse genes have a CpG island Cytosines are generally unmethylated in CpG islands associated with active genes

How does DNA methylation regulate gene expression? Main mechanisms by which the methylation of DNA can prevent the transcription of genes 1. The methyl group changes the recognition sequence a. Prevents proteins binding Ex. CTCF does not bind methylated DNA b. Attracts a different protein Ex. Kaiso binds methylated DNA 2. Attracts Methyl-binding domain proteins (MBDs) which recruit protein complexes that modify histones Methyl groups added by DNA methyltransferases

DNA methylation is added at specific sequences DNA methyltransferase 3a and 3b are the de novo methylases DNA methyltransferase 3L is an accessory protein De novo methylation primarily occurs in germline and early embryos De novo methylation can also occur in the adult

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Adult stem cells

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In certain tissues during aging Abnormal cells such as cancers Cell lines grown in culture

Methylation first occurs de novo 1. 2. 3. 4.

Methylated DNA Fully Methylated DNA DNA Replication Hemi Methylated DNA DNMT1 (Maintenance methylation, DNMT1 recruited to replication fork)

DNMT1 accumulates at DNA replication sites by binding to proliferating cell nuclear antigen (PCNA) and an E3 ubiquitin ligase (UHRF1) DNA methylation is the heritable mark

How are methyl groups removed? Passive DNA demethylation – occurs are several cell divisions “enzymatic process that removes or modifies the methyl group from 5mC. By contrast, passive DNA demethylation refers to loss of 5mC during successive rounds of replication in the absence of functional DNA methylation maintenance machinery.”

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5-hydroxymethylcytosine (5hmC) • First oxidative product in the active demethylation of 5mC • Prevalent in embryonic stems cells and in the brain • Reduced levels of TET1 and subsequently 5hmC cause impaired self-renewal of stem cells i.e. 5hmC is not just an intermediate

Histone modifications • DNA is wrapped around histones • Histone H2A, H2B, H3 and H4 are the core histone • These histones have tails that protrude from the nucleosome • These tails can be modified • Methylated, phosphorylated, ubiquitinalated etc • These modifications change the structure of chromatin • Looser = euchromatin = active • Tighter = heterochromatin = silent • The modifications are also recognised by other proteins

- Acetylation of lysines on histone H3 tails by Histone acetyl transferases (HATs) is associated with gene expression/active chromatin - Methylation of H3K4 on histone H3 tails by histone methyltransferases (HMTs) is associated with gene expression/active chromatin Absence of DNA methylation is associated with gene expression

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Removal of acetylation by Histone Deacetylases (HDACs) is associated with gene silencing

histone methyltransferases

Can histone modifications be inherited through cell cycle? - After DNA replication, the methylated nucleosomes are distributed equally to the - two daughter strands

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New MTCs re-establish the methylated domain. (Very like DNA methylation mechanism)

Learning outcomes/Summary • DNA methylation is the key mechanism underlying epigenetic memory • Methyl groups are added to cytosines when paired with guanine (CG) • DNA methyltransferases add methyl groups • DNA methylation can be lost passively (lack of maintenance methylation) • DNA methylation can be removed actively (TET enzymes and BER) • Some histone modifications likely contribute to epigenetic memory but we are not sure of the mechanism in mammals...