DNA modifications

What can "epigenetic clocks" be used to determine?

(Chronological or Biological) Age of a person based on age-dependent methylation changes in certain CpG loci

In terms of epigenetics what has been seen in some age-associated diseases? Give some examples of these diseases (5)

- Acceleration in DNA methylation age - Alzheimers - Cardiovascular disease - Cancer - Down syndrome

Describe the role of aberrant DNA methylation in cancer. (2)

- Activation of tumour promoting genes - MAGE, S100P - Silencing of tumour suppressor genes - VHL, MLH1

Describe how methylation levels change through development. (8)

- After fertilisation methylation levels of paternal genome drops rapidly - Maternal methylation levels also decline but slower - Methylation is erased - Following implantation, cells begin to differentiate and methylation levels of both increase - Highest levels of methylation in the epiblast - Aquire the epigenotypes - Methylation is fairly maintained throughout the adult lifetime - Primordial germ cells - methylation erased then re-established for specific gamete type

Give examples of drugs used to passively de-methylate DNA. (3)

- Azacytidine - Decitabine - Cytosine nucleoside analogous

What are the different types of disease that can arise due to abnormal methylation patterns? (4)

- Cancer - Autoimmune diseases - Metabolic syndromes - Neurological disorders

Why can 5hmC be a stable modification?

- Concentrated at transcriptional regulatory elements - Positively associated with transcriptional activity

What are the clinical signs of Fragile X syndrome? (6)

- Delayed development of speech - Hand-flapping - Poor eye contact - Hyperextensible joints - Large prominent ears - Long narrow face

Describe how Bisulfate Sequencing can be used to detect DNA methylation. (4)

- Doesn't affect methylated C, converts unmethylated C into U - During PCR amplification and sequencing, unmethylated C's will be read as T's - Allows for distinguishing between unmethylated T and methylated C - Use sequencing software to compare the new sequence to original template to determine

What are the roles of 5hmC? (3)

- Embryonic development - Cellular differentiation - Stem cell reprogramming

What two factors can be compared to determine accelerated ageing? What predisposes people to accelerated ageing? (3)

- Epigenetic age (DNA methylation levels) - Chronological age (time) - Infectious risks and exposure due to sanitary conditions

What are the different biological processes that DNA methylation plays a role in? (4)

- Gene regulation - Chromosome stability - Genomic imprinting - X Chromosome inactivation

Explain why anti-cancer (cytotoxic) chemotherapy drugs have been useful for demethylating to reactivate genes in cancer but not been successful for Fragile X Syndrome. (4)

- Has been successful in cell lines - Cells have to divide - Cancer - uncontrollable cell division - Neuronal cells affected by FMR1 gene mutation do not divide

How can cytosine nucleoside analogous passively de-methylate DNA?

- Incorporated into replicating DNA - Form covalent bonds with DNMTS - inhibiting them - Only works for dividing cells - during DNA replication

What are the four methods of detecting DNA methylation? (4)

- Methylation-sensitive restriction enzymes - Antibodies - dot blot / immunoprecipitation - Bisulfite sequencing - Nanopore sequencing

What has 5hmC been implicated in regulating? (3)

- Neurological plasticity - Immunology - Dynamic systems by the TET enzyme family

Describe the CGG repeats in the FRMR1 gene. (4)

- Numerous CGG repeat within the UTR (untranslated region) - Normally between 6-60 repeats - Full mutation is over 200 repeats plus methylation = silenced promoter - All upstream of ATG

Describe the role of the Polycomb-trithorax system in epigenetic systems. (4)

- PcG and TrxG group proteins are essential epigenetic regulators - Can maintain stable epigenetic memory of silent states (via PcG) and active states (TrxG) - Modify chromatin - Allows for poised states in pluripotent cells

What are two classic cellular memory systems?

- Polycomb-trithorax system - DNA methylation / modifications

What are CpGs? (2)

- Predominant site where methylation occurs - Cytosines separated by one phosphate group from a Guanine

What triggers DNA methylation? (4)

- Repetitive DNA - Histone modifications - Certain RNAs - Metabolites

Abberrant methylation of FMR1 once established during early development is ............. and ........... ................. (2) Methylation is established in early development before ....................................

- Stable AND somatically heritable - Differentiation of cell lineages

What are the two processes that TET enzymes can mediate via oxi-mC production? (2)

- Thymine DNA glycosylate (TDG)-mediated base excision repair (BER) - of 5fC:G and 5caC:G - Replication-dependent passive demethylation

Which area of the gene has the lowest probabilities of being methylated? Why? (2)

- Transcription factor binding sites - Interfere with DNMT trying to methylate a CpG

What is cytosine methylation? (2)

- Transfer of a methyl group from S-danosyl methionine (SAM) to the fifth position of cytosine nucleotides - Forming 5-methylcytosine (5mC)

Why are microarrays ideal for surveying thousands of DNA samples? (4)

- 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

Explain the potential for artificial transcription factors in decreasing methylation levels.

- Zinc-binding protein (ZF-VP64) to boost de-methylation events - Binds specifically to methylated CGG repeats only

How prevalent is Fragile X syndrome?

1:4,000 males and 1:8000 females

How many CpG sites are there in the human genome?

28 million

What is 'ATG'?

5' end of gene

............ is an intermediate step in the demethylation of cytosine

5hmC

How many CpG sites are methylated in human somatic cells?

70-80%

What is epigenetic inheritance?

Ability of daughter cell to retain a memory of the gene expression patterns that were present in the parent cell

What is Biological memory?

Can be defined as a sustained cellular response to a transient stimulus

Where on cytosine is the methyl group added?

Carbon 5

Where are most of the unmethylated CpG sites found?

Clustered in the CpG island located on the promotor region of genes

What is the role of Ten-eleven translocation (TET) deoxygenates in DNA methylation?

Converting 5-methylcytosine to 5-hydroxomethylcystosine

Where are the predominant sites of cytosine methylation?

CpG dinucleotides

What enzyme catalyses cytosine methylation?

DNA methyltransferases (DNMT1 and DNMT3a/3b)

What is the difference between DNMT1 and DNMT3a/b action? (2)

DNMT1 - maintenance of methylation patterns by replicating the hemimethylated CpG sites DNMT3a/b - de novo, new patterns of methylation

What are the limitations of Bisulfate Sequencing?

Does not distinguish between 5mC and 5hmC

What does deletion of the DNA methyltransferase genes result in?

Embryonic (DNMT1 and 3b) or Postnatal (DNMT3a) lethality

What can DNA methylation patterns be used to quantify?

Epigenetic age

Is DNA methylation higher in exons or introns?

Exons

What is Biological age?

Explains variation in biological status of individuals with the same chronological age

Is methylation heritable or not?

Heritable

DNA methylation is .......... at the early stages of development

High

How can methylation of the FMR1 gene be detected?

Isolate haemopoeitic cells from bone marrow

What is the role of Activation-induced deaminase (AID)?

Mediate deamination of 5hmC to 5hmU followed by TDG-mediated BER

What is Fragile X syndrome?

Most common cause of inherited intellectual disability - FMR1 gene

What effect can TET proteins have on thymine?

Oxidise thymine to 5hmU - change to uracil

What is the difference between passive and active demethylation? (2)

Passive - caused by inhibition of DNMT1 during cell replication Active - modulated by the DNA methylases

Where are CpG islands typically located?

Promoter region - 5' to TSS

How is 5hmc action different from 5mc?

Recruits different transcriptional factors, splicing factors, DNA repair proteins and chromatin regulators

What are CpG islands?

Regions of high CpG density that often lack methylation, found at promotors of most human genes

When 5hmc was found in the male pronucleus of mouse zygotes what was the theory?

Role in early demethylation of the paternal genome

Which has higher methylation levels, sperm or oocyte?

Sperm

What are the two different models of epigenitics? Describe them. (2)

Static / conserved - mediated by maintenance methylation Dynamic equilibrium - continuous de novo methylation and demethylation

Which enzyme excises 5hmU:G mismatches?

Thymine DNA glycosylase (TDG)

DNA methylation in regions near the ................................ is closely associated with suppression of gene expression

Transcription Start Site (TSS)

There is an .................. distribution of CpG dinucleotides in mammalian genomes and their modification states

Uneven

Cytosine methylation can be heritable in daughter cells but how can it be lost?

Via passive demethylation

Where is the FMR1 gene located?

Xq27.3

How many of the human CpG sites can be currently analysed by microarrays?

~2-3%