Epigenetic regulation

Question 1

What is epigenetics? Give 3 examples?

Answer 1

altering of gene expression without changing the primary DNA structure
- DNA methylation
- histone modifications
- non-coding RNAs

Question 2

What is the role of epigenetics in normal cells? Give 3 examples

Answer 2

• the compartmentalisation of a complex genome into active and repressed regions
• silencing of parasitic sequences
• X inactivation
• imprinting
• mainly controlled by DNA methylation and histone modification

Question 3

What is the epigenome

Answer 3

the epigenetic code of the DNA

Question 4

What is histone modification?

Answer 4

molecules attach to histone tails and affect chromatin condensation and DNAs ability to be transcribed

Question 5

What is the basic mechanism underlying DNA methylation?

Answer 5

• methyl groups added to cytosines make them into 5-methylcytosine
• catalysed by DNMTs

Question 6

How are methyl marks removed? how is it different to addition?

Answer 6

• multiple steps and slower
• TET enzymes drive multiple steps where the cytosine has multiple formations before going back to normal

Question 7

How is DNA methylation controlled?

Answer 7

• formed at gametogenesis and maintained through divisions
• DNMT1 is a maintenance DNMT that maintains methyl marks
• DNMT3A/B/L are de novo DNMTs that don’t require a template to add new methyl groups
• TET genes involved in demethylation
• KOs found role of DNMTs in mice that are embryonic lethal and alter methylation and imprintation

Question 8

How can DNMTs be altered in cancer?

Answer 8

• very rarely mutated
• often overexpressed

Question 9

Where does most DNA methylation occur in mammals?

Answer 9

• at CpG islands (some non CpG especially in ESCs)
• 50% of genes have them
• stretches of DNA with CG content over 60%
• usually demethylated and methylation is associated with cancer
• non CpG methylation occurs in ESCs and not fully understood

Question 10

Describe a method that can be used to test for methylation

Answer 10

• purify DNA and treat with bisulphide
• unmethylated cytosines will be deaminated into uracils
• methylated cytosines will remain to be measured

Question 11

Name 3 ways methylation inhibits transcription

Answer 11

• directly causing steric hindrance and stopping TF binding
• indirect effects via chromatin remodeling
• boundary elements

Question 12

How can methylation affect chromatin to inhibit transription?

Answer 12

when methylation occurs, methyl-binding domain proteins can recognise methyl groups and complex with histone deacetylase proteins and deacetylase the histones, pulling the DNA closer to further repress transcription

Question 13

What are boundary elements?

Answer 13

• markers that help to control the boundaries between methylated and unmethylated regions
• can affect the action of promoters
• helps to regulate gene transcription

Question 14

How are histone modifications controlled?

Answer 14

• change throughout development
• marks are added and removed by enzymes such as HATs and HDACs
• regulated by readers, writers and erasers

Question 15

Give an example of histone mark readers

Answer 15

• bromodomains
• recognise marks on the histones and signal for downstream TF and chromatin regulation

Question 16

Give an example of histone erasers

Answer 16

• TETs perform demethylation

Question 17

Give an example of histone writers

Answer 17

• HDACs, HATs
• methyltransferases, demethylases

Question 18

What are the 2 possible mechanisms by which DNA methylation and histone modificatoin may interact?

Answer 18

• DNA methylation driving histone modification
• histone modification driving DNA methylation
• evidence for both has been found

Question 19

How might DNA methylation direct histone modifications?

Answer 19

• DNA methylated regions recruit methyl-CpG-binding proteins and histone deacetylase
• this recruits histone methyltransferases that methylate the histones and stabilise the inactive chromatin

Question 20

How might histone modifications direct DNA methylation?

Answer 20

• inactive chromatin undergoes DNA replication
• histones recruit HP1 which recruits DNA methyltransferases to maintain DNA methylation throughout division

Question 21

Does histone methylation have activating or repressing effects?

Answer 21

• both
• H3K4 = activating
• H3K9 or H3K27 = repressive

Question 22

How does DNA/histone methylation co-operation turn off pluripotency genes in development?

Answer 22

• G9a histone methyltransferase induced H3K9me2
• also recruits DNMTs to induce DNA methylation

Question 23

What are chromsome domains?

Answer 23

• modular structures in chromatin that correlate with epigenetic marks
• each have distinct tsructural and functional properties
• for example polycomb domains control Hox gene silencing in drosophila

Question 24

How do epigenetic changes lead to X-inactivation

Answer 24

• long non-coding RNA Xist binds the chromosome to be inactivated
• DNA is highly methylated with repessive histone methyl marks too (H3K9me2, H4K27me3)
• Xist can attract chromatin-modifying complexes such as polycomb to mediate inactivation

Question 25

How do epigenetic changes lead to inactivation of parasitic sequences?

Answer 25

- 50% of the human genome is made of damaging repeats such as transposons that can insert into other genes - most 5-methylcytosine is found at these sites and transposon promoters are inactivated - prevents unwanted transcription

Question 26

What is genomic imprinting?

Answer 26

- mule female horse, hinny female donkey - parental origin of genes are different - imprinted genes only express one allele - phenotype dependent on parental origin - resets on passage through the germline

Question 27

What is the life cycle of DNA methylation in imprinting ?

Answer 27

- at the germline, de novo DNMTs add methyl marks - these are maintained through cell division by maintenance DNMTs - continues until gametogenesis where TETs remove methyl marks and new ones are applied

Question 28

What are the basic characteristics of imprinted genes?

Answer 28

- only expressed on one allele - often in clusters with imprinting control regions within them - antisense and ncRNAs are common - different methylation patterns on each allele

Question 29

What are two human tumours that can arise from incorrect imprinting?

Answer 29

- Wilm's tumour from incorrect WT1 imprinting (leads to poor MET) - ovarian teratomas caused by lac of paternal imprinting

Question 30

Give an example of a genomic imprinting locus.

Answer 30

- IGF2 encodes fetal growth factor and is only expressed on the paternal allele - H19 encodes an untranslated RNA that appears to act as a tumour suppressor and is only expressed on the maternal allele

Question 31

How do IGF2 and H19 interact in genomic imprinting on the paternal allele?

Answer 31

- on the paternal allele the IGF2 promoter interacts with an enhancer downstream of H19 - extensive DNA methylation of the boundary element between IGF2 and H19 means that CTCF cant bind and this can happen - 2 alleles like this can lead to wilms tumour

Question 32

How do IGF2 and H19 interact in genomic iprinting on the maternal allele?

Answer 32

- no hypermethylation of the boundary element - CTCF can bind here and create a boundary that stops the IGF2 promoter from accessing the promoter - allows H19 to use it instead - two alleles like this can lead to dwarfism

Question 33

What are epimutations?

Answer 33

- mutations that can interfere with the loss of boundary elements - seen in Wilm's tumour if IGF2 is overexpressed on both alleles - mutation that enhances the boundary element instead has 2 H19 alleles and leads to growth failure - silver-russell dwarfism)

Question 34

What are two theories on why genomic imprinting evolved?

Answer 34

Haig theory - imporinting lessens conflict between patnerla and maternal genomes by allowing a mixture to be expressed Barlow theory - methylation developed as a defense against foreign DNA and was evolved for control of development