DNA methylation

Question 1

Give an overview of DNA methylation?

Answer 1

Methylation takes place at the C5 position of the cytosine ring when cytosine is present in the CpG dinucleotides - in vertebrate/invertebrates
Can occur at CNG in plants
Modification is symmetrical - as CG on one strand is CG on the complimentary strand

It adds a bulky methyl group - that protrudes into the major groove of DNA - the major group is where many things bind
Leads to the profound effect on binding of proteins

Question 2

How does methylation occur during replication?

Answer 2

DNA methylation patterns are inherited upon replication
Newly synthesized daughter strands are hemi-methylated
Hemi-methylated DNA is converted to fully methylated state by Dnmt1 - DNA methyl transferase 1, during replication
“Perfect” epigenetic mark - we can tell the enzyme where to methylate the daughter strand as it has a map on the template

CpG dinucleotides that are methylated stay methylated
CpG dinucleotides that are unmethylated (e.g. central CpG) stay unmethylated – usually
The fidelity of Dnmt1 = around 95%
Increased to 99%, when associate with NSP1

Question 3

What is de novo methylation?

Answer 3

De novo methylation occurs at a high level during early embryonic development
Can occur at a lower level at other developmental stages
Carried out by Dnmt3a and Dnmt3b
They convert a completely unmethylated CpG dinucleotide into a methylated one i.e. no marker on the ‘template strand’ like with Dnmt1 in replication

Question 4

Describe the mammalian DNA methyltransferases?

Answer 4

The C-terminal catalytic domains are highly conserved
They all flip the base out of the DNA
The N-terminal domains are not well conserved

Dnmt1 has a PCNA interaction site - associated with the replication fork
Maintenance of DNA methylation patterns each time DNA replicates to maintain the epigenetic mark

Dnmt2 does not have a known function
Dnmt3a and Dnmt3b associate with a regulatory factor Dnmt3L - this directs the enzymes to where they want to be

Question 5

What happens to DNA following fertilisation?

Answer 5

De-methylation of DNA
Important following fertilisation - as sperm is very highly methylated
Here methylation is taken down to around 30% of normal levels
Erasing pre-existing methylation marks and then de novo will re-establish this

There are two types - passive and active

Question 6

Describe the two types of de-methylation of DNA?

Answer 6

Passive de-methylation
One strand fails to be converted to the fully methylated form at replication
This then becomes inherited in daughter cells

Active de-methylation
Methyl-cytosine removed from DNA via enzymatic activity - in the absence of replication
Occurs much more quickly than passive demethylation can occur
Very important during early embryonic development to re-set epigenetic marks
Two methods - either DNA repair or hydroxylation of the methyl group

Question 7

Give an overview of the genomic distribution of DNA methylation?

Answer 7

70-80% of all CpG dinucleotides are methylated
Mammalian genome 40% GC

We expect 5meC to be 4% of all bases but there is only 1%
The dinucleotide CpG is 4-5 fold under-represented in mammalian DNA - due to mutations

Question 8

Describe the mutagenic nature of 5-me-C?

Answer 8

Cytosine spontaneously deaminates
Methyl-cytosine deaminates to thymine (not repaired efficiently)
Non-methyl-cytosine deaminates to uracil (can be repaired well)
= losing some methyl-cytosine from the genome

Question 9

Describe the repair of mutagenesis in non-methyl-cytosine?

Answer 9

The G:U mismatches are repaired by uracil DNA glycosylase
Deamination C to U -> uracil DNA glycosylase -> base excision repair
The glycosylase clips the base from the sugar phosphate backbone
High fidelity

Question 10

Describe the repair of mutagenesis in methyl-cytosine?

Answer 10

CpG dinucleotides are lost from genomic DNA since the mutation is not efficiently repaired
C to T transitions account for 50% of the point mutations found in genetic diseases (germline mutations) - C to T transitions are also found in cancer (somatic mutations)
C to T transitions are 7 times more frequent in the male germline compared to the female because sperm is heavily methylated

Question 11

Describe the distribution of CpG dinucleotides within invertebrate and vertebrate genomes?

Answer 11

Invertebrates tend to have more unmethylated DNA - therefore the genes remain in tact as their mutations can be repaired more easily
Within vertebrates we have more methylated DNA therefore allowing more mutations
However, in the unmethylated regions we form CG island = promotors

Question 12

What are CpG islands?

Answer 12

~1 kb long, GC-rich, no CpG depletion, unmethylated in all tissues
Promoter regions of all housekeeping genes and about 40% of tissue-specific genes
CpG islands span the promoter and into the first exon
Some replication origins map to CpG islands

CGIs can be located at annotated transcription start sites, within gene bodies (intragenic), or between annotated genes (intergenic)
Orphan CGIs are sites of transcriptional initiation = associated with gene promotors

Question 13

How can we detect/map DNA methylation?

Answer 13

Bisulphite sequencing
More modern method - sodium bisulphite, pH 5.5 and 55°C
Causes the deamination of C to U but leaves 5MeC as C
Therefore we can detect methylated sites by sequencing - comparing to the wild type

Restriction enzymes
Msp I cuts CCGG regardless of methylation
Hpa II cuts CCGG only when it is unmethylated

Question 14

What is a feature of DNA methylation?

Answer 14

DNA methylation represses transcription
Broadly speaking - DNA methylation dampens illegitimate transcription (further 50x repression)

Directly - prevents transcription factors from binding as the methyl groups stick out of the major groove where binding sites are e.g. CTCF; Myc
However not all TFs have CpG in their recognition site
Indirectly - methylated DNA binding proteins bind to me-CpG and prevent TFs from binding/accessing the promoters

Question 15

What evidence was found to support there were methylated DNA binding proteins?

Answer 15

As the amount of DNA template is increased, the repression by DNA methylation is overcome - showing the repression is limited
Suggests a diffusible factor and not necessarily a mutation in the binding site
= non-specific methylated DNA can compete off the repressor
Suggests the repressor binds any methylated DNA

Question 16

What further tests were carried out in vivo in relation to MBPs?

Answer 16

A methylated DNA binding protein had been identified, MeCP1 - this binds methylated DNA only when there are sufficient methyl CpGs
= confirmation the repressor is MeCP1

Question 17

How are CpG islands methylated?

Answer 17

CpG islands are always unmethylated in the germ-line and in most somatic cells
CpG islands do become methylated on the inactive X chromosome and in some cancers
When the CpG island is methylated, its transcription is strongly repressed

Question 18

Describe CpG density and gene expression?

Answer 18

Normally methylation doesn’t regulate their transcription of 70% of genes
But it does affect 15% - weak CpG islands

Three classes of promoters:
CpG islands – one CpG every 10 bp and are unmethylated in all tissues of the organism
Exception to unmethylated – inactive X chromosome and some imprinted genes

Weak CpG islands – one CpG every 20-30 bp - become methylated
When these are methylated they are repressed (MBPs) and when they’re non-methylated you get transcription
Here DNA methylation can regulate expression during development

Low CpG promoters – one CpG about every hundred bp, weak/no binding MBPs = no effect of methylation on gene expression

Question 19

What different methylated DNA binding proteins exist?

Answer 19

Methylated DNA binding proteins were isolated by homology to the MBD of MeCP2
MeCP2 involved in heterochromatin formation in neurones
When mutated - neurodegenerative disorder rett syndrome

Protein involved in transcription repression - Kaiso and MBD1 and MBD2

MBD3 - mutated and doesn’t bind methylated DNA, but is part of the NURD complex (repressive chromatin remodelling complex)

MeCP1 activity equivalent to two different complexes: Kaiso and MBD2/HDAC

Question 20

What does the transcription repressor domain (TRD) do?

Answer 20

he TRD recruits histone acetyl transferases (deacetylases)
Deacetylation of histones helps to reinforce the repression
MBPs repress transcription by recruitment of the HDAC/Sin3A Complex

Question 21

What is MBD4 involved in?

Answer 21

Involved in the repair of mutations induced by the deamination of 5-me-C to T
It recognises the hemi-methylated DNA and the base pair mismatch
Glycosylase removes T base to allow repair of mutation by base excision repair
But this is not enough to prevent all mutations of 5-me-CpG

Question 22

How are CpG islands usually unmethylated?

Answer 22

The domain of demethylation extends far beyond transcription factor binding sites

H3K4me3 (positive histone mark) is an active chromatin mark - associated with histones
It inhibits de novo methylation - as it prevents the binding of DNMT3a/b
H3K4me3 is catalysed by SET1 - this is recruited by RNA pol II Ser5 phosphorylated CTD
Transcription causes deposition of H3K4me3 at promoters to prevent binding of DNMT3L and de novo DNA methylation

Question 23

What is the Cfp1 novel protein?

Answer 23

Cfp1 and H3K4me3 are enriched/tightly associate at CpG islands
The protein Cfp1 recruits the enzyme, Set1, that puts the positive mark on at CpG islands
Knock-down of Cfp1 leads to loss of H3K4me3 at CpG islands

This active chromatin conformation facilitates transcription
Active transcription is also a mechanism to prevent de novo methylation
= positive mark prevents de novo methylation