Kourosh L2

Question 1

where does DNA methylation occur

what is it catalysed by

Answer 1

CYTOSINE bases at C5 position of CpG
catalysed by: DNA methyl transferases in the presence of:
- S-adenosyl-L-methionine (AdoMet/SAM) cofactor
SAM is the universal methyl donor

Question 2

what are the 2 MAIN classes of DNMTs (DNA METHYL TRANSFERASES)
what is the other class, what does it do

Answer 2

DNMT3a and DNMT3b

others add methyl groups to daughter strand during DNA replication

Question 3

what is the role of DNA methylation

Answer 3

• regulating tissue specifc gene expression
• cellular differentiation
• genomic imprinting
• gene silencing
• X-chromosome inactivation

Question 4

how do CpG islands affect gene expression

Answer 4

1) regulation of chromatin structure
2) transcription factor bidning by:
- less nucleosomes
- often close to TSS
- usually encompass TF binding sites
- methylation of exon 1 helps recruit TFs

Question 5

how does DNA methylation regulate gene expression

Answer 5

modulate the chromatin state to control gene expression

1) Promoter methylation: hyper-methylation of CGIs in a gene promoter is associated with gene inactivation. Euchromatin= transcriptionally permissive, while heterochromatin =transcriptionally repressive
2) Gene body methylation: cancer: gene body CpGs are preferential sites for de novo methylation. This gene body methylation is associated with gene expression

Question 6

how does DNA methylation affect regulation of chromatin structure and transcription

Answer 6

• Recruitment of inhibitory transcription factors
• Disrupting binding to transcription factor binding sites
• Methylation of promoter causes gene silencing

STABLE SILENCING

Question 7

histone tail methylation inhibits what

Answer 7

DNMT

Question 8

how is DNA methylation detected

Answer 8

BS-Seq (Bisulphite-Sequencing)

Question 9

how does BS-Seq work

Answer 9

• DNA obtained
• sonicate DNA
• BISULPHITE CONVERSION: : any C in seq is bisulfite converted (REMOVED) UNLESS it has a methyl group attached
• PCR amplification to replace U with T
• sequencing gives us METHYLATION positions

Question 10

case study 3 epigenetic drift: twins

Answer 10

• studied locus specific differences in DNA methylation and histone acetylation of a large cohory of monozygotic twins
• older twins showed greatest differences in gene expression profiles

Question 11

case study 2 fragile X syndrome

Answer 11

• rare (1/4000 males, 1/8000 fem)
• mutation in FMR1 gene, which encodes FMRP protein
• CGG triplet repeat in 5’ UTR is expanded in the FMR1 gene
• normally repeated 5-40 times, in fragile X it is repeated >200 times
• expansion of CGG segment silences FMR1 gene
• hypermethylation of FMR1 promotor
• DEC FMR1 transcription O loss of FMR1 protein