BIOC2306 DNA methylation

which sequences of bases are methylated

CpG dinucleotides

where is CpG methylated

C5 of cytosine

what sequence can also be methylated in plants

CNG

what does it mean that the DNA methylation modification is symmetrical

both CG in coding and non-coding strand methylated at same time

how is DNA methylation inherited upon DNA replication

parent strand conserved in both daughter DNA (hemimethylated)

hemimethylated DNA recognit by Dnm1 which methylates daughter strand

what is maintenance methylation

restoration of methylation symmetry after DNA rep

role of Dnmt1

recognise hemimethylated DNA

bind and methylated C in daughter strand (maintenance methylation)

role of Dnmt3a or 3b

de novo methylation of CpG

when is de novo methylation prevelent

during embryonic development

what are Dnmt’s

DNA methyl transferases (DNA specific methylases)

which haploid cell is highly methylated

sperm cell

function of sperm demethylation

make sperm gene expression more ‘plastic’

structure of mammalian Dnmt’s

C terminal catalystic domains (v conserved)

N term not highly conserved

which Dnmt doesnt have a known function

Dnmt2

what is Dnmt3L

regulatory factor of Dnmt3a and 3b

which Dnmt associates with the replication fork

Dnmt1

(to maintenance methylation)

(assoc with sliding clamp, PCNA)

both methods of DNA demethylation

passive

active

passive demethylation

1 strand fails to be conserved in fully methylated form

inherited in daughter cells

how is passive DNA demethylation achieved

by removing Dnmt1 from the nucleus (proteasomal degre

what is active DNA demethylation

demethylation using enzyme activity a

active vs passive demethylation

much faster (enzyme vs no enzyme)

important to reset epigenetic marks

why is less DNA methylated than expected

5-me-C is mutagenic

what is the mutagenic nature of 5-me-C

can be spontaneously deaminated into thymine (unlike C that goes to uracil)

are there repair mechanisms for both GC and GT mismatches

yes

why is G-T mismatch repair less efficient than G-U

as uracil is an unusual base in DNA, unlike thymine

role of uracil DNA glycosylase

remove uracil base from DNA (deaminated cytosine) in base excision repair

what mutations are 50 % of point mutations in genetic disease

C-to-T transitions

where are C-to-T mutation transitions also observed

some cancers

why are C-to-T transitions 7x more likely in male germline than female

sperm more heavily methylated than oocyte

where are genes located in invertebrates/sea urchins

in blocks of unmethylated DNA

why is 5-me-CpG lost over time in genome

mutation causes loss in genome

what are CpG islands

regions of CpG dinucleotides in genome

unmethylated

~ 1 kbp long

span promoter and into first exon

at promoter region of all housekeeping genes

where are CpG (CG) islands often found

at promoters of housekeeping genes and 40 % of tissue specific genes

(some replication origins also map to them)

what are orphan CG islands

CG islands not associated with a promoter but still show transcriptional activity (sites of transc init)

2 ways to detect DNA methylation

restriction enyzmes

Bisulphite sequencing

what restriction enzymes are used to detect DNA methylation

Msp I cuts CCGG regardless of methylation

Hpa II cuts CCGG only when unmethylated

therefore observe restriction pattern (sizes)

how does Bisulphite sequencing detect DNA methylation

treat with sodium bisulphite and sequence

bisulphite converts C to U (which are sequenced as thymine)

but me-C are read as C

what percentage of transcription is affected by DNA methylation

only 15 %

what effect can DNA methylation have on transc

represses it

what form of genetic material is the most repressed form

chromatin with methylated DNA

How does DNA methylation directly inhibit transcription

by preventing TF binding

why is direct inhibition of transcription by DNA methylation limited

Not all TFs have CpG in their recognition site, so would have no effect

How does DNA methylation indirectly inhibit transcription

methylated DNA binding proteins bind to me-CpG and prev TF binding to promoters

what is the primary method by which DNA methylation inihibits transcription

indirectly

what is MeCP1

a methylated DNA binding protein (repressor)

are CpG islands methylated or unmethylated in most cells (somatic and germline)

unmethylated

when are CpG islands methylated (the exception)

on the inactivated X chromosome of females

so binding proteins can inactivate it

why is transcription not inhibited by DNA methylation for 85% of genes

not enough DNA methylation to recruit proteins

what determines if DNA methylation does or doesnt inhibit transc

CpG density

why do strong CG islands become weak CG islands over time

met-C lost over time via mutation

how do methylated DNA binding proteins inhibit transcription

bind methylated DNA and recruit HDAC/Sin3A complex

(histone deacetylase)

what DNA binding protein doesnt bind methylated DNA

(unlike others)

MBD3

what is MBD4 involved with (methylated DNA binding protein)

in repair of 5-me-C deamination mutations

glycosylation domain removes T base to allow base excision repair

(but not able to prev all muts)

why are promoter regions not methylated

prev de novo methylation due to present active chromatin mark, H3K4me3 (on histone tail)

active transc prevs too

role of Cfp1

binds at CpG islands and recruits H3K4 methyltransferase (Set1) that makes active mark (H3K4me3) that prevs de novo methylation