Lecture notes from a series of lectures in BIOL3015 Regulation of Gene Expression, epigenetics. Covering: DNA methylation, chromatin structure, epigenetic modifiers, non-coding RNAs, Epigenetics and disease, social environment, cancer and tumour supression
Epigenetics
Epigenetics
Literally means ‘on top of genetics ‘
Definition – Processes that include long-term stable changes in gene activity without
a change in gene sequence
Main 3 processes are: DNA methylation, Histone modification and non-coding RNAs
DNA methylation
Cytosine is methylated to 5-methyl Cytosine
90% of methylated cytosine is found as a dinucleotide CpG
Hemi methylated – one strand methylated
Mapping methylation status of genes
Two restriction enzymes used:
MspI – recgonises and cuts CCGG, will also cut CCMeGG
HpaII – only cuts CCGG is second C is not methylated
If 3 unmethylated CCGG both enzymes will cut all of them
If a site is methylated, Msp1 will cut all sites, Hpa2 will only cut non methylated
CCGG
Hence, different size bands are left
Can show if methylation is present
Analysis of methylation status of the globin gene
Erythroid cells produced the same number of bands of the same size hence no
methylation
Liver cells Msp1 got two bands whereas Hpa2 only had one band
If the gene transcriptionally active then the cells were unmethylated (Erythroid), but
if the gene transcriptionally inactive then the cells are methylated (Liver cells)
Methylation and transcription
If low levels of methylation in the promotor region – the gene active
high levels of methylation in promotor regions – gene inactive
Model for DNA methylation inhibition of gene expression
Transcription factor binding is blocked by DNA methylation
MeCP2 (Methyl CpG binding protein) Binds to methylated CpG and coats it to block
RNA polymerase
DNA methyl transferases (DNMTs)
DNMT 1:
Copies methylation marks from original strand of DNA onto new DNA
DNMT3a and 3b: de novo DNMT’s:
Responsible for establishing methylation marks
If removed in mice – they are lethal
DNMT2 not lethal in mice – unknown function
Search for DNA demethylase
Could not be found
Suggested that passive process could remove methylation
Dnmt1 does not replicate methylation marks, leaving hemimethylated DNA which in
the next round of replication will produce DNA without methylation
Active demethylation
, Hydroxy methyl cytosine (hmC)
Found in 1972
2009 Tet1 (ten-eleven translocase 1) found to convert 5mC to 5hmC)
Hydroxy form is thought to be intermediate in demethylation of DNA
Oxidation or deamination can then remove methylation
5hmC an intermediate in removal of 5mC
Low levels in the genome (less than 105 of 5mC i.e., short-lived entity)
Over expression of tet1 leads to decrease in 5mC, increase in unmethylated Cytosine
Tet 1 depletion leads to an increase in 5mC
Chromatin structure
Nucleosome structure
DNA loop wrapped around a core of histones
Histones are positively charged proteins – 8 histones
Nucleosome is then folded upon itself to make a solenoid structure/ 30nm fibre
Further looped and compacted to form 200 nm fibre which forms DNA in cells
DNase 1
Endonuclease that cleaves double-stranded DNA
Chromatin from erythroid cells added to increasing concentration of DNase 1
Run on gel, blot and hybridized with probes from gene of interest
Ovelbumin – transcriptionally inactive in cells – little or no digestion of gene –
condensed form so difficult for DNase 1 or RNA polymerase to access
globin gene – Active in cells – complete digestion of gene even in low
concentrations – easy for DNase 1 and RNA pol to gain access
Chromatin structure
Loose or tight
Transcriptionally active gene – unmethylated – relaxed structure – accessible to RNA
pol
Transcriptionally inactive – methylated – condensed structure – not accessible to
RNA pol
DNase 1 hypersensitive sites
Erythroid cells –
Located at the 5 prime ends of genes
Are nucleosome free
Initiation is reduced on DNA that is associated with nucleosomes
Transcription factors can recruit co-activators
Modify histone structure
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