Lecture 2 Flashcards
Where does DNA methylation happen in mammals?
In all tissues - but methylation profile is tissue specific
Mainly on CG dinucleotides (CpG) but also CHH (where H= anything except G) in some tissues (brain, placenta, embryonic SC)
What are CpG islands?
Region (300-3000bp) in promoters with higher than average CpG content, found in 60% of genes, and are usually un-methylated, and hence the gene is active.
Why are methyl-cytosines considered more mutagenic?
SPpontaneous Deamination of 5-mC will lead to thymine which if not repaired will lead to a mutation. Whereas spontaneous deamination of cytosine leads to uracil which will be repaired by mismatch repair
What type of genes have their CpG island methylated during development?
Stem cell master regulators when becoming differentiated cells that need to lose their pluripotency
How do the different techniques for assessing methylation profiles differ?
Their resolution
What techniques can be used to assess DNA methylation profiles?
1) Using methylation-sensitive restriction enzymes and then PCR with primers for the intact product ; only tells you about that site
2) Using anti-methylcytosine and/or anti-hydroxymethylcytosine antibodies to immunoprecipitate methylated DNA followed by next generation sequencing; only know that some site within that fragment is methylated
3) Bisulphide sequencing; Treat with sodium bisulphite and then use desulphonation (Deaminated, sulphinated and desulphinated). Cytosine converted to uracil and methyl-cytosine not affected. Sequencing will reveal C-to-T transition for un-methylated cytosines. Cannot distinguish between 5-mC and 5-hmC
How can you distinguish between 5-mC and 5-hmC in bisulphite sequencing?
TET-assisted bisulphite sequencing Glucosylate the 5-hmC TET oxidation of mC to caC 5-mC read as T whereas 5-hmC read as C Combine TET-assisted bisulphite sequencing and bisulphite sequencing to discover which are 5-hmC and which are 5-mC
What are core histones?
Four small and basic particles (H2A, H2B, H3 and H4), with C-terminus core with DNA wrapped round and N-terminus tails
What post-translation modifications can occur to histones?
over 100 exist however not all effect DNA expression
What effect does methylation of histone 3 have on gene expression?
Can be both repressive, e.g. Lys4, and activating, e.g. Lys 9.
How can post-translation histone modification affect chromatin structure?
Two possibilities
1) Disrupting contacts between nucleosomes in order to unravel the chromatin, by lessening the interaction between the histone and the DNA - e.g. acetylation of Lys neutralises the basic (positive) charge
2) Recruitment of non-histone proteins (Histone code hypothesis)
How does Epigenetics effect Honeybee Queens?
Queen larvae gets fed royal jelly throughout development
Royal jelly contains phenyl butyrate (a histone de-actetylase inhibitor)
Leads to chromatin being more open, loss of DNA methylation and more active genes
Can be mimicked by giving larvae methylation inhibitors
Why does opening the chromosome decrease methylation?
Activating the gene expression causes other machinery to bind blocking access to methyltransferases
Chromatin more open for demethylating enzymes to act.
Give some example proteins that can recognise different modifications to histone tails according to the histone code hypothesis
Methylation: Chromo, Tudor, PHD and MBT
Acetylation: Bromo
Phosphorylation: 14-3-3
What effect does methylation have on the histones?
Does not affect charge
Recruits proteins to affect chromatin structure
Associated with gene activation (H3K4me and H3K36me) and repression (H3K9me and H3K27me)
