epigentics - mehtlyation and assays Flashcards
Which protein is responsible for the maintenance of DNA methylation? A Dnmt1 B Dnmt2 C Dnmt3a D Dnmt3b E Dnmt4
Option A - DNA methyltransferase 1 which has a preference for hemimethylated DNA during DNA replication so can maintain methylation between generations
3a and 3b involved in denovo methylation
without 1 and 3, there is embryo lethality as its essential for early embryonic development
What is epigentics
Heritable chemical changes to DNA that affect permently switch off gene
Regulation through repression
Underpins celkualr differentiation, genomic imprinting disorders and x inactivation
Why is vitB9 foliac acid essential in foetus
It’s a cofactor for metylase actions
…
What are cpg islanss
Areas rich in CpGs that are unmethylaee and can be over 200bp long. Associated ei
They can act as promoters and associated with housekeeping genes which remain unmethyksred in germline and are genomically stable
Can you give examples of housekeeping genes which have methylated regions
Leptin - hasislnd overlapping with TSS and is switched on in adipose tissue to regulate appetite, body mass and fat deostiopn
GLUT4 has 2 distinct islands located at TF binding sites. If methylated GLUT4 transcription is doentegulated as the TF can’t bind
Which of these DMNT not present in mammal A DMNT 1 o B Dnmt2 C DNMT 3a D DMNT 1
Number 2 - present in drosphilla
1,3a abd 3b essential fo life
Mutants wow aloss of
Why do Cpg islands need to avoid being methylated?
Because the DMNTs are non specific. They have a DNA binding site that recognise any and every CpG so could theoretically methylate every dinucleotide they see
However CpG Islands have adapted mechanism to avoid being methylated
How do Cpg islands avoid being methylated?
They have boundary elemtns which wee proteins that can exclude and physically block DMNT from certain areas of DNA
> tether chromatin to structural component of nucleus
> attract Hat or TET to reverse effectof demtylation
>bind directly to a histone blocking it from being modified furthur
Example of this is the Sp1 protein which is a DNA bidnkgn protein that acts as a boundary element
Decribe the gernal epigenetic mechamsims
Methylation of Cpg on DNA
Methylation of histone
(acetlyation, ubiwuination, phosphorylation
This results in CHROMATIN Remodelling which ultimately cn regulate gene expression
How is X chromosome become inactive
NcRNA Xist is encoded from the XIC loci and can coat the inactive X will then recruit silencing factors to inactivate it which invoked chromatin remodelling and methylation of chromosome
Interesting as its one of the few RNA directedgene siloecong in mammals as that is uailly plants only
WHERE CAN 5hmC be found
quite high levels in embryonic Cells as it allows demtethylaation of genome which enable ESC to be pluripotent and differentiate into a cel
what does the thrifty phenotype result in
a reponse to the enivromnemt in poor malnutrition in whihc we can be metagoically efficietn and store energy
what are the symptoms of the reciprocal imprintning disorders Praderwilli and anglemann syndrome?
prader - parental deletion IGF2 or epigentic
> Darth vader so short and fat, hyperphagia, hypogonadism
anglemann - maternal deletion H19 so small thin, speech delay, laughter and jolly
how does the DNA methylation change through fertilsation and brith
passive (mum) or active (dad) demethylation at the zygote level allowing for ESC to be pluripotent but some epigenetic marks remain - evidence
embryogenesis DNA methylation will increase as cells differetiate. As we age is relatively constant and THEN there is global demethylation and specific methylation of CpG islands
> increase our risk of cancer as more likely for TS genes to be silenced like p53 gene oh nooo
how can we detect DNA methylation in a lab
use restriction enzymes (ones that cut umethylated and ones that cut methylated like Hpall and Msp1
use bisulphite modifications that will deaminate any C-U-T but much more slowly on a 5mC
methylation specific PCR using primers to do this
pyrosequencing that quantitatively monitors the real-time incorporation of nucleotides. i guess in combination with bisulfite
