epigenetic Flashcards
where does methylation take place one the DNA
at the cpg site (where a cytosine is followed by a guanine), it occurs on the cystestine on the lysine
where do the methyl groups for methylation come from?
deitry methyl donors
what is the cycle taht produced methyl
methionine metabolic pathway which is part of the one carbon pathway
how the methyl produced
methionine is converted to SAM. SAM donated its methyl group to make SAH which is then hydrolysed to homocytocine which is catabolised or remethylated to methionine
what is the enzyme involved in methylation
DNA methyltransferase
which DNMT are de novo and what does it mean
DNMT 3a and 3b and it means it can methylate unmethylated DNA
which DNMT are mainitance and what does this mean
DNMT1 and attached a methyl group to semi methylated DNA during replication
what is euchromatin
decondensed and transcriptionally active
what is heterochromatin
condensed and transcriptionally inactive
what does the nucleosome core consists of
3 copies of H2A,H2B, H3 and H4 making a histone Octamer, globular C terminals and N terminal tail
how many bp does a nucleosome consist of
146
what does it mean if nucleosome is acetylated
it is has a. ore open formation of DNA allowing for easier transcription
what does it mean if the nucleosome is deacteylated
less easy transcription
which enzyme deacetylates DNA
HDACs
which enzyme acetylates DNA
hats or KATs with actyl-coa as substrate
how many times can lysine be methylated
3
how many cytosines are methylated
3-4%
how does transcriptional repression happen
MeCP2 binds to methylated DNA and recreuits to HDCA1 for deatylation
also recuits DNMT which methylates the DNA
what enzyme add a methyl to lysine
KMT
how does DNA hypomethylation agent work?
it is a cytidine analogue. Methyl group cannot be added to the nitrogen on the 5 position. Also known to bind and insist DNMT
what is the example of tumour suppressor gene taht is switched off in cancer and what can it be switched back on by
P15INK4B inhibits cyclin dependant kinases and decitabine
what methylation is smoking related to
decreased methylation of coagulation factor II receptor like 3 gene- induces platelet activation
the methylation at what gene is thought to increase diabetes risk
5 gene loci
what is the relevance of epigenetic to modern medicine
-understanding disease medicine
-identifying biomarkers that might predict disease or inform us about prognosis
-using pigenetics to find new treatments
what is Mendels law
it states that in a diploid organisms there is an equal genetic contribution to offspring made by each parent
what is genetic imprinting
mono-allelic expression
what does maternally imprinted mean
maternally inactivated so paternally expressed
what does paternally imprinted mean
paternally inactivated so maternally expressed
why are genes imprinted
thought to be evolutionary as paternal genes tend to promote growth hand mothers tend to suppress
where in the chromosome can you see loss of genes due to imprinting
15q11-13
which imprinting disorder is maternal
anglemans
why is DNA methylation easier to study
there are multiple methods, it is better studied and its highly stable over time regardless off how it is treated and collected
what are genome studies
looking at a range of unspecific CpGs throughout the genome. Across Cpgs within report elements through out the genome
what epigenome wide studies
detailed analysis of specific regions/cpGs across the genome
what is gene/locus specific?
detailed analysis of specific region of genome
wha are the 6 techniques used in methylation testing?
-restriction enzyme digest
-DNA sequencing
-immunoprecipitation
-fragment size analysis
-bisulfite modification
-DNA microarray
how does cytosine extension assay work
HpaII restruction digests the unmethylated DNA
extension of the overhang using Taq polymerase and radio labelled cytosine
What do the results of cytosine extension tell you
more radio active cytosine the lower methylation
what are the advantages of cytosine extension
-cheap
-quanitifcation of methylation across the genome
-allows overall estimation of methylation between groups
what are the disadvantages
-limited to CpGs in HpaII sites
-dont know where in the genome methylation differences are
-time consuming
-uses radio labelled cytosine
what does MRE-Seq work?
cut with MSP1 and HPAII with one sample cut with just MSP1 and other just HPAII and then add adapters and then compare.
what are the advantages of MRE-Seq
-quantification of methylation across genome
-allows overall estimation of methylation between groups
-sequencing method should allow for detection where changes are in genome
what are disadvantages
-limited to CpGs in HpaII or other restriction sites
-expenisve
how does MeDIP work
DNA is sonificated and then denatured. 5 methylcytidine Ab is used for immunoprecipation
what two methods can you go onto to do with MeDIP
MeDIP seq and MEDIP-CIP
What can MeDIP-Seq be used for
can compare healthy cells to cancer cells
What can MeDIP-Seq be used for
can compare healthy cells to cancer cells
what are the advantages of MeDIP
- better knowledge of regions of the genome with methylation change
-good genomic coverage
what are the disadvantages of MeDIP
- doesnt tell you the magnitude of methylation
-cant resolve single CpG methylation changes
-expensive
what are the steps of covering cytosine to uracil in bisulphite modification
denature DNA and fragment it at 95 degrees and then do a conversion step by incubated it with sodium bisulphate at 65 degrees. Then do desulphitization which makes cytosine uracil.
what is the differences between DNA with methylated and unmethylated with bisulphate modification
methyklateed has cytosine and unmethkated goes to uracil to thinene
what are the advantages and disadvantages of whole genome bisulphite sequencing
-coverage of the whole genome
-exopensove
-lacks some precision
what are the advantages/disadvantages of illumina infinitum methylation EPIC Bead chips
- not always informative for distal regulators elements
highly reproducible
precise methylation quantification
why measure methylation of specific genes
-interetested in a specific disease related gene
-hypotheiss driven research
-replication of findings
-biomarker of exposure
-predicitive disease biomarkers
which methodology is most commonly used for gene specific methylation
bisulphite modification
what are the advantages of methylation specific primers
relatively cheap
quick to develop
doesnt require specialist equipment
what advantage does bisulphate have over MSP
a lot more qulaitive
how do you do histone modification assays
Carry out DNA digestion by Mnase, then fragment DNA, then look for the specific histone modification that you want by using the antibody. Then fragments are pulled down again and sequenced.
what is the histone code
post translational modification of key aa of the histone
what does closed and open chromatin refer to
closed- trasncriptianallly silent
open- transcriptionally active
