Landsberger: Lecture XXII Flashcards
DNA Methylation: Detection Strategies and Function
What are the 2 roles of DNA methylation?
inhibitor of gene transcription
maintenance of genome integrity
When we looked at the phenotype of the DNA methyl transferase de novo mutants, what was found?
genomic instability and activation of transposons
What does it mean to maintain genomic integrity?
chromosomes do not break and para-CpG sites do not get activated
How was it proven that DNA methylation is important despite drosophila and C. elegans not showing signs of DNA methylation?
cell cultures were treated with inhibitors of DNA methyl transferase (5-azacytidine) and these inhibitors caused global or general demethylation of the genome
Xenopus oocytes were the main models used
Why were Xenopus oocytes the main models used?
Xenopus have some advantage in understanding the role of DNA methylation
What were they looking to be methylated in culture before injecting into Xenopus oocytes?
the plasmid, which could be methylated or not methylated by enzyme Sss1 methylase (prokaryotic enzyme that methylates any C-G dinucleotide that is in the sequence)
In the experiment, what happens when the promoter is methylated?
reportr gene is expressed
What does methylation do?
silences gene expression
Why did they use the oocytes of Xenopus?
Xenopus is a toad, and the female has many oocytes, which are 1 mm in diameter and can be seen without difficulty
What is the dark part of the Xenopus oocyte called?
animal pole
Where is the nucleus located in the Xenopus oocyte?
between the animal pole and the vegetal pole
What does the nucleus of the oocyte store?
material required for the 1st division after fertilization
What happens after the oocyte gets fertilized?
it has 12 divisions that are made by alternating S/G2/M phases without a G1 phase
What can be concluded since the oocytes do not undergo a G1 phase?
cells exploit all the material that is present in the vegetal pole in order to obtain the first 12 divisions
What is it called when DNA is transcribed and injected directly into the nucleus?
blind method since we cannot see the nucleus, but we know that it is in the white line of the oocyte
What is the only thing that cannot be evaluated using Xenopus oocytes?
replication of DNA unless a ss DNA is introduced
the oocyte still has chromatin because it contains a large amount of histone proteins
When are histones synthesized?
S phase
Can you study post-translational modification in transfected cells?
no
What was found through the analysis of the Xenopus oocyte and the trafected cell lines?
methylation of the vector leads to inhibition of transcription from pole 1, pole 2, and pole 3 so it is a general effect
What is the direct model?
model proposed by Adrian Bird that answers how the methylation is occuring
methylated TF → methyl group overruns major groove → methyl group interferes with binding site → TF does not bind → repression
called direct model because the methyl group directly blocks the binding of TF
Describe how DNA methylation occurs:
it is known the mthyl group overhangs into the major groove of DNA
TF interact with DNA into the major groove by using 𝛼 helices to interact with sequence-specific DNA
Adrian proposed that if we have a promoter, we will have some CG (as indicated by the lollipop)
if the binding sites of TF are not methylated, the TF can bind and the gene is expressed (White lollipop)
How can the direct model be proved?
using the electrophoretic mobility shift assay
How does the electrophoretic mobility shift assay work?
probes (piece of promoter that contains a piece of the binding site for factor) is put into a tube with different samples (not methylated with no factor, methylated with no factor) and the migration is observed
expected outcome: if factor can bind to the promoter or sequence, DNA will be delayed because it is heavier so it will not travel as far
*this was done in vitro
What assay can we use to prove a diseased cell is methylated in vivo?
using a chromatin precipitation assay, followed by PCR sequencing to see if the sequence of interest has been bound (or rtPCR, which is more expensive)
What is a chromatin precipitation assay?
ChIP (antibody-based technology used to selectively enrich specific DNA-binding proteins along with their DNA targets)
What were 2 limitations to the direct model?
CG dinucleotides are much less abundant than expected, so we cannot say transcription is off since the TF is not directly inhibited by the methyl group
sometimes TF were able to bind to the recognition sequence even if it was methylated
What is the indirect model?
if DNA is not methylated, gene is expressed
What are methly binding proteins?
proteins that bind to DNA because it is methylated not because of a sequence
this will cause the inhibition of TF and gene expression
Which model proposed by Adrian Bird was right?
Indirect model
What did Adrian Bird call the bands?
NCP1 and NCP2 (or MeCP1 and MeCP2: stands for methyl CpG binding protein 1 & 2 depending on the shift)
What 2 domains are found in MeCP2?
methyl binding domain
transcriptional repressor domain
MBP and the chromatin structure are ____.
2 independent epigenetic mechanisms that cross-talk
*therefore, no methylation leads to chromatin that is more open
**methylation leads to the recruitment of MBP and a closed chromatin structure
Why is splicing affected by defects in DNA methylation?
splicing is regulated due to consensus sequence
fast polymerase → no splicing → select splicing on strong consensus sites
this makes no sense
