Lesson 18 Flashcards
why are both HpaII and MspI methylation sensitive?
both of them recognize CCGG
how does MspI relate to HpaII?
MspI functions to control the HpaII digestion
what was used in the 90s to determine if a gene or a promotor of a gene was methylated?
radioactive probes
what technique permits to understand if a specific fragment of DNA has a different molecular weight after hybridization?
southern blot
what does it mean if digestions is identical?
there is no difference in methylation between the two samples
describe the difference in ribosomal DNA methylation between vertebrates and invertebrates:
vertebrates have more methylation in ribosomal DNA then invertebrates
what are the four main issues with the radioactive probes?
- depend on restriction enzymes
- most of the restriction enzymes don’t have the control
- we have problems looking at all the GC in which were interested because they might not be part of the palindrome sequence
- it is time consuming - radioactivity is never ideal
what must we do in order to study DNA methylation?
we first have to treat the samples and then there is the amplification
what happens if the DNA is unmethylated and we try to perform PCR?
I don’t detect the fragment of 200 bp: if we digest and there are two cuts, after the denaturation there will be one strand and then there is the annealing of primers and the DNA polymerase will replicates, but at the end I don’t detect any signal because in case the DNA is not methylated and therefore the enzyme will cut, but the fragment will not be amplified by PCR because the primer (blue) is outside of the restriction sites
what are the two limitations of combined restriction enzymes with PCR?
limited by restriction enzymes and incredibly sensitive and can give false positives
what is used to verify restriction enzymes + PCR when testing for methylation?
quantitative PCR → can detect if it is occurring once in a while or if its actually methylated
while the restriction enzyme technique solves the issue of undigested DNA, what is it still limited by?
the number of restriction enzyme available → we need to look for a technique that permits to detect CG dinucleotide if it’s methylated or not
what is the idea of the bisulfite treatment?
a technique that cytosine by cytosine will tell if a cytosine is methylated or not and how often it occurs the methylation of that site
what does sodium bisulfite work on?
only on single stranded DNA → we have to make sure that the DNA is completely single strand because if we have repetitive DNA rich in CG it will be tough to denature or it might re-nature easily and bisulfite will not work and the DNA will appear methylated, but actually it’s not
what are the steps of bisulfite treatment?
reduce it in small pieces (sonication, fragmentation) → denaturation via heating to 95 C (single strand DNA) → treatment with sodium bisulfide at very low pH. If you are interested in knowing if a promoter is methylated
or not → amplification by PCR the sequence of interest (after the bisulfite treatment) and that we can perform a Sanger sequencing in automatic version
how does bisulfite cause deamination?
Instead of a natural deamination it’s a sodium bisulfide-induced deamination of cytosines. All the cytosines are converted in uracil apart those
that are methylated → if they are methylated they are protected
what are some caveats to bisulfite treatment?
denaturing is very strong bisulfite can’t distinguish between methyl cytosines and hydroxymethyl cytosines
if you want to understand that a specific sequence is methylated, what is the best thing to do?
do not sequence generally, but clone the fragment → we obtain an indication of the frequency of the methylation cytosines by cytosines of the region in which you’re interested
what is COBRA?
combined bisulfate restriction analysis
how does COBRA work?
get the DNA → denature → bisulfide treatment → PCR amplification → digestion the product of amplification → run it in a gel and you can also hybridize it to a probe in order to quantify the ratio between different fragments
describe the process of bisulfite treatment followed by methylation sensitive PCR:
We design two sets of primers
for the PCR: in the first tube you put your couple of primers in which one is common and amplifies unmethylated DNA while in the other one will amplify the DNA only if it’s methylated (the C is maintained)
what is the best way to obtain a global approach?
if you want to understand how many genes can be prognostic in advanced cancers we have to do an unbiased approach, so an omics approach
what type of disease is cancer?
combination of a genetic disorder and an epigenetic disorder
why did we start looking and global approaches?
we wanted tis know which are the sequences that get methylated and wanted to know if they provide functions as biomarkers for the disorder
why is methylation observed in cancer?
in some cases it is easier to do a diagnosis and to understand the state of a tumor looking at methylation
although it is not perfect, what is considered the gold standard?
bisulfite
what three things are omic techniques based on?
- the base conversion (bisulfide or a modification of it if we have to distinguish between hydroxyl or methyl cytosine)
- endonuclease digestion (restriction enzymes)
- affinity enrichment.
what is the idea of MRE-Seq?
we are going to select restriction enzyme that are methylation sensitive
how does MRE-Seq work?
you digest DNA with restriction enzymes and that we expect is that methylated fragments aren’t cut and so longer whereas fragments cut are short. Then we select the size of the fragment that we want to sequence
when generating the library for MRE-Seq, which part is read?
When we generate the libraries, we make them consider the extremities and they are not methylated. By reading inside, we expect that there are restriction sites that haven’t been digested. This give you an indication of the methylation
what does affinity chromatography use?
can either use antibodies or substrates that are recognized by specific enzymes → we are looking for strong interactions because the surface is complimentary
how does MeDIP seq work?
We fragment the DNA, then we have a sort of chip without a cross link (because we aren’t looking for protein-DNA interaction, but something that is covalently bind to the DNA.) We
then sonicate the DNA, to reduce in tiny fragments and denature. Finally, we treat them with an antibody against the methylated cytosine
what is the major concern of MeDIP Seq?
if in fragment that we are sequencing there is more than one cytosine, we can’t know where the methylated cytosine is
what is the idea of MBD (methyl binding domain) Seq?
they look at the methyl-DNA binding domain of the reader (proteins that can bind to methylated sites) that have a high affinity for high methylated DNA (more than 1 site)
how can we control our results using MBD Seq?
MBD can bind to highly or less methylated DNA → if we treat with low
abundance of any CL, we are going to see that first the fragments with few methyl groups are release, then if we increase CL, we have the release of medium level of methylation, with highest concentration all of them
what is a disadvantage of MBD Seq?
it does not tell you where the methylation occurs
what two techniques can be combined to give a better idea of methylation?
MeDIP and MRE-Seq
why is global methylation not too good when looking at a whole tissue?
whole tissue does not take into account that it is made by several kinds of cells that may have different epigenetic signals
what does looking at single cell level allow for?
doesn’t have the coverage that normal methylation has, but we can sort the cells that we are mores interested in
describe the methylation levels in unicellular eukaryotes:
do not have DNA methylation
describe the methylation levels in C. elegans:
no DNA methylation, no DNA transferase
describe the methylation levels in D. melanogaster:
tiny amount of methylation
describe the methylation levels in invertebrates:
intermediate level of methylation - as we saw with ribosomal DNA but it is not a biomarker
describe the methylation levels in vertebrates:
high level of methylation
describe the methylation levels in plants:
highest level of methylation - more than humans
basal transcription is given by the presence of what?
the presence of the transcriptional apparatus and the regulatory elements (the promotor) - no transcriptional factors involved
describe transcription in prokaryotes:
most of the transcription is basal → there is one RNA polymerase that recognizes a promotor that is common to all promotors
describe the growth of E. coli if there is lactose and glucose present:
they firstly use glucose and have a growth stage (I), after consuming all the glucose, they have a stationary phase
during which they produce the enzymes necessary for the lactose metabolism, and after that a second growth stage (II) in which they use lactose as nutrient
why do humans not have basal transcription?
In our cells, we can’t not afford basal transcription because we have to make sure that in some cells some genes are not expressed therefore, we have to keep everything down
why do our cells use methylation?
it helps us to add layers and to communicate chromatin compaction and helps us in adding differences in working as dimer in transcriptional regulation
what did scientists discover when they focused on the study or regulatory elements?
that, in general, if a gene is a housekeeping gene (=always expressed) it does not have a lot of methylation. On the contrary genes that are diversely expressed, were diversely methylated depending on the condition. There was a very good correlation between DNA methylation → silencing (no transcription) and no methylation → transcription
what three things were discovered when they studied DNA methyltransferase in embryonic stem cells?
- Methyltransferase 1 was an important enzymes for DNA methylation
- Methyltransferase 1 cannot be the only enzyme because if it was the only one enzyme, we would have 100% loss of methylation
- Embryonic stem cells can survive and replicate without DNA methylation, and they found out that the cells that were lacking methylation (and most of times they were going to apoptosis) were not able to differentiate
describe the zygote in relation to methylation:
if we look at the zygotes that is quite methylated - when we start to have fist division, we have a big wave of loss of DNA methylation. It’s not all gone, there is a little methylation,
describe the methylation of the paternal vs maternal genome:
the paternal genomes is highly methylated; the sperm is highly methylated and the oocyte have sort of average level of DNA isolation → the paternal genome is very quickly methylated while the maternal instead is slowly methylate
how will methylation differ as cells differentiate?
the pattern will depend on the destiny of the different cells
During development we have changes in DNA methylation, there is loss of specific region and also gain of methylation during post-natal developments, in late fetal development there is an increase of DNA methylation, while enhancers are losing it. What occurs after birth?
we start to gain methylation in neurons, depending to neuron subtypes → There is wave of wave of demethylation, and wave of methylation, but regulatory sequences are little methylated
describe methylation levels in tumors:
methylation is particularly low
