Lesson 18

Question 1

why are both HpaII and MspI methylation sensitive?

Answer 1

both of them recognize CCGG

Question 2

how does MspI relate to HpaII?

Answer 2

MspI functions to control the HpaII digestion

Question 3

what was used in the 90s to determine if a gene or a promotor of a gene was methylated?

Answer 3

radioactive probes

Question 4

what technique permits to understand if a specific fragment of DNA has a different molecular weight after
hybridization?

Answer 4

southern blot

Question 5

what does it mean if digestions is identical?

Answer 5

there is no difference in methylation between the two samples

Question 6

describe the difference in ribosomal DNA methylation between vertebrates and invertebrates:

Answer 6

vertebrates have more methylation in ribosomal DNA then invertebrates

Question 7

what are the four main issues with the radioactive probes?

Answer 7

1. depend on restriction enzymes
2. most of the restriction enzymes don’t have the control
3. we have problems looking at all the GC in which were interested because they might not be part of the palindrome sequence
4. it is time consuming - radioactivity is never ideal

Question 8

what must we do in order to study DNA methylation?

Answer 8

we first have to treat the samples and then there is the amplification

Question 9

what happens if the DNA is unmethylated and we try to perform PCR?

Answer 9

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

Question 10

what are the two limitations of combined restriction enzymes with PCR?

Answer 10

limited by restriction enzymes and incredibly sensitive and can give false positives

Question 11

what is used to verify restriction enzymes + PCR when testing for methylation?

Answer 11

quantitative PCR → can detect if it is occurring once in a while or if its actually methylated

Question 12

while the restriction enzyme technique solves the issue of undigested DNA, what is it still limited by?

Answer 12

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

Question 13

what is the idea of the bisulfite treatment?

Answer 13

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

Question 14

what does sodium bisulfite work on?

Answer 14

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

Question 15

what are the steps of bisulfite treatment?

Answer 15

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

Question 16

how does bisulfite cause deamination?

Answer 16

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

Question 17

what are some caveats to bisulfite treatment?

Answer 17

denaturing is very strong bisulfite can’t distinguish between methyl cytosines and hydroxymethyl cytosines

Question 18

if you want to understand that a specific sequence is methylated, what is the best thing to do?

Answer 18

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

Question 19

what is COBRA?

Answer 19

combined bisulfate restriction analysis

Question 20

how does COBRA work?

Answer 20

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

Question 21

describe the process of bisulfite treatment followed by methylation sensitive PCR:

Answer 21

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)

Question 22

what is the best way to obtain a global approach?

Answer 22

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

Question 23

what type of disease is cancer?

Answer 23

combination of a genetic disorder and an epigenetic disorder

Question 24

why did we start looking and global approaches?

Answer 24

we wanted tis know which are the sequences that get methylated and wanted to know if they provide functions as biomarkers for the disorder

Question 25

why is methylation observed in cancer?

Answer 25

in some cases it is easier to do a diagnosis and to understand the state of a tumor looking at methylation

Question 26

although it is not perfect, what is considered the gold standard?

Answer 26

bisulfite

Question 27

what three things are omic techniques based on?

Answer 27

• 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.

Question 28

what is the idea of MRE-Seq?

Answer 28

we are going to select restriction enzyme that are methylation sensitive

Question 29

how does MRE-Seq work?

Answer 29

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

Question 30

when generating the library for MRE-Seq, which part is read?

Answer 30

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

Question 31

what does affinity chromatography use?

Answer 31

can either use antibodies or substrates that are recognized by specific enzymes → we are looking for strong interactions because the surface is complimentary

Question 32

how does MeDIP seq work?

Answer 32

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

Question 33

what is the major concern of MeDIP Seq?

Answer 33

if in fragment that we are sequencing there is more than one cytosine, we can’t know where the methylated cytosine is

Question 34

what is the idea of MBD (methyl binding domain) Seq?

Answer 34

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)

Question 35

how can we control our results using MBD Seq?

Answer 35

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

Question 36

what is a disadvantage of MBD Seq?

Answer 36

it does not tell you where the methylation occurs

Question 37

what two techniques can be combined to give a better idea of methylation?

Answer 37

MeDIP and MRE-Seq

Question 38

why is global methylation not too good when looking at a whole tissue?

Answer 38

whole tissue does not take into account that it is made by several kinds of cells that may have different epigenetic signals

Question 39

what does looking at single cell level allow for?

Answer 39

doesn’t have the coverage that normal methylation has, but we can sort the cells that we are mores interested in

Question 40

describe the methylation levels in unicellular eukaryotes:

Answer 40

do not have DNA methylation

Question 41

describe the methylation levels in C. elegans:

Answer 41

no DNA methylation, no DNA transferase

Question 42

describe the methylation levels in D. melanogaster:

Answer 42

tiny amount of methylation

Question 43

describe the methylation levels in invertebrates:

Answer 43

intermediate level of methylation - as we saw with ribosomal DNA but it is not a biomarker

Question 44

describe the methylation levels in vertebrates:

Answer 44

high level of methylation

Question 45

describe the methylation levels in plants:

Answer 45

highest level of methylation - more than humans

Question 46

basal transcription is given by the presence of what?

Answer 46

the presence of the transcriptional apparatus and the regulatory elements (the promotor) - no transcriptional factors involved

Question 47

describe transcription in prokaryotes:

Answer 47

most of the transcription is basal → there is one RNA polymerase that recognizes a promotor that is common to all promotors

Question 48

describe the growth of E. coli if there is lactose and glucose present:

Answer 48

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

Question 49

why do humans not have basal transcription?

Answer 49

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

Question 50

why do our cells use methylation?

Answer 50

it helps us to add layers and to communicate chromatin compaction and helps us in adding differences in working as dimer in transcriptional regulation

Question 51

what did scientists discover when they focused on the study or regulatory elements?

Answer 51

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

Question 52

what three things were discovered when they studied DNA methyltransferase in embryonic stem cells?

Answer 52

1. Methyltransferase 1 was an important enzymes for DNA methylation
2. Methyltransferase 1 cannot be the only enzyme because if it was the only one enzyme, we would have 100% loss of methylation
3. 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

Question 53

describe the zygote in relation to methylation:

Answer 53

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,

Question 54

describe the methylation of the paternal vs maternal genome:

Answer 54

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

Question 55

how will methylation differ as cells differentiate?

Answer 55

the pattern will depend on the destiny of the different cells

Question 56

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?

Answer 56

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

Question 57

describe methylation levels in tumors:

Answer 57

methylation is particularly low