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Regulation of Gene Expression > Landsberger: Lecture XXII > Flashcards

Landsberger: Lecture XXII Flashcards

DNA Methylation: Detection Strategies and Function

1
Q

What are the 2 roles of DNA methylation?

A

inhibitor of gene transcription

maintenance of genome integrity

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2
Q

When we looked at the phenotype of the DNA methyl transferase de novo mutants, what was found?

A

genomic instability and activation of transposons

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3
Q

What does it mean to maintain genomic integrity?

A

chromosomes do not break and para-CpG sites do not get activated

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4
Q

How was it proven that DNA methylation is important despite drosophila and C. elegans not showing signs of DNA methylation?

A

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

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5
Q

Why were Xenopus oocytes the main models used?

A

Xenopus have some advantage in understanding the role of DNA methylation

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6
Q

What were they looking to be methylated in culture before injecting into Xenopus oocytes?

A

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)

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7
Q

In the experiment, what happens when the promoter is methylated?

A

reportr gene is expressed

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8
Q

What does methylation do?

A

silences gene expression

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9
Q

Why did they use the oocytes of Xenopus?

A

Xenopus is a toad, and the female has many oocytes, which are 1 mm in diameter and can be seen without difficulty

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10
Q

What is the dark part of the Xenopus oocyte called?

A

animal pole

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11
Q

Where is the nucleus located in the Xenopus oocyte?

A

between the animal pole and the vegetal pole

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12
Q

What does the nucleus of the oocyte store?

A

material required for the 1st division after fertilization

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13
Q

What happens after the oocyte gets fertilized?

A

it has 12 divisions that are made by alternating S/G2/M phases without a G1 phase

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14
Q

What can be concluded since the oocytes do not undergo a G1 phase?

A

cells exploit all the material that is present in the vegetal pole in order to obtain the first 12 divisions

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15
Q

What is it called when DNA is transcribed and injected directly into the nucleus?

A

blind method since we cannot see the nucleus, but we know that it is in the white line of the oocyte

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16
Q

What is the only thing that cannot be evaluated using Xenopus oocytes?

A

replication of DNA unless a ss DNA is introduced

the oocyte still has chromatin because it contains a large amount of histone proteins

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17
Q

When are histones synthesized?

A

S phase

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18
Q

Can you study post-translational modification in transfected cells?

A

no

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19
Q

What was found through the analysis of the Xenopus oocyte and the trafected cell lines?

A

methylation of the vector leads to inhibition of transcription from pole 1, pole 2, and pole 3 so it is a general effect

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20
Q

What is the direct model?

A

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

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21
Q

Describe how DNA methylation occurs:

A

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)

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22
Q

How can the direct model be proved?

A

using the electrophoretic mobility shift assay

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23
Q

How does the electrophoretic mobility shift assay work?

A

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

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24
Q

What assay can we use to prove a diseased cell is methylated in vivo?

A

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)

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25
What is a chromatin precipitation assay?
ChIP (antibody-based technology used to selectively enrich specific DNA-binding proteins along with their DNA targets)
26
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
27
What is the indirect model?
if DNA is not methylated, gene is expressed
28
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
29
Which model proposed by Adrian Bird was right?
Indirect model
30
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)
31
Methylation inhibits ___.
transcription through readers of methylation using in part chromatin structure and nucleosome activity
32
How long does it take dsDNA to fully assemble into a detectable nucleosomal structure after injection?
4 hours
33
What happens when GAL4-VP16 combine?
activate transcription
34
What happens when GAL4-VP16 combine with chromatin?
it is more condensed and blocks transcription activation
35
What was the 1st methyl binding protein identified (reader)?
MeCP2
36
What is the methyl binding domain in MeCP2 important for?
region necessary to bind the DNA that has at least 1 symetrically methylated CpG dinucleotide
37
What 2 domains are found in MeCP2?
methyl binding domain transcriptional repressor domain
38
What is the cause of Rett syndrome?
mutation in MeCP2
39
Where is MeCP2 located?
everywhere but it gets localized very heavily on methylated pericentromeric DNA
40
Which assay is used to prove that a protein is interacting with another protein?
Co-immunoprecipitation
41
What can the reader of methylation do?
assemble into methylated DNA through the interaction with histone deacetylases
42
If a piece of DNA is methylated, readers are ___ & lead to a ____.
If a piece of DNA is methylated, readers are bound & can lead to a deacetylated chromatin structure.
43
What can compete with Histone 1 for binding to the DNA?
MeCP2
44
What does Prof. Landsberger believe in terms of MeCP2?
MeCP2 is abundant and probably organizes into compact chromatin MeCP2 is the reader of DNA methylation, it binds everywhere, it generates and represses transcription through the recruitment of histone deacetylase
45
How does MeCP2 work in neurons?
it can organize into special chromatin structures that is not dependent on histone deacetylases, but instead dependent on the ability to substitute histone H1
46
What other readers make-up the family started by MeCP2?
MBD1, MBD2, MBD3, MBD4, MBD5, and MBD6
47
What is the protein that brings DNA methlytransferase 1 onto hemimethylated DNA?
Uhrf1 (also called Np95)
48
How can TF bind to methylated DNA?
they have ZFN
49
Describe MBD1:
contains a binding domain, zinc fingers, a long sequence, and the transcriptional repression domain
50
What is MBD1 able to bind?
methylated DNA, so it is a reader of methylation also non-methylated DNA by using zinc fingers *TRD can repress transcription from both methylated and non-methylated DNA because it depends on where the protein is found
51
What other thing is MBD1 capable of?
repressing transcription by recruiting histone methyl transferase
52
What are MBD1 mutations associated with?
autism
53
What is MBD2?
protein that belongs to the chromatin remodelling complex and contains histone deacetylases *bind to methylated DNA, they recruit or they are a part of a family of proteins that modify the chromatin structure
54
What is NuRD?
nucleosome Remodeling Deacetylase
55
What exclusively binds to the NuRD complex mutually?
MBD2 and MBD3
56
What are mutations in MBD1, MBD2, and MBD3 always involved in?
cancer
57
What is different about MBD4?
it contains a MBD but no TRD, but instead a glycosylase domain (important to repair DNA)
58
What is MBD4 important for?
protein that protects our genome from the damage that occurs during the deamination of methylated DNA using the glycosylase activity to remove the base so the DNA can be fixed
59
What would occur if a null animal for MBD4 is made?
tumor progression because the direct mutations would not be fixed
60
MBD1, MBD2, & MBD3 bind to methylated DNA →
condensed chromatin structure
61
How can the binding of MBD1, MBD2, & MBD3 lead to condensed chromatin structure?
recruiting histone deacetylases, histone methyl transferase, ATP-dependent remodeling complexes
62
w does DNA methylation inhibit gene expression>?
recruits methyl binding protein and the methyl binding protein recruits proteins that condense the chromatin structure
63
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
64
mutation in MBD1 leds to...
neurological alteration or problem for cancer
65
mutation in MBD2 leads to...
cancer or a problem with hemtopoeisis
66
mutation in MBD3 leads to...
neurological issues and cancer
67
Methylation is regulating gene expression and if it regulates gene expression, ___
there are disorders that are very much affected by the gene expression: cancer and neurons disorders as neurons require perfect regulation of gene expression
68
If DNA is more compact, ____.
it will recombine less, it will be less accessible to nuclease, it doesn't permit the activation of transposons, proviruses, etc. *DNA methylation is involved in protecting our genome stability
69
What does a gene body mean?
RNA polymerase transcribes in exons
70
What was discovered about DNA methylation in gene bodies?
DNA methylation is more present in gene bodies when the genes are active
71
What happens if we do not have proper DNA methylation?
lots of spurious transcription takes place from the bodies of the genes
72
What happens if we have defects in DNA methylation?
we have defects in spliceosome because everything is affected: transcription, condensation, chromatin structure (which in turn affects methylation)
73
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*
74
Why is transcription slow?
due to methylation in the body of the gene (by MBP) will bind and compact chromatin structure leads to the slowing of transcription
75
Long story short, what does methylation and the alteration of methylation affect?
initial transcription and splicing

Regulation of Gene Expression (28 decks)

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