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Regulation Of Gene Expression > Lesson 21 > Flashcards

Lesson 21 Flashcards

1
Q

in the zygote, the DNA is replicated through the first division - what type of DNA is obtained?

A

hemimethylated → one strand is methylated the other is not, methylation occurs after DNA replication

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2
Q

describe the methylation pattern DNA follows as the zygote goes from 2 molecules to 16 (4 divisions)

A
  • After the 1st division ⇒ 2 molecules of DNA (both hemimethylated).
  • After the 2nd division ⇒ 4 molecules of DNA (2 hemimethylated and 2 not methylated).
  • After the 3rd division ⇒ 8 molecules of DNA (2 hemimethylated and 6 not methylated).
  • After the 4th division ⇒ 16 Molecules of DNA (2 hemimethylated and 14 not methylated), and so on.
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3
Q

is demethylase activity passive or active during development?

A

both → since we have a rapid division of cells I those moments we have a dilution, but an active demethylation activity is also happening to improve the loss of methylation

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4
Q

which genome is missing methylation in one of its cycles, paternal or maternal?

A

paternal

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5
Q

why can we assume we need to look for demethylases?

A

we can anticipate that a loss of DNA methylation in our cells often occurs without replication, so there is an active mechanism of demethylation

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6
Q

DNA methylate has which types of activity?

A

both maintenance and de novo enzyme

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7
Q

on average, what percentage of methylated cysteins is different between cell divisions?

A

on average 5%

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8
Q

what do we need to methylate DNA?

A

need methyl groups that come from S-Adenosyl methionine (SAM)

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9
Q

what is an easy way to know if the DNA is methylated or not?

A

we can label the donor methylation groups with radioactive molecules → now we use fluorescence to accomplish the same goal

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10
Q

how can we distinguish between a de novo and maintenance activity?

A
  • de novo: we have to provide non methyaotied DNA (if we find methylated DNA that means there the enzyme was able to perform methylation)
  • maintenance: we have to provide hemimethylated DNA to see if it becomes fully methylate
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11
Q

what is the issue with testing methylation activity in vitro?

A

there are no competitors so it does not replicate the body well

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12
Q

what are two ways to put our cells in an environment where they have to choose in order to adequately analyze methylation?

A
  • put both unmethylated and hemimethylated DNA in our tube and see which one is methylated first
  • put them in singles tubes and them measure how much radioactivity was incorporated - tells us which enzyme is more or less efficient giving the mechanism
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13
Q

describe the two parts of DNA methyltransferase 1:

A
  • a long N-terminal region that does not contain any catalytic activity
  • a C terminal region containing catalytic activity
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14
Q

what is the function of the N terminal of DNA mehtyltrasnferase?

A

regulate the activity of the enzyme

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15
Q

what is proliferating cell nuclear antigen (PCNA)?

A

also known as replication fork targeting sequence → also called side clamping, sort of a ring molecule that goes around DNA to stabilize the polymerase during replication

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16
Q

what type of activity does DNA methyltransferase 1 have?

A

maintenance → enzymes prefer to methylate hemimethylated DNA

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17
Q

although DNA methyltransferase 1 is expressed in all cells, where is it most common?

A

more expressed in ones that are dividing

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18
Q

describe the two types of domains that DNA methyltransferase presents in:

A
  1. interacts with transcriptional factors or chromatin remodeling activity
  2. bind transcription factors that do not interact directly with DNA, therefore brining the enzyme to the DNA
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19
Q

describe the activity of DNA methyltransferase 1:

A

incredible processive in its activity: it can methylate long stretches of hememethylated dna to make sure that we maintain the state of methylation

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20
Q

what is DNA methyltransferase 1 unable to do?

A

unable to methylate C followed by any nucleotide different than G (it would not be a palindromic sequence)

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21
Q

what occurs if there is a full knockout of DNA methyltransferase 1 in mice?

A

they do not survive, proving that the maintenance of DNA methylation is essential for development

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22
Q

in the full DNA methyltransferase 1 KO mice, what did they find, indicating that there was some other methylation enzyme present?

A
  • there was still 5% methylation
  • if proviruses were introduced, there still was methylation (cells do it to protect themselves from the expression of parasitic sequences)
23
Q

what was found if they removed DNA methyltransferase 1 selectively in the CNS?

A

evidence of defects in neuronal survival and maturation

24
Q

disease in humans most often have what types of DNA methyltransferase 1 allele knockout?

A

either hylomorphic or hypermorphic - either leads to a less function or more functional protein that is detrimental to the cell

25
Q

what are mutations of DNA methyltransferase 1 related to in physiology?

A

ataxia, dementia, and hearing loss - required for proper brain function

26
Q

when the EST sequence of DNA methyltransferase 1 was searched, what was discovered?

A

DNA methyl 2, DNA methyl 3a, and DNA methyl 3b

27
Q

what are ESTs?

A

expressed sequence tags - sequences that, before the whole genome sequence, came from sequencing the RNAs

28
Q

describe DNA methyltransferase 2:

A

very short enzyme (~450 aa) with only the catalytic domain

29
Q

when studying DNA methyltransferase 2, what function did they discover?

A

it is highly conserved but has no de novo or maintenance activity → it is a tRNA methyltransferase: devoted to tRNA methylation and important for protein synthesis

30
Q

describe DNA methyltransferase 3 a and b:

A

they are not two splicing isoforms but come from two different genes → have a catalytic domain in the C terminal, and the N terminal is devoted to regulating the activity of these enzymes and establishing where they should go

31
Q

what do DNA methyltransferase 3 a and b methylate?

A

CH

32
Q

what occurs when there is a KO of DNA methyltransferase 3a in mice?

A

most of the methylation pattern is preserved but we see neuron developmental issues → it is essential for setting relevant marks for the mouse germline

33
Q

what clinical issue are associated with DNA methyltransferase 3a mutations?

A

acute myeloid leukemia - drive the carcinogenic process

34
Q

what occurs when there is a KO for DNA methyltransferase 3b in mice?

A

it is embryonic lethal → most important enzyme in establishing our pattern of methylation sooner after the blastocyst

35
Q

what do we see with a lack global DNA methylation?

A

a lot of chromosomal instability and aberrant proliferation

36
Q

what is DNA methyltransferase 3b recruited by to centromeric and pericentromeric satellite repeats for methylation?

A

CENP-C

37
Q

what is CENP-C?

A

a histone variant (epigenetic signal) with a specific long terminal domain on H3 tag can recognize only the pericentromeric DNA, so it is important to tag the centromere on the chromosome

38
Q

what is centromeric DNA made of?

A

repetitive DNA (same for the pericentromeric region)

39
Q

when there was a completely KO of DNA methyltransferase 3, what occurred?

A

phenotype is more severe and they die earlier → suggests that there is not a full redundancy of 3a and therefore the 2 enzymes have their specialized function o methylating de novo specific sequences

40
Q

what do rodents have that humans don’t?

A

DNA methyltransferase 3c

41
Q

what is DNA methyltransferase 3L?

A

no catalytic activity, but is still important for our pattern of DNA methylation → interacts with unmethylated H3K4 directing DNA methylation to chromatin regions with this epigenetic mark (interacts with other enzymes - DNA methyltransferase 3a and 3b)

42
Q

what is the function of Uhrf1 (aka NP95)?

A

can recognize the DNA when it is hemi-methylated - binds there and can recruit DNA methyltransferase 1 to maintain the pattern

43
Q

where are CpH methylations most commonly found?

A

we find abundant CpH methylations (enrichment) in embryonic stem cells, neurons (more abundant in adults than fetal neurons), and in immune system cells

44
Q

what is CpG methylation associated with?

A

repression in gene expression and even transposons

45
Q

what happen if methylated bases are deaminated?

A

they can be fixed by repair mechanisms - NOT IN ANIMALS

46
Q

what are the functions of the three TET enzymes?

A

TET1 oxidizes the methylated C leading to hydroxy-methyl-cytosine, which can be further oxidized by wither TET2 or TET3

47
Q

what is the most important TET enzyme?

A

TET1 → important for starting the demethylation process

48
Q

what happens if there is a mutant devoid of TET1?

A

increase in methylation levels of cytosine in our DNA → equilibrium between methylated and demethylated (equilibrium between TET and DNMTs)

49
Q

what does TET3 KO lead to?

A

post-natal lethality

50
Q

what does a single mutation of either TET1 or TET2 lead to?

A

viable or with minor phenotype

51
Q

what does a dysregulation of TET enzymes lead to?

A

cancer, learning, and memory

52
Q

can methylation and hydroxymethyaltion occur in the same region?

A

yes - hydroxymethylation occurs before methylation

53
Q

how does the rate of methylation differ?

A

is different depending on the genomic region in which it is occurring - more stable in some regions such as in promotors

Regulation Of Gene Expression (28 decks)

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