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Regulation of Gene Expression > Landsberger: Lecture XVI > Flashcards

Landsberger: Lecture XVI Flashcards

Chromatin Affects Gene Expression - DNA Methylation

1
Q

Epigenetics is a nonstable code: it needs __, __, & __.

A

writers, readers and modifiers

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2
Q

Every epigenetic mechanism has to be _____.

A

transmitted and read by specific proteins

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3
Q

What is the PHO5 promoter?

A

it is a promoter that has nucleosomes that are positioned precisely so that the TATA box is always assembled into nucleosomes so that TBP (TATA binding protein) cannot bind → promoter is off

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4
Q

PHO5 promoter is generally ___ in promoters.

A

blocked

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5
Q

Since nucleosomes are repressive, they need to be removed prior to transcription. What does this establish?

A

chromatin remodeling is a prerequisite and not a consequence of gene transcription

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6
Q

Why is chromatin important?

A

keeps genes repressed so that they are activated only when it is necessary

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7
Q

Can chromatin participate as an epigenetic signal?

A

no

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8
Q

Why are nucleosomes important?

A

they can condense DNA and participate in gene expression

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9
Q

Can chromatin induce gene expression?

A

yes

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10
Q

What are the 2 reasons Grunstein and Winston decided to use yeast for their experiments?

A

simple model

only 2 copies of histones (humans have many)

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11
Q

Grunstein and Winston created a yeast strain completely devoid of H4 but carried the gene on a plasmid. What regulated this gene’s expression?

A

an inducible promoter

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12
Q

What did the yeast strain’s expression rely on?

A

galactose: activated the gene

glucose: deactivated the gene

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13
Q

H4 expression is shut down →

A

H4 histone level in strain is low → nucleosome density is reduced → less dense chromatin → linker DNA size increased

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14
Q

Summarize the experiment by Grunstein and Winston:

A

yeast was modified to have deleted both H4 alleles and supplied by stable extrachromosomal plasmid where the H4 gene was under the control of inducible GAL1 promoter

growth in presence of galactose → H4 is expressed

growth in presence of glucose → H4 not expressed

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15
Q

How do you check after 3 generations what happens to gene expression?

!!

A

If I don’t have a canidate gene, I have to use a global approach

If I have a canidate gene, I have to use a single approach

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16
Q

What is the best current global approach?

A

analyzing gene expression using RNA-seq

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17
Q

What could be concluded from the experiments of Grunstein and Winston?

A

some gene’s nucleosomes are an integral part of regulatory mechanisms

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18
Q

closed chromatin →

A

gene is not transcribed

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19
Q

open chromatin →

A

gene is transcribed

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20
Q

How can cells modify the chromatin structure on a gene?

A

through ATP dependent chromatin remodeling complexes or by epigenetic mechanisms

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21
Q

ATP dependent chromatin remodeling complex

A

make DNA more accessible

can alter the position or structure of nucleosomes through ATP hydrolysis

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22
Q

Post-translational modifications (PTMs) of histone’s tails (epigenetics)

A

modifications are induced to recruit proteins that open or closed chromatin

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23
Q

Introduction of histone variants (epigenetics)

A

resemble canonical histones, with a few different aa and they are characterized by specific structural and functional properties

the tags tell the cycles that a specific region of the chromatin must be highly transcribed or must be repressed

readers read the specific histone variants

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24
Q

What can ATP dependent chromatin remodeling complexes induce?

A

nucleosome sliding

nucleosome ejection

H2A-H2B dimer ejection

H2A-H2B dimer replacement

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25
Q

Nucleosome Sliding

A

region which is hidden by a nucleosome becomes accessible to TF

ex: nucleosome can go into linker DNA

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26
Q

Nucleosome Ejection

A

all nucleosomes are removed and so the DNA becomes accessible

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27
Q

H2A-H2B dimer ejection

A

nucleosomes are made by DNA wrapped around the octamer

the tetramer made with H3 and H4 is sufficient to have DNA wrapping around but without H2A and H2B, DNA has less contacts so TFs have more access

H2A and H2B removal make the chromatin more transparent for the binding of TFs

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28
Q

H2A-H2B dimer replacement

A

after the removal of the dimer, it is possible to insert histone variants

29
Q

What can ATP remodeling complexes do?

A

work in both directions (open and close chromatin)

30
Q

What can ATP remodeling complexes be involved in?

A

every DNA metabolic process and neurological disorder and cancers

31
Q

What are SWI-SNF (BAF) complexes?

A

typical ATP dependent chromatin remodelling complexes involved in human tumors

32
Q

What is a rhabdoid tumor?

A

a very aggressive pediatric tumor that affects the brain, kidneys and other soft tissues

33
Q

A deletion in one of the subunits needed for ATP dependent chromatin remodeling complex assembly results in what?

A

100% of rhabdoid tumors

34
Q

100% of rhabdoid tumors have a deletion in what?

A

SMARC5B (SNF2) gene: the lack of protein causes the disassembly of all the related complexes and at least 1 ATP dependent chromatin remodeling complex is always involved

35
Q

How can ATP dependent chromatin remodeling complexes reach target DNA?

A
  1. be recruited by TF, which have access to chromatin thanks to PTMs
  2. randomly: they slide nucleosomes and if a TF is there, it will have the chance to bind
  3. readers of PTMs
36
Q

How can we test which genes are affected by a specific ATP dependent chromatin remodeling complex?

A

ChIP-Seq can be used to see which complexes are bound, but this will not allows us to known if expression is affected

37
Q

How can we see if gene expression is affected after doing an analysis with ChIP-Seq?

A

the best approach is using a knock-down using siRNA or shRNA (short-hairpin RNA)

cells are transfected or infected with siRNA or shRNA expressing vector and we check if the gene is a knock down via RT-PCR or WB (or both) and then RNA-SEq is performed unless we have a candidate gene we want to check

38
Q

What are the most common modifications on histone tails?

A

phosphorylation → on Ser, Thr, and Tyr

acetylation → on Lys

methylation → on Lys and Arg

ubiquitination → on Lys

39
Q

What was the first PTM studied on a histone?

A

acetylation, which is an epigenetic signal

40
Q

Histone acetylation correlate with _____.

A

transcriptional activation

41
Q

How does acetylation exert its effect on transcription?

A

acetylation neutralizes the + charge of histones so the tails become less positively charged and the contacts between the nucleosomes are reduced

42
Q

Are all positive charges neutralized in Lys residues in the presence of acetylation?

A

no, only 1 or 2 so it cannot be the main model (this artificial effect only occurs in vitro)

43
Q

What does acetylation do?

A

creates sites for the recruitment of other proteins

44
Q

What are some examples of different readers of different PTMs?

A

bromodomain: reads acetylated Lys (not only in histones…the human proteome contains more than 40 proteins with bromodomain)

chromodomain: reads methylation

45
Q

What is the histone code hypothesis?

A

all PTMs on a specific nucleosome must be considered

different combinations of “functional groups” added to the histone tails form specific signals to attract or repel protein complexes directly involved in transcriptional regulation

46
Q

Why is the combination of PTMs on a specific nucleosome important?

A

the same modification can mean either condensing or opening…it depends on what is close by

47
Q

Do prokaryotic cells use DNA methylation?

A

yes

48
Q

What are the 2 bases that get modified in prokaryotic cells?

A

cytosine and adenine

49
Q

When does DNA-methylation occur?

A

post-replication

50
Q

What is the enzyme that methylates DNA called?

A

DNA methyl transferase

51
Q

How does DNA methyl transferase work?

A

uses a donor of a methyl group called SAM (S-adenosyl methionine)

cytosine can be methylated on the 5’ carbon and the adenine gets methylated on the 6’ carbon

52
Q

What is the role of DNA methylation in prokaryotes?

A

protect the stability of the genome

53
Q

How is DNA methylation by prokaryotes used to protect the genome?

A

avoids the cut from restriction enzymes

distinguish between replicated vs non-replicated DNA (defense mechanism) → cell cycle control to separate the 2 chromosomes into 2 daughter cells

discriminate DNA strands for mismatch repair (defense mechanism) to protect the genome

54
Q

Is DNA methylation involved in gene expression?

A

sometimes

55
Q

How can DNA methylation affect replication?

A

when replication starts, the origin is hemimethylated → SeqA binds to the origin and can interact with a protein on the membrane → membrane grows → invagination occurs ensuring the 2 genomic DNA will go to 2 different cells

56
Q

How is DNA methylation involved in DNA repair?

A

when DNA needs to be repaired, it knows which strand to correct using DNA methylation

57
Q

Where are DAM methylation sites located?

A

along the chromosome

58
Q

What are the nt of DNA methylation sites?

A

GATC

59
Q

Describe the figure:

A

there is a missense mutation and the geometry of the double helix is altered

the mutation is recognized by MutS and it recruits MutL and MutH on the mutated DNA

this complex with endonuclease activity interupts phosphodiesterase bonds in order to degrade DNA

the system moves and loops to the closest parental strand and hydrolyzes it and generates the nick on the opposite strand

the hemimethylated state of DNA allows the repair machinery to recognize which strand carries the mutated nucleotide

*mechanism controls gene integrity

60
Q

Where is the genome methylated in mammalian cells?

!!

A

cysteine on the 5’ carbon on the CG dinucleotide

it can also occur on a C followed by another nucleotide ( C A or T, generally A)

CG methylation (most abundant) or CH methylation (C followed by another nt that is not G)

61
Q

What are the techniques available to detect DNA methylation?

A

thin layer chromatography (TLC): used to understand if the genome contained methylated nucleotides

high performance liquid chromatography (HPLC): used to understand if adenine is sensitive

restriction enzymes (HpaII and MspI): can recognize palindromic sequences that contain sites for methylation

62
Q

What is a problem with the TLC technique?

A

not very sensitive: since it is impossible to lead large amounts of nt on a thin layer, if the methylation is not high in abundance, it won’t be detected

does not give sequence information since the DNA was 1st hydrolysed to single nucleotide level

63
Q

How can we distinguish if a region is methylated or not and between different types of methylations?

A
64
Q

What are the enzymes involved in DNA methylation (writers, readers, and erasers)?

A
65
Q

What are the consequences of DNA methylation?

A
66
Q

What is the role of DNA methylation in cells?

A
67
Q

What happens if DNA methylation does not proceed properly?

A
68
Q

What are some disorders associated to DNA methylation?

A
69
Q

What do HpaII and MspI recognize?

A

CCGG but they have different sensitivity for methylation

HpaII is CG methylation sensitive and it cuts CG if it is NOT methylated

MspI is CH methylation sensitive

Regulation of Gene Expression (28 decks)

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