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MBG 4110 Test 1 > Lecture Panel 1 > Flashcards

Lecture Panel 1 Flashcards

1
Q

What is facultative heterochromatin?

A

Regions that exhibit heterochromatic packaging in a subset of cells
Can be converted to euchromatin
Example: sub-telomeric genes

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2
Q

What is constitutive heterochromatin?

A

Regions that are in a heterochromatic state in all cells, all the time
No gene expression, never becomes euchromatin, not accessible, silenced all the time
Example: Mating type loci in S. cerevsiae

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3
Q

What is a nucleosome?

A

There are repeating units consisting of 146 bp of DNA
Two of each of the four core histones: H2A, H2B, H3, H4
Core histones form an octamer
Core histones form the core of nucleosome

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4
Q

What does chromatin consist of?

A

Nucleosomes forming a jointed chain (beads on a string) and the string is DNA
The beads can be very compact (heterochromatin) or more loosely spaced and relaxed (euchromatin)
Several direct methods (MN sensitivity assay; EM microscopy) can distinguish the euchromatic or heterochromatic structure of chromatin in the fibre

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5
Q

Why are epigenetic marks important?

A

They are used to determine state of chromatin

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6
Q

How does a MN sensitivity assay work?

A

1) Isolate chromatin
2) Digest with Micrococcal Nuclease (endonuclease, which makes cuts in DNA that is not protected by proteins) for various lengths of time (time course with nuclease)
3) Separate the digested DNA on agarose gels

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7
Q

How do we get the results from an MN (Micrococcal nuclease) sensititvity assay work?

A

We get DNA bands of about 150, 300, 450, and in increments of 150 nucleotides
Certain regions of DNA are more sensitive to digestion with a Micrococcal nuclease
What is not exposed are the parts of DNA that were covered by nucleosomes

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8
Q

What does the results from an MN sensitivity assay indicate?

A

Chromatin has simple, repetitive structure with spacing of sites accessible to nuclease digestion
Compaction of chromatin varies at different loci

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9
Q

What is a southern blot?

A

Separate DNA by size and a probe is added to tag specific DNA

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10
Q

What are the technical aspects behind an MN assay

A

Micrococcal nuclease is an ends-nuclease
Binding of proteins to DNA protect it from the endo-nuclease
DNA that is tightly packaged in proteins is less sensitive to MN as compared to open DNA
–> This characteristic is the foundation of the so called “nuclease sensitivity assay”
The more exposed DNA, the more sensitive to MN it is, and vice-versa
Heterochromatin is less sensitive to MN than euchromatin
Bands in a mutant appear earlier in mutant, because chromatin is less compact in the mutant, which makes sense

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11
Q

What are histones? What do histones have? What are the characteristics of histone?

A

Histones are small proteins (10-12 kDa)
Rich in lysine (K) and arginine (R)
Histones are positively charged and interact with negative DNA
Extensively modified by various post translational modifications (PTMs)
These PTMs carry epigenetic information

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12
Q

What is the histone made up of?

A

H1: Linker Histone (bends DNA between nucleosomes)
H2A, H2B, H3, H4: Core Histones (they build the nucleosome)

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13
Q

What is the DNA methylation epigenetic mark?

A

5meCpG is an epigenetic mark associated with heterochromatin

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14
Q

What is the importance of DNA methylation?

A

Methylation is critical in regulating gene expression

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15
Q

What are DMTs? What do they do? What is their importance?

A

DNA Methyl Transferases and they catalyze the methylation of cytosine
Extensive methylation of DNA will prime and maintain formation of heterochromatin

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16
Q

Where is DNA methylation absent?

A

DNA methylation is absent in budding yeasts

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17
Q

How does cytosine methylation affect gene expression?

A

1) Directly: by altering DNA binding protein’s affinity to binding sites –> a protein won’t be able to bind to methylated cytosine
2) Indirectly (and more importantly) through supporting heterochromatin –> usually associated with gene silencing

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18
Q

What are the DNA methylation patterns seen in mammals?

A

1) Erased in germ line cells
2) Re-established in very early development (pre-implantation)
3) Maintained throughout the remainder of development and the lifespan of the organism
4) Transmission of 5mCpG marks is coupled to DNA replication
5) DNA methylation is tightly coordinated with the Histone PTMs

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19
Q

Where and how are 5meCpG islands found? What do these form?

A

5mCpG islands are found in clusters of multiple repeats of short symmetrical sites
5mCpG islands form blocks of heterochromatin, which are found around promoters and their function is to turn off promoters

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20
Q

What are some important facts about non methylated CpG sites?

A

Not all CpG sites are methylated
Non methylated CoG islands can serve as weak bi-directional promoters for RNA polymerase II
They produce RNAs that can have various effects

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21
Q

Explain the structure of histone PTMs

A

alpha helices: conserved domains
Variable non-structured N-terminal regions (N-tails): Heavy modifications are exposed to massive PTMs
Core histone fold: Form the core and three alpha helices interlock to form the octamer
Conserved domains found in all histones

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22
Q

What is the function of the core histone?

A

The core histone builds the nucleosome

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23
Q

What is the structure of the core histone fold?

A

Pertruding ends
No structure
There are lysines, that can be modified

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24
Q

Explain the experimental procedure used to determine the function of the N-termini of the histone. What was the outcome and conclusion

A

1) Isolate chromatin
2) Remove H1
3) Digest the exposed Histone N-termini with trypsin
Outcome: Loss of histone N-temrini does not disrupt nucleosomes, the structure is lost, but nucleosomes are still present
The conclusion: The N termini of the histone “core” is not needed for the nucleosome structure and stability

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25
Q

What does H1 contribute to?

A

H1 contributes to fibre stability, not nucleosome stability
Nucleosomes remain intact when H1 is removed

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26
Q

What is important information to remember about histones?

A

Among the most conserved proteins in eukaryotes
They are some of the most variable proteins in terms of post-translational modifications of their N-termini (PTMs)
It is these PTMs that have a functional significance
Some of the most modifications are found in the N-termini of the histones

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27
Q

What are epigenetic marks?

A

Most functional and structural differences between euchromatin and heterochromatin are due to biochemical modifications of histone and DNA
In other words, epigenetic marks are modifications found on histones

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28
Q

Where are methylated CpG islands found?

A

Heterochromatin DNA

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29
Q

What are some Epigenetic marks (PTMS) on the Histone N-termini? What is their purpose?

A

Acetylation of K and R (euchromatin): Allow gene expression
Methylation of K and R (euchromatin and heterochromatin): signals formation of heterochromatin, or for DNA to be transcribed, ti depends on what residues are methylated
Phosphorylation of S and T: damaged DNA
Mono-ubiquitination: damaged DNA
Sumoylation
ADP- ribosylation

30
Q

What is the histone code?

A

A combination of N-terminal histone PTMs can serve as a “barcode” (this is encoded information) that is recognized by other proteins, including histone modifying enzymes
In other words, the histone code is the code or information found on a single histone

31
Q

How much information can be stored in histones?

A

Lots of information

32
Q

What are some messages that will found on histone tails within the nucleosomes?

A

1) My DNA can be transcribed (H4-K16Ac): euchromatin
2) My DNA is transcribed (H3-K4Me3 (trimethylated): euchromatin, and the promoter is ready to be transcribed –> triple methylation: DNA has been transcribed
3) My DNA is damaged (phosphorylation)
4) DNA is ready to be transcribed (H3-K4-Me)
5) DNA must not be transcribed (H4-K16- deacetylated in yeast, but H3-K9-Me in metazoan)
6) DNA is being repaired, do not replicate (phosphorylated)
7) DNA has just been replicated and chromatin is being rebuilt on it (put an epigenetic mark to distinguish)
8) DNA has not been replicated yet
9) I am a nucleosome in a stem cell, do not disturb, decide what to do later

33
Q

What is a reader, writer, eraser, and mover?

A

Reader: a protein that recognizes a specific epigenetic mark
Writer: an enzyme that catalyzes the incorporation of a specific PTM on a specific residue of a histone –> promoters or other sites of chromatin compete for the mover
Eraser: Enzyme that removes specific PTM from a specific residue histone
Movers: Slide nucleosomes along DNA or displace a nucleosome

34
Q

How do writers, erasers, readers and movers interact with epigenetic marks?

A

A single PTM or a combination of PTMs of the N-termini can be specifically recognized by readers
Certain modifications (for example acetylation at a certain position, say H3-K9 may predispose a similar modification of the adjacent nucleosome).
In this situation, the reader can recruit writers or erasers
The writers or erasers can incorporate a PTM that will recruit the same or different readers
Such combinations of epigenetic marks and the interplay with modifiers dictate the state of chromatin, its maintenance, inheritance and perturbations

35
Q

What are histone variants?

A

There are variants to the five histones encoded by different genes
In different organisms, there are different sub-sets of histone variants
These variants contain minor amino-acid alterations, but have a substantially different function
There are rare histone variants that occupy specific positions of the genome in specific situations

36
Q

What are the histone variants and their roles (3)?

A

CENP-A/cse4: H3 variant, which is an epigenetic marker of the centromere
H2A.Z/H2AV: Transcription/double strand break repair
H2A.X: Double strand break repair/meiotic remodelling of sex chromosomes (H2A histone variants are H2s that have been replaced during DNA repair)

37
Q

What is the co-ordination of epigenetic marks?

A

Individual PTMs and the methylation of DNA influence each other and establish a dynamic but stable chromatin structure

38
Q

What is the importance of epigenetic marks being co-ordinated?

A

The co-ordinated PTMs support each other and prevent “erosion” of the state of chromatin
They also facilitate the rebuilding of the same chromatin structure after DNA replication
The dynamic state of chromatin allows for epigenetic change if the balance of writers, erasers, and readers change at the locus

39
Q

What is an example of how epigenetic marks are co-ordinated?

A

H3 is methylated at K9, K14, K27
H3 can be methylated on K4, K9, K14
These same lysines can also be acetylated
When H3-K9 is methylated, H3-K14 is also methylated
When H3-K9 is methylated, H3-K4 is deacetylated
When H3-K9 is unmethylated, H3-K4 is acetylated
These are co-ordinated epigenetic marks which serve to stabilize DNA

40
Q

How are epigenetic marks co-ordinated? Include HP1 in your explanation

A

H3-K9 Methylation is a critical epigenetic mark in heterochromatin in metazoan
H3-K9 co-ordinates the deacetylation of histones and the methylation of DNA through the binding of HP1 (Heterochromatin Protein 1)
In turn HP1 recruits HDAC, HMTs and DMTS to set up and spread heterochromatin at the locus
The methylated DNA recruits readers that recruit writers, including a H3-K9 HMT writer

41
Q

What is HP1? Where is it found? What does it contain? What do the domains do?

A

HP1 is a reader of the H3K9 epigenetic mark
Found exclusively on heterochromatin
Homologues of HP1 are found in most eukaryotes
It contains two distinct domains: Chromo-domain and the shadow domain
Chromo domain: Directly reads the H3-K9-Me epigenetic mark
Chromo Shadow Domain: Recruits other proteins: HDAC, H3MT, and DMT (These proteins cannot act at the same time)
Mutations in HP1 result in the loss of gene silencing (loss of heterochromatin)

42
Q

What is the genetic evidence to suggest the connection between DNA Methylation and H3-K9 Methylation?

A

2 mutants with defective gene silencing:
Both mutants had no 5mCpG methylation on the unsilenced genes (no methylation on active genes)
The identified mutants were dubbed dim2 and dim5
dim2: disrupts DMT gene
dim5: disrupts H3K9-MT gene
Overall, the loss of histone H3 methylation abolishes CpG methylation

43
Q

Explain how The coordination epigenetic marks take place

A

Loss of co-ordination = loss of stability
Image to make sense of

44
Q

How are the molecular mechanisms of coordination performed?

A

1) 5mCpG (methylated DNA) recruits a Methylated DNA binding protein (the readers MeCP or MBP) –> MeCP is a reader which binds to 5mC
2) MeCP recruits HDAC, which is an erasers –> HDAC binds to MeCP
HDAC deacetylates the adjacent histones
Deacetylated histones are then methylated by DMTs
3) H3K9 Me (methylated by DMTs) recruits HP1
4) HP1 recruits another HDAC
5) HDAC deacetylates nearby nucleosomes
–> Deacetylated histones are methylated and the spreading of histone deacetylation/methylation
After this deacetylation, methylation can now occur
The HP1 recruits HDAC and DMTs
6) Deacetylated histones are methylated:
–> H3K9 Me recruits HP1
–> HP1 recruits another DMT
–> DNA is methylated, recruits MeCP and recruits HDAC
This process is heritable, which means the epigenetic modifications are passed onto progeny and all the information will be passed onto another cell
The readers recruits an eraser or writer

45
Q

What do movers do? Where are movers present?

A

Chromatin Remodeling Factors (MOVERS) catalyze ATP dependent nucleosomes sliding along DNA or their disassembly and reassembly
Movers are present in limited amounts in most cells –> they are recruited to chromatin via Histone PTMs that direct them to the locus to be modified

46
Q

What is important to note about nucleosomes?

A

The position of a nucleosome determines the access of a transcription factor DNA
The density of nucleosomes over certain region of DNA regulates heterochromatin formation, we need to move nucleosomes to make them denser and build up heterochromatin
During DNA damage nucleosomes need to be removed from DNA to allow repair factors activity, and then Brough back when the repair is complete

47
Q

How does ATP dependent chromatin remodelling work?

A

Chromatin Remodelling Complex: Mover and needs ATP
Chromatin remodellers rearrange nucleosomes to make room for the binding of a transcription factors or allow the binding of a writer or erasers

48
Q

What can chromatin remodellers do?

A

Chromatin remodellers can loosen the grip of the nucleosome and wrinkle DNA to allow for the binding of a protein to DNA
OR they can
Move the whole nucleosome away to make room for the transcription factors

49
Q

What is the compaction of chromatin and chromatin fibres important for?

A

Well, it is an intermediate to build up heterochromatin
Necessary to regulate gene expression

50
Q

What do chromatin loops do?

A

Build higher order structures and facilitate the assembly of active transcription or replication complexes

51
Q

How are chromatin loops formed?

A

With the participation of CTCF and Cohesion
Cohesion: Holds DNA and keeps chromatin together, the 2 strands of DNA are held together by cohesion.
Cohesion grips DNA and holds it, while CTCF directs cohesion to form the loop seeing during replication, and CTCF has many functions
CTCF: Directs cohesion to hold DNA

52
Q

How is the chromatin structure within the loop controlled?

A

Controlled via other loops
Chromatin boundaries delineate one domain from another and allows for fine regulation of gene expression

53
Q

What are TADs?

A

3D structures formed from multiple replication loops coming together

54
Q

How are chromatin globules formed?

A

Chromatin looping between active genes and regulatory elements and clustering of honest the site of active transcription facilitates the formation of globules

55
Q

How do genes cluster together?

A

Active genes clusters associate with other expressed genes in active neighbourhoods while inactive genes cluster in silent neighbourhoods

56
Q

What can multiple loops clustering establish?

A

Multiple loops can cluster to establish close topological association of distal regions of the same chromosome and maintain euchromatic or heterochromatic nature of chromatin at these domains
DNA replication is not a linear phenomenon

57
Q

How do many loops work together?

A

Many loops work together to bring many active promoters together to create a highly transcribed euchromatic domain in the nucleus

58
Q

What do TAD’s make up?

A

TAD’s make up the epigenetic landscape of the nucleus and different cell types will have different epigenetic landscapes
Weak interactions between domains but together they form a very stable structure

59
Q

How do active and silent neighbourhoods associate?

A

Active and silent neighbourhoods associate in cis and in trans to form larger active and inactive compartments

60
Q

What does nuclear organization show?

A

Nuclear organization reflects clustering of active and inactive loci in distant coparents forming a fractal globule

61
Q

How are chromatin loops formed?

A

1) Loops of chromatin that defines a domain of chromatin with clearly defined boundaries
2) The protein CTCF plays a critical role in the formation of chromatin loops
3) The control of binding and pairing of CTCF proteins determine the high order structure of the chromosomes

62
Q

Explain CTCF in terms of chromatin loops

A

Different CTCF binding sites are methylated in different cell types, causing CTCF to form different loops in different cell types, which contributes to the epigenetic landscape
CTCF is a DNA binding protein, that binds to non methylated DNA and when binding site of CTCF is methylated it cannot bind DNA
CTCF molecules can form dimers, which form DNA loops

63
Q

What can CTCF and Cohesion do in terms of chromatin loops?

A

Distal sites can be brought closer together by the loops formed by CTCF
Cohesion forms the loops, and CTCF can bring distal sites together
Cohesion and CTCF work together to form the loops
Cohesion and CTCF are found in heterochromatin and euchromatin
Found everywhere
TADS are formed and found everywhere

64
Q

What is the process for detecting methylated DNA?

A

1) Sodium Bisulfphite converts unmethylated cytosines to uracils
Methylated cytosines are not converted
2) PCR converts the uracils to thymines (C –>T conversions ) during reaction
3) Parallel sequencing of DNA from treated and untreated samples identifies the position of the unmodified cytosines
–> The unmodified cytosines are the methylated ones
Overall, methylated cytosines remain the same in both samples, but unmethylated cytosines convert to thymine in second sample

65
Q

What is chromatin immunoprecipitation?

A

A technique for the analysis of the binding of proteins to DNA in vivo

66
Q

How does chromatin immunoprecipitation work?

A

1) Cross link protein DNA and proteins with formaldehyde
2) Lyse the cells and shear chromatin into small pieces (breaking DNA into pieces)
3) Extract cross linked complexes
4) Immuno precipitate with antibodies against the protein of interest (for example Protein C3) –> This is for the analysis of histone modifications
5) Denature proteins, reverse cross-links, release DNA and can attempt to purify specific DNA

67
Q

During chromatin immuno precipitation how can we analyze histone modification?

A

Immuno precipitate with antibodies against a specifically modified histone, for example H3-K4- Me

68
Q

How do we analyze long range chromatin interactions?

A

By CCC (Chromatin Conformation Capture)

69
Q

What are the steps in CCC?

A

1) Cross link chromatin with formaldehyde, which allows the conformation of chromatin to be captured
2) Isolate the chromatin
3) DNA is digested with an endonuclease and there will be chromatin disruption, the ends will be repaired, but DNA fragments that have been in proximity remain cross-linked
4) Dilute the extract DNA and ligate the DNA ends, dilute sample at a very high level in order to prevent the ligation of DNA pieces that are diluted
–> Only ends that are close to each other will ligate
5) Uncross link and PCR
–> Detect the ligation product by quantitative PCR and then reverse the cross-links
6) Analyse the produced DNA with multiple pairs of primers

70
Q

How can we detect TADS?

A

CCC

71
Q

Overall how does ChIP work?

A

1) Cross link bound proteins to DNA
2) Isolate chromatin and shear DNA
3) Precipitate chromatin with protein specific antibody
4) Reverse cross link and digest protein
5) Ligate P1 and P2 adaptors to construct fragment library

MBG 4110 Test 1 (6 decks)

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