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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
What does H1 contribute to?
H1 contributes to fibre stability, not nucleosome stability Nucleosomes remain intact when H1 is removed
26
What is important information to remember about histones?
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
27
What are epigenetic marks?
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
28
Where are methylated CpG islands found?
Heterochromatin DNA
29
What are some Epigenetic marks (PTMS) on the Histone N-termini? What is their purpose?
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
What is the histone code?
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
How much information can be stored in histones?
Lots of information
32
What are some messages that will found on histone tails within the nucleosomes?
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
What is a reader, writer, eraser, and mover?
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
How do writers, erasers, readers and movers interact with epigenetic marks?
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
What are histone variants?
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
What are the histone variants and their roles (3)?
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
What is the co-ordination of epigenetic marks?
Individual PTMs and the methylation of DNA influence each other and establish a dynamic but stable chromatin structure
38
What is the importance of epigenetic marks being co-ordinated?
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
What is an example of how epigenetic marks are co-ordinated?
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
How are epigenetic marks co-ordinated? Include HP1 in your explanation
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
What is HP1? Where is it found? What does it contain? What do the domains do?
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
What is the genetic evidence to suggest the connection between DNA Methylation and H3-K9 Methylation?
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
Explain how The coordination epigenetic marks take place
Loss of co-ordination = loss of stability Image to make sense of
44
How are the molecular mechanisms of coordination performed?
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
What do movers do? Where are movers present?
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
What is important to note about nucleosomes?
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
How does ATP dependent chromatin remodelling work?
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
What can chromatin remodellers do?
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
What is the compaction of chromatin and chromatin fibres important for?
Well, it is an intermediate to build up heterochromatin Necessary to regulate gene expression
50
What do chromatin loops do?
Build higher order structures and facilitate the assembly of active transcription or replication complexes
51
How are chromatin loops formed?
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
How is the chromatin structure within the loop controlled?
Controlled via other loops Chromatin boundaries delineate one domain from another and allows for fine regulation of gene expression
53
What are TADs?
3D structures formed from multiple replication loops coming together
54
How are chromatin globules formed?
Chromatin looping between active genes and regulatory elements and clustering of honest the site of active transcription facilitates the formation of globules
55
How do genes cluster together?
Active genes clusters associate with other expressed genes in active neighbourhoods while inactive genes cluster in silent neighbourhoods
56
What can multiple loops clustering establish?
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
How do many loops work together?
Many loops work together to bring many active promoters together to create a highly transcribed euchromatic domain in the nucleus
58
What do TAD's make up?
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
How do active and silent neighbourhoods associate?
Active and silent neighbourhoods associate in cis and in trans to form larger active and inactive compartments
60
What does nuclear organization show?
Nuclear organization reflects clustering of active and inactive loci in distant coparents forming a fractal globule
61
How are chromatin loops formed?
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
Explain CTCF in terms of chromatin loops
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
What can CTCF and Cohesion do in terms of chromatin loops?
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
What is the process for detecting methylated DNA?
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
What is chromatin immunoprecipitation?
A technique for the analysis of the binding of proteins to DNA in vivo
66
How does chromatin immunoprecipitation work?
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
During chromatin immuno precipitation how can we analyze histone modification?
Immuno precipitate with antibodies against a specifically modified histone, for example H3-K4- Me
68
How do we analyze long range chromatin interactions?
By CCC (Chromatin Conformation Capture)
69
What are the steps in CCC?
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
How can we detect TADS?
CCC
71
Overall how does ChIP work?
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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