Lecture Panel 3 Flashcards
What is totipotent?
Become any cell type
What are multiple progenitors?
Differentiate into a subset of cell types
What are differentiated cells?
Convert into pluripotent stem cells
What are pluripotent cells?
Differentiate in developing embryo
What is an IPS?
Induced Pluripotent Stem Cell
When does a gene show variegation?
Genes that go from euchromatin to heterochromatin exhibit gene silencing or some degree of variegation
What is important about the phenotype of variegation?
Phenotype is heritable through multiple cell divisions
How long does the phenotype of variegation last?
Can last for the duration of a multi-cellular organism
What happens when a gene goes from heterochromatin to euchromatin?
The gene is “active”/ displays the expected phenotype so ewe know that the genetic information (DNA sequence) is not the cause of the phenotype
What is the mechanism of heritable gene silencing?
Epigenetics
What is genetic information?
The primary DNA sequence
What is epigenetic information?
1) DNA Methylation
2) Histone Modifications
3) 3D chromatin organization (Loops, TADS)
What is important about the epigenetic information?
1) It is heritable, and more flexible than the information on DNA, which means that changes are allowed and that epigenetic marks can change
2) Governs tissue specific global gene expression, meaning it governs the expression of all genes, and allows for gene expression to be established after cell division
What are the disadvantages of using S. cerevisiae as a model organism?
No DNA Methylation and no HP1
What are the advantages of using S. cerevisiae as a model organism?
1) Variety of Histone PTMs
2) Homologous genes between yeast and humans
3) Similar regulation of cell cycle, DNA replication, and possibly chromatin structure?
How does yeast grow and replicate?
Yeast undergoes a standard eukaryotic cell cycle which is G1 –> S –> S2 –> M –> Cytokinesis or back to G1 and cycle begins
When describing the cell cycle for yeast what happens in the phases?
In G1: A bud is produced when the cell is ready to divide and it is a single round cell
In S: The bud grows, but DNA replicates and epigenetic marks are copied
In G2: Bud Grows
In M: Cell divides
Why do some yeast cells not divide?
There is a gene in yeast that regulate mitosis and in cells that do not divide, this gene is inactive
How does S. cerevisiae grow and divide?
Either as diploid or haploid cells
Why are haploid cells easier to study?
One set of chromosomes, means that if there is a mutation, the phenotype will show up right away because there is nothing to hide it.
How do cells become haploid?
Under low nutrient conditions diploid cells sporulate and produce haploid cells
How do two haploid yeast cells become a diploid yeast cell?
When cells don’t like the environment, they will go through meiosis but before this the cells try to find a mating partner
Each haploid cell expresses ONLY a or alpha, which are the mating type genes
Each haploid cell expresses only an a or alpha pheromone, and only an a or alpha pheromone receptor –> For example, mating type a releases a pheromones, but has an alpha pheromone receptor
The mating type genes, a or alpha, determine the mating type of the cell
How does mating between two haploid yeast cells occur?
Mating only happens between a and alpha haploid cells to produce a diploid cells
Mating type a recognizes alpha, and vice versa
What is the process by which two haploid yeast cells mate to form a diploid yeast cell?
Via Shmooing
1) Display of preference: a recognizes alpha through the pheromone release of alpha pheromones from alpha cell, and alpha pheromones bind to alpha pheromone receptors on a cell
2) Shmooing: a and alpha come close together
3) Mating: a and alpha attach together
What does it mean when there is dysregulation of mating genes and what happens?
Dysregulation of mating genes is when a yeast cell expresses both mating types, a and alpha
If this occurs, yeast cells will shmoo but not mate
What can happen in wild type yeast? Why does this process happen?
Wild-type yeast cells can switch mating types during haploid growth (a to alpha)
The mother cell express an a mating type but also has alpha genes that are not expressed (silenced).
When the mother cell divides, the daughter cells produced express alpha mating type (because the mother had these alpha genes). These daughter cells do have a genes (from the mother), but these a genes are silenced
Why can yeast cells switch mating types?
Genes for a and alpha are both found in the genome of all yeast checks, but only one of these genes are expressed and the other is silenced
If both genes are expressed, the cell may shmoo but never mate
Between Mata and Matalpha compare the differences and similarities according to mating, sporulation, the pheromone released and the receptor the cell type has
Mating: both Mata and Matalpha
Sporulation: Neither Mat and Matalpha spore
Mata releases a pheromone
Matalpha releases alpha pheromone
Mata has an alpha receptor
Matalpha has an a receptor
Describe where the two mating genes are located and what they are called?
The mating genes (a or alpha) are positioned in two mating type loci (HMR and HML) near the ends of chromosome II
Describe how the yeast mating type genes are expressed
The mating genes (a or alpha) positioned in HMR and HML are never expressed (they are silenced)
Any other gene that is inserted into these loci are not expressed (position effect)
How is the mating type of haploid cells in yeast determined?
The mating type of haploid cells is determined by the translocation of the a or alpha genes to the MAT locus and is expressed from this position (MAT locus)
How does translocation occur? Explain the process
It occurs by a copy and paste mechanism
An HO endonuclease is required for switching to occur
Wild type haploid HO can switch: Homothallic
Mutant ho yeast cannot switch: heterothallic
What happens if HMLa/HMR alpa are expressed?
If either are expressed, the cell cannot mate.
The cell may shoo but not mate
Describe the expression patterns of the mating type loci for S. cerevisiae
HML alpha and HMRa are constitutively silent loci, meaning they are never expressed, but the loss of silencing and expression of both a and alpha will prevent the cells from mating, cells will shoo but not mate
HML alpha and HMRa store the genes that specify the mating type
The MAT loci is constitutively expressed, which means it is alpha or a, not both
What does the MAT loci do?
The MAT loci determines if the cell is a or alpha
The MAT locus makes 2 transcript (a or alpha required for mating), but not both
What keeps HMLalpha and HMRa silent? What are these genes?
Four genes, that when mutated produced shmoos, which means these genes are required for silencing at HMR a and HML alpha:
SIRs (silent information region)
Silencers
Abf2 and Rap1
Explain the genes that keep HML alpha and HMRa silent
SIRs: Mutations in SIRs cause HMLalpha and HMRa to be expressed, leading to shmoos
Silencers: cis- elements and two of them: E (essential), I (important), silencers set up the epigenetic landscape of the cell
Explain silencers
Silencers are cis elements, and cis elements are DNA sequences that direct some type of phenotype
Silencers set up the epigenetic landscape of the cell
Silencers contain ACS (binds to ORC), as well as binding sites for the two proteins, Abf1 and Rap1
What is SIR2?
Histone deacetylase
What is special about ORC, Abf1 and Rap1?
They bind the proteins encoded by the SIR genes
What does silencing at HMRa and HMLalpha require? How is this process initiated?
Silencing requires silencers (E and I)
Histone deacetylation initiates at these silencers and spreads(wave of deacetylation spreads in both directions) into HMRa leading to the formation of heterochromatin (highly compact heterochromatin formed from histone deacetylation) and gene silencing at our silencing loci
What is the process of silencing at HMRa and HMLalpha?
1) In yeast, ORC recruits SIR1
2) Rap1 and Abf1 recruit a heterodimer of Sir3 and Sir4
3) Sir3 and Sir4 can bind to the deacetylated tail of histone4
4) Silencer recruits a reader and an eraser to an epigenetic mark
What are elements of the essential silencer? What are elements of the important silencer?
Essential: ARS317, RAP1, ABF1
Important: ABF1 and ARS318
What is the critical mass of epigenetic factors?
Need all 3 elements (ACS, RAP1, ABF1) of the silencer to recruit all the proteins needed to silence the gene
What do the SIR proteins do?
SIR2: Histone Deacetylase
SIR1: brings Sir2p to ACS
SIR3/SIR4: bring Sir2p to the Abf1 and Rap1 binding sites, but they also bind to deacetylated histones, in particular to the deacetylated H4 tail-K16
How do the SIR proteins spread and deacetylate histones?
Sir2 deacetylates the histones in the nucleosome next to the silencer
Sir3/4 bind to the deacetylated nucleosome and brings more Sir2, and the cycle occurs again
How does SIR spread over HMRalpha?
Once the acetylate group is removed, Sir3 and Sir4 can bind and recruit another sir2, which leads to a chain reaction leading to the deacetylation of many histones
However, the availability of Sir proteins limit the spreading of the chain reaction, and the chain reaction can also be stopped by large protein blockages and other boundaries
Overall what is the spreading of SIRs and histone deacetylation required for?
Spreading of SIRs and Histone deacetylation are critical for the silencing at HMRalpha and HMLa
The fact that HMRalpha and HMLa are NEVER active indicates that their repressed state is heritable, which is epigenetics
How is gene silencing established?
Gene silencing is established by specialized DNA elements called “SILENCERS”
Silencers are much larger in higher eukaryotes, found in CpG islands
How is silencing spread to neighbouring regions?
Silencing is spread to neighbouring regions by the spreading of histone modifications
Communication between readers, writers, and erases can lead to spreading of heterochromatin
What is one of the hallmarks of silent chromatin?
H4-K16 deacetylation
H3K9- Me is an epigenetic marker for heterochromatin in higher eukaryotes
How do we investigate the significance of histone deacetylation?
Get a mutation that mimics a certain post translational modification (Glutamic Acid, Q mimics/looks like acetylated lysine, as Q has same size and charge)
What happens when we replace an acetylated lysine with glutamic acid?
Genetic screen would determine the phenotype
Genetic screen would indicate that there was shmooing in the yeast, suggesting that silencing is disrupted
How is a suppressor screen performed with histone mutants?
1) Subsitution mutations in histones H3 and H4 have been used to perform mechanistic studies on histone modifications
K –> Q mutations mimics the acetylated of K-residuses, it has been found that mutations disrupt gene silencing
2) Look for secondary suppressor mutations that restore silencing in the H3, H4 K –> Q mutants
These mutations will identify the genes that encode proteins that interact with this particular residue on the histones
How do we perform a suppressor screen for gene silencing in yeast?
1) Create a mutation that disrupts gene silencing (function)
2) Produce a cell culture with the mutant cells
3) Treat this culture with a mutagen so that every cell contains about one additional mutation in any gene
4) Identify a cell that has regained the function, which is gene silencing (WT)
–> This cell now has a second mutation that surpasses the effect of the first mutation
5) Identify the gene that carries this second mutation to figure out which gene interacts with the first gene
How do we see the effects of histone mutations on gene silencing?
By the colour of a colony
Explain how we see the effects of histone mutations on gene silencing
ADE2: catalyzing a step in the synthesis of adenine, colonies with active ADE2 appear off white
If ADE2 is inactivate by a mutation, cell’s appear red, ADE2 is silenced
If ADE2 is inserted into a mating type locus, the gene is silenced and the colonies appear red
If this silencing is impaired, ADE2 is expressed and colonies appear white
If the silencing is restored, the cells will appear red once again
What happens when ADE2 is inserted into the mating type?
So, if there is no mutation on lysine (not Ac or Me), it is the wild type and under normal circumstances, ADE2 will be silenced, and appear red. Normally, ADE2 when inserted into the mating type loci should appear red
But, if there is a mutation on lysine and lysine is replaced with glutamate acid, there is a mutation and there is no longer silencing, and ADE2 is expressed, so cells will appear white. ADE2 is no longer silenced, but ADE2 should be silenced
How can we identify the genes that act through H4K16? Explain the process
A genetic screen is performed
1) Insert ADE2 in the mating type locus HMRa, and we see red colonies to suggest there is silencing of ADE2
2) Mutate K16 –> Q16 of histone 4, we see white colonies, suggesting ADE2 is expressed, and loss of silencing
3) Mutagenize (second mutation) for the whole genome and look for red colonies, to indicate the second mutation suppresses the first mutation, and ADE2 is silenced again
–> some cells would have H4Q16 reverted to H4K16, ADE2 is silenced
–> Any other mutation indicates a gene that works through H4K16, we have identified a suppressor mutation of H4Q16
How are SIRs and RAP1 involved in ADE2 silencing?
SIRs and RAP1 are the proteins needed to silence the mating type loci
If ADE2 is in the mating type locus, the cells would look red
1) Mutate H4K16 to H4Q16, ADE2 is expressed, silencing is lost, and white colonies (mutation so no silencing)
2) Mutagenize (perform second mutation/mutate the mutation), some cells will be red again indicating ADE2 is silenced
These red colonies that appear again have a mutation that supresses the effect of H4Q16
How can the gene that encodes the suppressor mutation be identified?
1) Get the red colony with restored silencing
2) Transform the cells with a library of plasmids, each plasmid expresses one of the genes found in S. cerevisiae
3) Each yeast cell acquires one plasmid
4) Look for white colonies that have again lost the ability to suppress the ADE2 gene
–> These colonies carry the wild type allele of the mutant suppressor (which means they do not have the mutant suppressor to suppress ADE2 gene)
5) Isolate the white plasmid, and sequence the gene, we have identified the gene that carries the suppressor mutation
What are the different suppressor screens found using ADE2?
1) Normally, Sir3 binds to H4K16: No mutation, ADE2 is silenced, colonies appear red
2) H4K16 is mutated to H4Q16, Sir3 cannot bind to H4Q16, so ADE2 is expressed, colonies appear white because ADE2 is active and not silenced
3) H4Q16, but sir3 is mutated, and because sir3 is mutated, sir3 can bind to H4Q16, leading to silencing –> suggests that the mutation in sir3 is the suppressor mutation, and supresses H4Q16’s ability to activate ADE2
Explain how Sir3 interacts with H4K16
Gene silencing requires an interaction between Sir3p and the H4K16 non acetylated
Acetylation of H4K16 (H4Q16) prevents an interaction with sir3p and allows the formation of euchromatin (active genes)
How do both SIR3 and SIR4 bind to H4?
After deacetylation by Sir2, the deacetylated H4K16 recruits Sir3 and Sir4 to recruit more Sir2 to deacetylate H4K16 and other acetylated Histone K’s on the histones on the next nucleosome to recruit more Sir3/Sir 4, and the cycle continues.
This is the mechanism of heterochromatin spreading (gene silencing spreading)
Which enzyme acetylates H4K16 to counter the establishment of heterochromatin
Screening of suppressors found that SAS2 encodes a H4K16 acetyl transferase
Explain Sir3, Sir2p, Sir3p, and Sas2p
Sir3 is a suppressor mutant that restores the function lost by the H4-K16Q mutant
Sir3p is a reader of H4-K16Ac
Sir3p/Sir4p recruit the HDAC Sir2p
Sir2p is the eraser of K16Ac
Sas2p is a H4-K16Ac writer
What are the functions of telomeres?
1) Protect the ends of linear DNA molecules from DNA exonucleases
2) Prevent fusion of chromosomes
3) Facilitate complete replication at the ends of linear DNA molecules
4) Act as a depository for factors that maintain genomic stability –> many proteins involved in DNA repair are idling near telomeres
5) Ensure complete replication of chromosome –> shortening telomeres following replication, associated with aging
6) Telomeres can sense aging, the integrity of the genome, and sense the proliferation state of the cell
What are telomeres?
All eukaryotic chromosomes end with a highly repetitive stretch of DNA, known as the telomeres
What do the repetitive sequences of telomeres generate?
Unusual DNA structures:
T-loops
R- loops
G-quadruplex DNA
These are also known as secondary structures, and may cause the replication fork to pause
What is the telesome/sheltrin?
Telomeres are wrapped in a large non-histone complex
What are the sub telomeres?
Contain genes that are frequently, but not always silent (facultative heterochromatin)
In metazoans, they are involved in development, so sub telomeres can be turned on or off
Contain histones, that are tightly condemned into heterochromatin
Describe the telomeric structures
Rap1: repressor activator protein, and acts as a silencer. Sir3 can bind to Rap1
Fork may stop at TRF2 which is a unique secondary structure found in shelterin
R loop: fork may also stop here
t loop: has affinity for certain proteins that protect and maintain the telomere
There are some isolated telomeric repeats that will still bind shelterin and in yeast, these isolated telomeric repeats can still bind Rap1
Sub-telomeres can recruit many weak silencers
In yeast what are the majority of pause sites for the replication fork?
Protein complexes/blockages
Explain the structure and function of sub-telomeres?
Subtelomere can either be euchromatin or heterochromatin depending on the needs of the cell
Sub-telomeres are conserved and repetitive, but not as conserved as telomeres
Sub-telomeric repeats are scattered around in the sub-telomere
What do yeast telomeres contain?
Telomere repeat, which is TGGG, but in humans it is T2AGbeta
TGGG repeats are found in subtelomeric DNA as well
TG3 repeats bind Rap1, which means Rap1 can bind to both the telomere and sub-telomere
ACS and Abf1 binding sites are found in the sub-telomere
What is the importance of Rap1, ACS and Afb1 clustering in the telomeres?
Rap1, ACS and Afb1 allows for the recruitment of Sir proteins, which leads to the weak silencing effect, depending on the availability of silencing factors
Cell expresses a limited number of silencing factors, which creates competition for binding sites, which mean silencing factors are the limiting factors
How are genes silenced at telomeres?
Sub telomeric genes in yeast are silenced
The silencer is actually the telomere itself
However, Histone Acetyl-Transferases (HATs), counteract the silencing at telomeres, and in particular HATs counteract the SIR2 HDAC activity, which means they counteract the histone deacetylates
What is Position Effect Variegation or the Telomere Position effect?
It is the odd behaviour that the silencing at the sub-telomeres is meta-stable, which means sub-telomeric genes convert their state (silenced versus active) one about every 20 generations
Other words, activation/deactivation of a gene depends on the position of the gene, not the promoter of the gene itself
How can the establishment and spreading of silencing occur at telomeres?
1) Rap1 binds to the telomere and recruits Sir2
2) Histone deacetylation spreads at HML/HMR and is counteracted by SAS2 and other HATs (HATs add acetylation back to histone marks)
Explain position variegation
The competition between SIRs and HATS (and many other anti-silencing and silencing factors) produce meta-stable alternating of silenced or active states of the genes –> these states persist for many cell generations
If ADE2 is inserted in the telomere, the colonies display red (silenced ADE2) and white (active ADE2) segments
What do we learn from meta stable phenotypes?
Weak cis regulatory elements determine epigenetic landscapes of the cel l
Genes that regulate epigenetic conversions
How are regulatory cis elements identified?
These elements are cloned next to a reporter gene and then we measure the activity of the reporter
What is the process of how we identify regulatory cis-elements?
1) Insert a reporter gene next to the telomere
2) Insert a candidate cis-regulator (proto-silencer or anti-silencer) between the telomere and the reporter gene
3) Insert another or multiple candidate cis-regulator (porto-silencer or anti-silencer) next to the gene distal to the telomere –> Distal gene: gene far away from telomere
4) Insert another candidate cis regulator (boundary) next to the gene
5) Prepare strains with each combination of cis-regulatory elements
6) Grow a culture with each of the recombinant strains
7) Measure the proportion of cells in which the reporter gene is silent
8) Identify the function of the inserted elements
What are proto silencers?
Proto-silencers are weak silencers that relay and enhance signals by strong silencers
Our silencer is in the telomere, our reporter gene is next to the telomere, and our proto-silencer is on the other side of the reporter gene
What are anti silencers?
Anti-silencers reduce silencing and reduce stability of the heterochromatin domain
Silencer is telomere, we have an anti-silencer as well that will reduce the stability of heterochromatin domain
What are insulators AKA Chromatin boundaries?
They prevent silencing of the reporter gene by physically preventing/blocking the spread of silencing factors (CTCF, large protein complex)
What are examples of proto-silencers?
Silencers: Clusters of ACS and binding sites for Abf1p, Rap1
Porto-silencers: Isolated ACS, binding sites for Abf1p and Rap1p
How is silencing relayed?
Abf1 and ACS elements relay and enhance the spreading of silencing away from the telomeres
They act as “proto- silencers”
Where are transposons located?
In yeast they are found in subtelomeric regions (heterochromatin silencers transposons)
What are metabolic genes? Where are they found? What do they help?
Genes that are silenced in the presence of nutrients and active in the absence of nutrients, they aid in adaption and plasticity. They help a cell adapt to changes in its environment. Metabolic genes are found in sub0telomeric regions
Where are telomeres found? How are they found
The telomeric clusters are found in the nuclear periphery and they tend to cluster together in the periphery of a cell’s nucleus
What happens when cells start aging?
When cells start aging, some of this clustering is lost because aging cells are worse at maintaining heterochromatin stability
How do parasites use telomeric variegation?
Evade immune responses
In human fibroblasts and yeasts how does telomeric chromatin sense aging?
Young: Heterochromatin is compact, there is telomere clustering at the cell periphery, as aging occurs, replication and telomeric DNA damaged, and reduced H3/H4 production there is less telomere clustering and less heterochromatin clustering at the cell periphery
How do cells sense ageing?
Shorter telomeres: arrest cell division: senescence –> both aging yeast cells and senescing mammalian cells have shorter telomeres
Less compact heterochromatin: sensescence –> both aging yeast cells and senesing metazoan cells have less compact chromatin
Sensing of genome instability: Many DNA repair factors are stored in the subtelomeric regions, they are released upon DNA damage
Supression of transposons: Transposons are important for the formation of heterochromatin blocks that regulate gene expression
What are transposons?
Harmful genetic elements that are mobile
In S. cerevisiae there are a small number of transposons, found in subtelomeric regions where heterochromatin is abundant
In metazoans, there are millions of transposons found in heterochromatin regions
Explain the Position Effect Variegation in Drosophila
A chromsome rearrangement inverts a chromosomal segment and positions a euchromatin gene (white) close to the centromere
White is responsible for the synthesis of a red eye pigment
White is now at the boundary between centromeric heterochromatin and euchromatin
How doe Position Effect Variegation in Drosophila occur?
The repositioning of white causes variegated (patchy) pigmentation of the eye
This pattern is caused by infrequent conversions between a silenced and active state of white
What is PEV dependent on?
The position of white
What is observed during PEV in Drospophila?
White: Mutant
Red: Wildtype
White at the centromere: PEV, the gene that should be silenced is now expressed in the telomere
The white gene is involved in the production of pigment in drosophila eyes and the phenotype of the eye depends on position
What phenotypes cause eye colour
W+/W+ or W+/W-: Red eyes (WT)
w-/w-: Deletion mutation, white eyes
PEV: The white gene has been moved to a different position and next to the centromeric heterochromatin
What happens during irradiation?
X-rays are applied to the gene. Our W+ moves position and becomes W(v): variegated allele
Explain the position effect?
There is a chromosome inversion that juxtaposes the white gene next to centromeric heterochromatin
At this position the white gene is expressed only in some facets (the red ones) of the eye
All facets have identical genomes
What happens for PEV when we replace the white gene with beta galactoside?
There would still be a patchy type phenotype, but would be blue instead of red/white because this is a position effect
The gene that is found in that position doesn’t matter, it is the position that matters
If white is replaced with another gene, this gene will be expressed in a variegated fashion
In PEV what happens when the white gene is moved back to euchromatin?
When the white gene is moved back to euchromatin, it works (activated)
Therefore, the genetic information (DNA sequence) is not the cause of the phenotype
Therefore the mechanism of silencing must be epigenetic
Why does PEV happen?
Heterochromatin formation over the gene is due to the “position effect” meaning that chromatin in the neighborhood determines the state of expression
Uncertainty: The variegated silencing fo the gene is caused by “oozing” of the heterochromatin proteins over the hypothetical heterochromatin-euchromatin boundary
“Oozing” is the spreading of heterochromatin by mechanisms similar to the Sir dependent spreading in yeasts
When discussing PEV what is important to note?
Cells containing limiting amounts of heterochromatin factors
Competition for these limiting heterochromatin factors is leading to alternative states of individual loci
How do we identify limiting factors of heterochromatin factors?
1) Genetic screens in Drosophila with a variegated white gene’s are conducted
–> Mutagenize fly embryos with transposons or irradiation
–> Grow the fly population to adulthood
–> Look for flies with a specific phenotype
2) Loss of patched eyes (completely white or completely red) in a white variegated strain indicates loss of genes that regulate PEV or gene silencing
What types of variegated alleles have been classified?
Su(Var): Supressors of variegation (mostly red eye)
E(Var): Enhancers of variegation (mostly white eye)
There are lots of Su(Var) and E(Var) in the loci >100
What are the Su(Var) genes?
Su(Var)2-5: encodes HP1 (a key component of heterochromatin, interacts with H3-K9Me)
Su(Var)3-9: encodes H3-K9 Methyl-transferase
Both of these genes encode proteins related to H3-K9Me histone mark
In insects the methylation of H3K9 is the critical modification that leads to the establishment and maintenance of the repression of white variegated alleles
What do mutations in HP1a lead to? What are the Su(Var) genes? What is the role? Discuss TPE
Mutations in HP1a lead to weaker gene repression
Su(Var) 3-9 are histone methyl transferases that methylate H3-K9
TPE is also present in drosophila and the function is more or less the same
What is the importance of the Su(var) and E(var) genes?
Homologoues of Su(var) and E9var) genes have been identified as the key regulators of many epigenetic transactions and are mutated in cancers and various rare genetic disorders
These genes encode key readers, writers, and erasers of epigenetic marks
The discovery was the foundation of modern epigenetics
What has been determined from PEV and TPE?
1) Formation/maintenance of heterochromatin is critical for the silencing of genes
2) Heterochromatin is inaccessible to transcription factors
3) Hallmarks of heterochromatin (DNA methylation, histone methylation) apply to this silenced state of the variegated genes
What is model 1 for the mechanism of epigenetic conversions?
PEV at the pericentric heterochromatin of Drosophila. This is a spill over hypothetical chromatin boundary
1) Pericentric chromatin recruits heterochromatin factors and nucleates a silenced domain
2) Heterochromatin factors modify histones (Deacetylation, H3K9 methylation) in nearby nucleosomes
3) Modified histones recruit HP1 and additional heterochromatin factors, which modify the histones in the next nucleosome
4) The spreading of heterochromatin continues until a chromatin boundary (a large protein complex bound to DNA or a hap in the nucleosome array) is reached
5) A euchromatin domain is established and delineated by the boundary
6) The removal of the boundary leads to the loss of delineation and creates a metastable locus
Explain a metastable locus
Metastable loci can either become heterochromatin or euchromatin
There are several limiting factors that limit how far heterochromatin/euchromatin can spread; this creates a dynamic equilibrium between heterochromatin and euchromatin
The variegation phenotype occurs due to an epigenetic instability by creating a metastable locus that can lead to a gene being turned on or turned off
Whether the gene is turned on or off is determined during development and when the DNA is replicated it will remain on or off (does not change after development
Is model 1 for epigenetic conversion correct?
Heterochromatin is spread linearly over a boundary
If the spreading of heterochromatin is linear:
1) Gene A is inactivated more frequently that Gene B
2) Gene A will always be OFF when Gene B is OFF
But, Gene A is ON in cells when Gene B is OFF
What is the model for Discontinuous spreading: Chromatin bending and proximity of heterochromatin domains, chromatin boundaries, proper silencers and a spill over of chromatin factors
1) A gene is positioned close to a proto silencer
2) The distal porto-silencer flips over to come in proximity to a strong silencer
3) Heterochromatin factors spill over from the silencer and are retained by the proto-silencer
4) The gene next to the porto-silencer is now silenced (OFF)
5) The genes between the silencer and the porto-silencer remain ON
How does the model for Discontinuous spreading: Chromatin bending and proximity of heterochromatin domains, chromatin boundaries, proper silencers and a spill over of chromatin factors work?
1) Sir3p/sir4p are recruited to the telomere by Rap1. Sir4p recruits Sir2p and initiates a cascade of histone deacetylation
2) The spreading of deacetylation is counteracted by HATs
–> Regulating the bending of DNA can control which genes are inactivated, can loose this effect with age due to dispersion of heterochromatin increasing with age
3) A metastable locus is established by the competition between HATs and Sir proteins
4) Bending the chromatin fibre brings a distal locus in proximity to the Sir-protein cluster. Sir2p initiates the spreading of histone deacetylation
5) The spreading is restricted by chromatin boundaries
6) Sochastic establishment of this 3-dimentional structure generates a meta-stable locus
When the distal locus is brought close to the silencer, silencing factors can jump to distal sites
Heterochromatin may get weaker as it spreads away from the silencer due to the competition of factors
–> creates epigenetic instability of variegated phenotype
What are the areas that have telomeres clustered together believed to be?
The telomeres that cluster together in the nuclear periphery are believed to represent a sub-domain in the nucleus with highly condensed heterochromatin
The alternative recruitment of a locus in these domains allow for alternative states of the locus
What is the model to show how epigenetic variations by the recruitment into a nuclear heterochromatin domain?
1) Gene B exits the perinuclear heterochromatin domain, turning “on”
2) Gene A enters the perinuclear heterochromatin domain, turning “off”
3) Both genes enter the perinuclear heterochromatin domain, turning “off”
4) Both genes exit the perinuclear heterochromatin domain, turning “on”
Need to be able to push the genes into or out of the heterochromatin to turn them on or off, regulated by 3D chromatin structure and has significant consequences for how genes are expressed in the cell
Transitions between any of these states provide for cell to cell variations in the expression of Gene A and Gene B
What did we learn from yeast about the switching of an epigenetic state?
Searched for mutations in genes that reduce the frequency of switching at the telomere
Found that
CAF1 and ASF1: Chaperones associated with the replication fork
Rrm3p: DNA helicase that helps the fork to resume elongation after pausing
Tof1p: a component of the Fork Protection Complex at paused replication forks
What is the model to explain how replication coupled epigenetic switch at stalled fork?
During DNA replication the chromatin is completely disassembled and reassembled
Replication forks temporarily pause at tightly bound proteins
Genes can use this opportunity to change the epigenetic code of the locus
If gene A switches and gene B does not, we will have independent variegation of the two neighbour genes
Explain the role of Rrm3p at stalled forks in the loss of epigenetic marks
When the fork is paused, the histones ahead of the fork are no longer available to be shuttled behind the fork
The impaired histone rebuilding mechanism is impaired when the fork is paused, which leads to a 50/50 chance of building either heterochromatin or euchromatin, regardless of what type of chromatin was in that position before
What are trypanosomes? How do they look?
Trypanosomes are blood parasites that cause the sleeping sickness
VSG coats the parasite and exposes themselves to the immune system
While the immune response towards the VSGx builds up, the parasite switches to another VSG
There are about 1000 VSGs and most VSG genes are subtelomeric
VSG encodes variable surface lipoproteins
Can’t be effectively countered by our antibodies since the surface protein keeps changing
There is no cure and very hard to treat
Why should we care about TPE?
Due to VAR genes in Plasmodium
Plasmodiums are blood intracellular parasites that live in erythrocytes and they cause malaria
Explain VARs and the immune system
VARs are positioned at the subtelomers
They are expressed and transported to the surface of the erythrocyte
While the immune response towards VARx builds up, the parasite switches to another VAR
There are about 60 VARs
They switch in a very coordinated fashion so that only one VAR is expressed at a time
Explain the VAR genes in plasmodium. Be sure to include the TPE in your explanation
Most VSG and VAR genes are positioned in the sub-telomere and are silenced and variegated by a TPE mechanism that is sitar to TPE in yeast
The subtelomerers of Plasmodium and Trypanosomes contains assembles of proto silencers, anti silencers, and insulators that comprise a very sophisticated and seemingly undestructible switching machine
If we suppress switching, the immune system will overwhelm the parasites
Explain the VSG and VAR genomes
Telomeres act as a silencer for all these genes
These pathogens Plasmodium and Trypanosoma use the telomere position effect as a weapon against the host
What is the importance of TPE?
FSHD is a rare genetic disorder that is caused by the shortening of the D4Z4 repeat at the 4q35 locus next to the telomere of chromosome IV
What is the importance of D4Z4?
D4Z4 repeat probably acts as a chromatin boundary
The shortening of the D4Z4 repeats expose the telomere distal genes
The shortening of D4Z4 is causing repression of DUX4 a gene encoded within the repeat, again linking TPE to FSH
All of these damages prevent development of certain muscles
Genes are brought closer to the telomere when the D4Z4 gene is shortened (occurs in FSHD)
The genes are still expressed, but the balance between them is disrupted leading to epigenetic instability and impaired development of musculature
