Lecture 3 - Epigenetics in animal development Flashcards
1
Q
What are the homeobox genes?
A
- Transcription factors that control segmental patterning duing development
- Control the expression of genes in the antennapedia complex and the bithorax complex
- Have the same rearrangement in the genome as they do in the timing of the expression of the body parts
- When one of them is expressed in a particular segment, the expression of all of the others is switched off
2
Q
How does the activation of the HOX genes fit the pattern for epigentic mechanisms?
A
- early acting transcription factors set up the expression of the HOX genes and are then switched off
- HOX gene expression is maintained throughout development (segment specific profile)
3
Q
What was the resultant phenotype of the hometotic mutants?
A
- Homeotic muants had alteractions in cell fate due to the Hox loss of function or the de-regulation of Hox gene expression
- e.g. antennapedia mutant has a mis expression on Antp - antennae to legs tranformation
- Antp should be on in the first leg segment not where the antennae should be
4
Q
What is the Pc mutant?
A
- wild-type males only have sex combs on the 1st legs
- but in the polycomb (pc) mutant have extra sex combs on the 2nd and 3rd legs
- take on the characteristic of the 1st leg
5
Q
What are the two main classes of regulatory proteins that maintain HOX gene expression?
A
- Polycomb group (PcG) proteins: maintain repression
- Trithorax group (Trx) group: maintain activation
These are antagonistics and compromises functionally distinct proteins
e.g. When Anyp is expressed the trithorax proteins will keep expression on in the leg segment and plycomb group proteins wi.ll keep everything else off
Different in each of the segments
6
Q
What are the features of the polycomb group (PcG) proteins?
A
- conservation of function across species and kingdoms
- targets 100s of genes
- two principle classes of multi-protein complex
- polycomb repressive complex 1
- polycomb repressive complex 2
7
Q
What are the features of the polycomb repressive complex 2?
A
- complex of four main proteins
- H3K27 methyltransferase (D: Enhancer of Zeste (E(z))
- WD40 protein (D:Extra sex-combs (ESC))
- Histone binding (D: Caf1/p55)
- Zinc finger, VEFS (D: supressor of zeste12 (SU(Z)12))
- multiple versions
- main activity of PRC2: mathlation of H3K27, repressive mark
- WD40 for protein-protein interactions
8
Q
What is the activity of the H3K27 methyltransferase of PCR2?
A
- adds a H3K27me3 repressive mark
- H3K27R variant (Lys-to-Arg) mimics loss of the protein (E(z)) as Arg can’t be methylated
- lethal if everywhere (have to express in certain cell types/tissues)
- methylation neccessary for repressive activity
9
Q
What are the features of polycomb repressive complex 1?
A
- broadly four protein complex
- chromodomain (D:polycomb (Pc))
- Zn Finger SAM domain (D: polyhomeotic (PH))
- Ring Finger (D: Posterior sex combs (PSC))
- E3 ubiquitin ligase (D; Ring1 (dRing1))
- Chromodomain protein binds H3K27me3 mark
- Monoubiqitination of H2AK119
- Zinc-finger involved in DNA binding
10
Q
Outline the models for how PRCs inhibit transcription
A
- hundreds of targets
- don’t all repress in the same way
- some genes require just PCR1/2
- Some polycomb targets in vertebrates also become methyated at the DNA level
- Inhibition of transcription (H3K27me3 and binding of PRC1 i)
- H2AK119-ubiquiting mark - blocks transcription by stalling RNA polymerase II
- Chromatin compaction
- Recruitment of DNMTs
11
Q
List the models for how PRCs inhibit transcription
A
- PCR2, H3K27me3 ->
- Inhibitiion of transcription with PCR1 blocking Pol II
- H2AK119 ubiquitination by PRC1
- Chromatin compaction
- Recruitment of DNMTs by PRC2
12
Q
How are PRCs targeted to silenced genes?
A
- in drosophila, polycomb response elements (PREs) have been discovered in the promoters of PRC regulated genes
- but no equivelent founf in vertebrates or plants (could recognise CpG islands?)
- recuitment is likely to be via DNA binding proteins and/or noncoding RNA
13
Q
Give an example of a long ncRNA that is involved in PRC2 recruitment?
A
- HOTAIR is a long ncRNA involved in PRC2 recruitment at vertebrate HOX genes
- forms secondary structure recognised by protein complexes
- recruits two distinct complexes
- PRC2 - adds repressive H3K27me3
- KDM1A-coREST (histone demethylase)- removed H3K4me3 (active mark)
- trans-acting long nc RNA
- transcribed from the HoxC cluster and represses HoxD expression
14
Q
How do the drosophila and vertebrate hox genes relate?
A
- vertebrates have duplications and amplifications of the hox gene clusters
- maintain patterns of expression within the cluster
15
Q
What is the evidence for HOTAIR acting as a scaffold to recruit PRC2 and KDM1A-coREST-REST complex?
A
- HOTAIR interacts with the histone deacetylase LSD1 (KDM1) and EZH2 (H3K27 methyltransferase)
- EZH2 is a mammalian equivelent of Drosophila Enhancer of Zeste (E(z))
- 3 different cell lines:
- Fibroblast
- IP:LSD1, IgG
- HeLa cells with a LSD1-FLAG tag (for immunoprecipitation with an antibody)
- IP:FLAG-LSD1, EZH2,IgG
- HeLa cells (naturally produce LSD1)
- IP: LSD1, FLAG
- Fibroblast
- Antibodies against: LSD1, FLAG (to check flag isn’t having a non-specific effect), EZH2, IGG (non-specific specum as negative control)
- Take out cell extract
- Immunoprecipitated proteins with an Antibody
- Use RT-PCR to see if HOTAIR was precipitated alongside (U1 as negative control (ncRNA component of splicosome)
Results - what RNA was retrieved with what IP
- Fibroblasts
- LSD1 IP: high HOTAIR, U1 non detectable (rules out non-specific interactions)
- IgG IP: low levels HOTAIR, ND U1
- FLAG-LSD1 HeLa Cells
- FLAG-LSD1 IP: high HOTAIR, low with U1
- EZH2 IP: high HOTAIR, low U1
- IgG IP: low HOTAIR, low U1
- HeLa Cells
- LSD1 IP: high HOTAIR, ND U1
- FLAG IP: low HOTAIR, NDU1
When FLAG (LSD1) or EZH2 are immunoprecipitated, can bring out HOTAIR as well. LSD1 and EZH2 bind HOTAIR.
16
Q
Aside from LSD1 and EZH2, what else does HOTAIR interact with?
A
- Generated biotinylated HOTAIR RNA and controls
- GFP RNA
- antisense-HOTAIR fragment
- Biotin label used to purify HOTAIR RNA and the controls by RNA pull down
- Performed western blot on proteins of interest
- EZH2
- LSD1
- CoREST
- TEST
- G9a (H3K9 methyltransferase)
- CDYL (involved in other examples of REST dependent silencing)
- All candidates present in the cell extract (5% input)
- HOTAIR RNA is associated with:
- EZH2
- LSD1
- CoREST
- REST
17
Q
What is the experimental evidence that EZH2 and LSD1 interact, and that the interaction is dependent on HOTAIR?
A
- IP of EZH2 from fibroblasts and check association with LSD1 (WB of LSD1) (IgG - control, EZH2 western blot as + control)
- IP of LSD1 from fibroblasts and check association with EZH2 (WB of EZH2) (IgG as control, LSD1 as positive control)
- Use RNA interference with siRNAs corresponding to HOTAIR (or GFP negative control) to reduce HOTAIR abundance and see if still get IP of EZH2 or LDS1 with the other proteins
- check input to make sure the proteins are actually in the cell extracts
Results
- EZH2 immunoprecipitates with LSD1 (and vice versa) so long as HOTIP RNA is present
18
Q
What has experimental evidence shown about HOTAIR?
A
- Interacts with histone deacetlyase LSD1 (KDM1) and EZH2 (H3K27 methyltransferase) [IP of LSD1 and EZH2, HOTAIR with rtPCR)
- Interacts with EZH2 (PRC2 component), LSD1, CoREST, REST [biotinylated HOTAIR pull down, western blot on proteins of interest]
- EZH2 and LSD1 interact and the interaction is dependent on HOTAIR [IP of EZH2 and western blot of LSD1 (and the reverse), siRNAs corresponding to HOTAIR)
19
Q
What is the involvement of HOTAIR in cancer?
A
- Highly upregulated in metastatic breast cancers
- qPCR of HOTAIR abundance in patients
- Does too much HOTAIR transcipt repress targets it shouldn’t?
20
Q
What did the artificial upregulation of HOTAIR demonstrate about its targets?
A
- gemone-wide ChIP
- demonstrated that get altered PRC2 occupancy by HOTAIR over-expression
- many new targets are genes normally associated with inhibiting breast cancer progression
- HOXD10
- PGR (progesterone receptor)
- PCDs (protocadherin cell adhesion molecules - normally involved in keeping cells together, if upreg. HOTAIR, PCDs are represssed, leading to a greater migratory capacity of cells)
- Looked at what gains an Me3 and that links with a metastatic phenotype
21
Q
What are the features of the trithorax group (trxG) proteins?
A
- involved in maintaining the active state of Hox gene expression
- mutants mimic loss-of-function Hox mutations
- variety of biochemical functions including
- chromatin remodelling (nucleosome sliding)
- histone methyltransferases (H3K4) and acetlyases
- greater diversity than in the PcG group
22
Q
How do TrxG proteins act?
A
- Antagonise the repressive action of PcG complexes
- TrxG: H3K4me3 and H3K6me2 inhibit PcG H3K27me3
- TrxG H2K27Ach inhibits PcG H3K27me3
23
Q
How are TrxGs recruited to their target sites?
A
Two main ways by which TrxG complexes can be targeted to target sites:
- Interaction with unmethylated CpG rich sequences via Zinc-finger domains (MLL proteins have H3K3 methyltransferase activity)
- Interactions with transcription factors or polymerase associated factor 1 (PAF1)
24
Q
Outline the recuitment of TrxG complexes to target sites by HOTTIP
A
- HOTTIP ncRNA is expressed from the 5’ end of the HOXA cluster
- coordinates the activation of HoxA9-13 genes
- Acts as a scaffold to bring in active marks
- noncoding RNA interacting with adapter proteins
25
What is the experimental evidence that HOTTIP results in HoxA expression?
* Supression of HOTTIP by RNAi results in a reduction in HoxA expression (siGFP as control - wouldn't expect an effect)
* measured levels of HoxA9-13, HoxA7, HOTTIP by qRT-PCR
* See gradient of supression with highest of the genes most closely located to HOTTIP (A13)
26
What is the model for the activation of HoxA genes by HOTTIP?
1. HOTTIP interactions with WDR5 (trxG protein) [test by immunoprecipitating WDR5 and see is HOTTIP is assocated with western blot) to recruit MLL1
2. MLL1 is a H3K4 methyltransferase
3. H3K4me3 is an active histone mark
4. chromosome looping brings together HoxA9-13 and HOTTIP
5. Allows MLL1 to methylate cluster of nucleosomes
**Used chromatin capture confirmation**
1. look at which regions of the chromosome are close together
2. When HOTTIP is present, HoxA genes are clustered together
3. When HOTTIP supressed, lose the gathering
27
What are the characteristic features of stem cells?
1. capable of self renewal
2. capable of entering distinct differentiation pathways
28
What are the molecular signatures of 'stemness'
* Can compare transcipt profiles in ES cells, adult stem cells and commited cells
* transcription factors Oct4, SOX2 and Nanog are stem cell master regulators
29
What are the advantages/disadvantages of transcription profiling?
* can tell us what genes are on and off
* can't tell why a gene is off
* active repression vs. lack of activating proteins is not distinguished
30
How can the global chromatin status of ES cells be assessed?
* Immuno-labelling of chromatin proteins [antibodies against different characterisic chromatin marks and proteins]
* FRAP (fluorescent recovery after photobleaching)
* ChIP (chromatin immunoprecipitation) [look at specific protein-DNA interactions]
31
How did Meshoreer et all demonstrate the hyperdynamic plasticity of chromatin proteins in pluripotent ES cells?
Compared chromatin structure during ES cell differentiation
* ES cell -\> depletion of leukemia inhibitory factor (LIF) -\> neural progenitor cells
* Take ES cell and trigger differentiation into NPC by deplition of LIF
* Looked for specific marks between ES cells and NPC
* DAPI staining
* Oct4/Nestin (stem cell marker/NPC marker)
* HP1alpha (heterochromatin associated protein)
* Results
* Oct4 - expected to be on in ESC
* Nestin on in NPC
* HP1alpha - (binds H3K9me3 repressive mark) more in NPC, more closed chromatin
* Limitation: no quantification
* Also looked at distribution of HP1 amd H3K9me3
* see increase in repressive chromatin marks in NPC cells, during differentiation. More active state in ESC
* This was then quantified by:
* comparing area of H3K9me3 labelling: higher in NPC
* western blot of Oct4, H3K9, AcH3, AcH4, H3 of ESC, 24hrs, NPC
* Found: During differentiation H3K9 methylation mark increased, AcH3/4 (active) depressed
* Overall losing active marks and gaining represive marks
Suggests that chromatin is re-organised globally to a more repressive state as ES cells differentiate and lose their potency
32
How was the compacting of chromating during differentiation looked at using a more dynamic technique?
Used Fluoresence recovery after photobleaching (FRAP)
1. black region of cell with a laser, destroy protein of interest and label
2. Measure time taken for the fluoresent label to recover by the dynamic movement of fluoresence into that area
1. Did FRAP in cells expressing HP1a-GFP (binds inactive marks)
2. Measured time taken to recover in ESC, 24hr, NPC
3. in ESC the recovery is faster, suggesting that the architechural chromatin proteins are hyperdynamic and bind loosely to chromatin in ES cells
33
What are the differences between pluripotent and differentiated cells?
**Pluripotent cells**
* hyperdynamic, have loosely bound chromatin proteins
* not yet commited to any differentiation pathway
**During differentiation**
* Chromatin-remodelling (compacting), transcriptional silencing
**Differentiated cell**
* immobilised chromatin proteins
* fixed patterns of gene expression
34
What is the process of a genome-wide chromatin immunoprecipitation?
1. Looking for protein-DNA interactions in vivo
2. Treat cells with formaldehyde to crosslink proteins to DNA
3. Random shearing of DNA
4. Immunoprecipitate with an antibody to recognise the protein of interest which will also immunoprecipitat any linked DNA
5. Reverse crosslinking, purify and analyse immunoprecipitated DNA by:
1. Sequencing
2. qPCR detection with primers for candidate loci
3. Hybridisation on microarray
35
What are the results from genome-wide chromatin immunopreciptations of histone modifications in ES cells?
* found things not normally found in differentiated cells
* **Bivalent chromatin structures** in the promoters of some developmentally important genes (have both active and repressive marks):
* high levels of acetylated H3 and H4 - usually acssocaited with active trascription
* high levels of H3K4me3 - usually associated with active transcription
* high levels of H3K27me3 - associated with repressed chromatin
36
What are the differences in the chromatin profiles in ES vs. differentiated cells?
In stem cells the lineage specific genes are not expressed but in a 'poised' state ready for either activation or full repression.
Allows rapid response when differentiation is triggered by key transcription factors.
For example:
Early embryonic developmental regulator gene (e.g oct4) expressed in embryonic stem cell (active marks: H3K9ac, H3K4me) But has repressive marks added when differentiated (H3K27me)
Hematopoietic and Neural-associated developmental regulator gene (e.g. Ikaros/Math1) in a 'poised' stae in ES cell (expressing both active: H3K9ac, H3K4me; and repressive: H3K27me marks). In a lymphocyte, where the hematopeitic developnmental regulator should be on but the nerual-associated gene should not, these express just the appropraite active or repressive marks.
37
As PCR2 recognises H3K27me (present as a repressive mark in developmental genes in embryonic stem cells); are the polycomb group proteins important in maintaining stemness?
* PcG mutant stem cells can still self-renew and express Oct4, Sox2 and Nanog (do not normally have repressive marks)
* Lack of EED (mammalian PRC2 component) has been reported to be associated with spontaenous differentiation of ES cells (removal of the adder of repressive marks leading to differentation)
* Strong evidence that PcG proteins are required for the correct differentaition and lineage commitment - keep genes off that need to be off, allowing commitment to the correct pathway
38
How are trithorax group proteins involved in stem cell maintenance?
Assessed gene expression profiles by microarray
* looked at gene expression in stem cells when they differentiate (ES cells induced to differentiate with retinoic acid (RA))
* Looked at the expression pattern during differentiation, specifically looking for genes that were being turned off during differentiation
* Oct4 and Nanog go down as expected
* WDR5 (TrxG member) also goes down - thought to be required for stem cell maintenance
39
What happens if Wdr5 (thought to be involved in maintaining stem-ness like Oct4 and Nanog (stem-cell regulators)) levels are supressed in stem cells (as seen in differentiated cells)?
* WDR5 levels were supressed in stem cells using RNAi for Wdr5 using small hairpins (sh) which can direct the destruction of target mRNAs
* Also had 'scrambled' as control - shRNAs that do not correspond to Wdr5
* Suppressing WDR5 levels results in a global reduction in H3K4me3 (active mark)
* Looked at levels of (antibody staining in western blot):
* WDR5 (to confirm levels are reducd)
* H3K3me3 (overall active mark) - reduced
* H3 (loading control)
* Wdr5 involved in maintaining active marks in stem cells
40
What is the experimental evidence that WDR5 is required for stem cell self renewal (similar to oct4)?
* stem cell assay - plate up and look for colony formation when Oct4 and Wdr5 levels reduced by shRNA (against just the vector)
* Oct4 knock down stem cells do not self renew (measure CFU)
* WDR5 knock downs don't self renew
Indicates that trithorax acitivty is requred for SC maintenance and renewal
41
How does WDR5 act?
WDR5 coorperates with Oct4 (TF, stem cell master regulator) to mediate ES self revewal
* WDR5 interacts with Oct4 and acts on the Oct4 gene and various other targets in ES/iPS cells
* Helps to recruit thrithorax complex to these regions to lay down active marks and keep oct4 levels up
* if remove WDR5 lose the ability of oct4 to regulate its own expression and levels of oct4 drop dramatically
