BMS334 Epigenetics Flashcards
What are epigenetic mechanisms?
- Create molecular environment that shape accessibility of genes to transcription machinery
- Regulate levels of gene transcription and sensitivity to change by extrinsic DNA binding transcription factors
- Epigenetics do not change the underlying DNA sequence
What do epigenetic mechanisms regulate?
Epigenetic mechanisms regulate growth, development, and maintenance of physiological homeostasis across the life course
- Development and tissue homeostasis
- Adaptive and maladaptive responses to environmental factors
What molecules are involved in epigenetic mechanisms?
- DNA methylation of CpG dinucleotides
- Covalent Modifications of nucleosomal histones within chromatin
- Non-histone proteins that generate, recognise or remove DNA methylation, histone modifications
- Non-coding regulatory RNAs
Give an example of the epigenetic mechanism that directly acts on promotors or enhancers
- Steroid hormones e.g, ecdysone (potent regulators of gene transcription) bind to its TF receptor (ecdysone receptor) which undergoes a conformational change that allows entry to nucleus. It can then bind to recognised DNA and recruit proteins which stabilise the interaction between the RNA polymerase and the promotor regions of the ecdyson receptor
How does metylation of CpG dinucleotides result in gene regulation?
- Methyl group added to cytosine bases using DNA methyltransferases.
- Changes the interior surface of the major groove of DNA which changes the ability for TF’s to recognise the DNA surface
- It produces recruitment sites for DNA binding proteins to mask the major groove stopping direct contact to DNA. This blocks the ability of DNA to be recognised, read and decoded
How does the DNA sequence of promotor regions allow the regulation of gene expression?
Promotor sequences in genes are rich in these nucleotides. Whether a gene is active is often determined by the methylation state of these CpG nucleotides
How can the methylation of core histones regulate gene transcription?
In transcriptionally silent chromosomes, there are histones that are highly methylated. The combination of methylated histones and methylated cytosine residues shuts the locus down
How can the acetylation of core histones regulate gene transcription?
Transcriptionally active chromatin has a much more open structure (nucleosome free regions of DNA) and the nucleosomes are instead hyper acetylated. These negatively charged acetyl groups help the DNA to remain accessible to TFs by maintaining the chromatin in an uncondensed state
What part of the core histones are modified?
The N terminal tails of core histones are targets of a wide range of different histone modifications by enzymes that function as modification “writers”.
What is the role of histone Acetyltransferases?
Histone Acetyltransferases add acetyl groups to multiple lysines in the N-terminal tails of core histones
What is the role of Histone Methyltransferases?
Histone Methyltransferases add methyl groups to specific lysines or arginines in the N-terminal tails of core histones
What is the role of Histone kinases?
Histone kinases add phosphoryl groups to Serines / Threonines of core histones
How are histone modifications recognised?
Recognised by proteins with modification-specific binding domains – modification “readers”
What are acetylated histones recognised by?
Proteins with bromodomains
What are methylated histones recognised by?
Proteins with chromodomains
What are modification erasers?
If there are no proteins bound then these modifications can then be selectively removed
- For acetylation, the protein to remove it is histone deacetylases and for methylation, the enzyme is histone demethylases
How can long non coding RNAs regulate gene expression?
- Can be several KB in length and full of complex tertiary structures e.g. hair pin loops. These loops can interact with chromatin regulatory proteins.
- These RNA protein complexes can impact on the structure and function of the chromatin of which they are apart
Give an example of a long non coding RNA that regulates gene expression
For example, the X chromosome inactivation RNA called Xist. Has complicated hair pinned loops which allow interaction with proteins which allow selective shutting down of one of the two X chromosome’s in female cells
Give examples of short non coding RNAs
MicroRNA
PiRNA
How can Micro RNAs regulate gene expression?
Micro RNAs are present in the cytoplasm and interfere with the transaction of existing mRNA that is complementary to the micro RNA
- Can also lead to the destruction of the mRNA once hybridisation occurs
Where are piRNA found?
Nucleus
What is the role of piRNAs?
piRNAs have complementarity with nascent mRNAs. These RNAs encode transposable elements which controls the expression of transposable elements in the genome.
How do piRNAs control gene expression of transposable elements?
- They suppress the expression of transposable elements RNA through by recruitment of PIWI proteins to transposon loci and promote formation of transcriptionally silent heterochromatin
- They also act as nucleation signals for chromosome modification enzymes which shut the transposable mechanism down
- piRNA that are exported from the nucleus can also act in the cytoplasm to promote inhibition or degradation of transposable mRNA
What is meant by epigenetics ensuring a robust phenotype?
Epigenetic mechanisms guide development and ensure robust, stable phenotypes are produced through reliable mechanisms that regulate expression of the genome according to a predictable schedule.
What is meant by epigenetics ensuring plasticity?
Epigenetic mechanisms are also flexible and sensitive to physiological signals, which may originate within an organism or as a consequence of changes in the external environment
What kind of changes can the phenotypic plasticity of epigenetic mechanisms lead to?
This can either be beneficial changes or create capacities for the emergence of maladaptive chronic disease states
What are the two types of phenotypic plasticity?
Reaction norms
Polyphenisms
What are reaction norms in relation to phenotypic plasticity?
Reaction norms are ranges of phenotypic characteristics that are continuous and proportional to the environmental stimulus
Give an example of reaction norms in relation to phenotypic plasticity
Phenotypic Plasticity engenders beneficial adaptations that can improve fitness. Used resistance training to develop muscle hypertrophy. This is driven by PGC1alpha TF. The more resistance training, the more PGC1alpha, the more muscle hypertrophy
What did Ruas et al, 2012 discover about PGC1-alpha?
- PGC1-alpha has two regulatory elements with one being responsive to resistance training which leads to a form of PGC1 alpha that leads to hypertrophy.
- If over express this form of PGC1 alpha in mice then muscle size increases.
- If do different training e.g. running then there is a different form of PGC1alpha activated and increases the metabolic function of the muscle and allow it to function over long periods of time
- Saw that gene expression changes with muscle loading and unloading and saw the change in DNA methylation of the regulatory agents around these genes
What are polyphenisms?
Discrete transformation of phenotypic characteristics from one type to another in response to a threshold quantity of environmental factors being exceeded
- Switching between discrete alternative forms in response to changes in environmental stimulus
How are daphnia an example of polyphenisms?
- In response to a predator Daphnia change shape to give a spikey helmet to keep the predators away. The survival of these daphnia is much higher than those without.
- However, to get these helmets the daphnia must be in a predatory environment that release chemical stimuli and induce the change
How are butterflies an example of polyphenisms?
- When the animals develop in the dry season they are brown but if develop in the wet season then the animals have eye spots on their wings.
- This is linked to the production of levels of ecdysone. These eye spots are driven in an ecdysone dependant way by Distal-less TF expression.
- In wet season, there is much more Distal-less expression meaning that the eye spot phenotype is much stronger
How is epigeneitcs linked to healthy ageing in humans?
Human behaviours and environmental exposures modules the activities of epigenetic mechanisms and promote chronic diseases
What was the dutch famine?
- Rationed food, as little as 500 calories a day.
- The babies conceived during the last three months of the famine were born with a low birth weight and these people then had an increased chance of chronic diseases such as coronary heart disease and schizophrenia
How was the phenotype of children conceived in the critical period of the dutch famine explained by epigenetic mechanisms?
- Set of epigenetic modification revealed through analysis of blood samples
- Can see the pattern of methylation changes. If conceived in the critical period, there is an increased level of methylation in some loci and decreased in other loci in comparison to unexposed sibling. This shows that exposure to the famine induced long lasting epigenetic changes to the genome
How can the epigenetic phenotype of children conceived in the critical period of the dutch famine explained?
- This can be explained by the thrifty phenotype hypothesis: if developing under adverse conditions, metabolism attacks any nutritional sources available – this includes in laying down excess fat.
- The metabolism is set in the period of adversity and still does this even when adverse conditions have been removed
How are chromosomal regions organised in the nucleus?
- Chromosomal regions are condensed into distinct functional regions located on the periphery of the nucleus.
- When this is the case, the domains are full of transcriptionally silent genes
- Towards the centre, there are other domains that are associated with morphological structures that are defined by TFs e.g. RNA polymerase II.
- These are called chromosomal domains and are topologically organised
What kind of tools do we need in order to analyse the structure function relationships within the nucleus?
- Biochemical methods for defining and analysing structures e.g. Protein and nucleic acid purification and sequencing techniques
- Genetic techniques for perturbing gene function and linking biochemical changes to phenotypes – model organism studies. These allow us to define distinct functions of genes
- Computational tools for analysing genomic / epigenomic data. This has allowed the above two tools to be brought together – analyse the complex molecular interactions
What are the three main methods of detection of methylated DNA sequences and histone modifications?
- Treatment of genomic DNA with Restriction Enzymes that selectively recognise
and cleave DNA containing methylated OR unmethylated CpG dinucleotides - Chromatin immunoprecipitation with antibodies recognising specific covalent histone
modifications - Bisulfite sequencing analysis of genomic DNA to map and quantify
methylated CpG sites
What was the limitation of using resitiction enzymes that selectively recognised methylated or unmethylated CpG dinucleotides?
Was very limited as often focused on a specific gene or nucleotide pair - not genome wide
How can restriction enzymes be used to investigate specific DNA methylation?
- Restriction enzymes HpaII and MSP1 recognise CCGG
- However, HpaII will only cleave if there is no methylation at either C. MSP1 cleavage is blocked if the outer C is methylated but not the inner C. In mammalian genomes, methylation is limited to CG sites so MSP1 is affectively unaffected by methylation.
- Can therefore digest genomic DNA from different tissues with MSP1 and HpaII to create different fragments and create a southern blot
- Use probe specific for promotor of gene of interest
- If it is methylated, it will be cut by Msp1 (does not discriminate between modified CpGs) but it won’t be cut by HpaII (which does not cut methylated CpGs).
- The probe will reveal the length of the fragment and therefore whether it has been cleaved
- Using this approach allows mapping of distribution of methylated CpGs across a section of DNA providing there are specific probes
Give an experiment that used restriction enzymes to investigate the methylation of a certain gene
Lu and Davies, 1997
- Focused on the promotor of the Tissue Transgutaminase gene
- Msp1 digest of genomic DNA from the Hela cells gives a 6Kb fragment whereas HpaII gives a 9Kb fragment. Shows that the CCGG sequence is methylated in the hela cells as it is not being cleaved by HpaII
- If treat Hela cells with 5-AzaCytosine (inhibitor of DNA methyltransferases – prevents methylation from occurring). The fragment produced by HpaII disappears and the MspI fragment appears - Methylation was decreasing
- This method allows investigation of the abundance of methylation in specific locations
How can antibodies specific for covalent modifications be used to detect DNA and histone modifications?
MeDIP
- Used anti-5-methylcytosine antibody to select out fragments of nucleic acids that contain methylated CpG and leave behind the Unmethylated DNA.
Variations of this technique
- Chromatin immunoprecipitation with histone modification-specific antibody, e.g. anti-acetyl-histone antibody. Selectively isolate these modifications and then use large genome sequencing
- These can then be mapped back to the reference genome and the locations of the modified histones can be assigned to specific locations within the genome
How is MeDIP carried out?
- Purified genomic DNA is sheared randomly into fragments.
- Fragments are converted into a library with primer sequences attached for DNA sequencing
- DNA fragments are incubated with an antibody that binds specifically to 5-methylcytosine
- Antibody-DNA complexes are immunoprecipitated
- Each fragment of immunoprecipitated DNA is sequenced and the DNA sequences are mapped to the genome to identify the location of methylated CpGs
How can MeDIP be used to create a methylation map of the genome of that cell?
This technique is usually carried out on a tissue sample so will therefore contain multiple cells (each having two copies of each DNA sequence). Can count the number of times each fragment is sequenced to see how much a specific sequence is methylated in the tissue compared to another fragment. If a specific fragment is being sequenced a lot compared to others then can make a map of the methylation levels over the genome. The more times sequenced, the more methylation of that locus in that tissue sample
How can MeDip be used to investigate cancer?
MeDip gives a genome wide methylation profile
- Can then compare the methylation across the genome in different tissues e.g cancer cells v non cancer cells
- E.g Can see different methylation fluctuations between fibroblasts and colon cancer cell lines. Methylation is an indicator of silencing, applying that that cancer cells have genes that are active that aren’t active in the fibroblasts – these could be oncogenes
How can methylation profiles be use to predict gene transcription in different cell types?
e. g. Methylation profile of NF-kappa B
- Abundantly expressed in white blood cells and involved in regulation of inflammatory response and T cell activation
- Can compare the transcript abundance and methylation profile in different tissue types. Can see that there is similarities between the profiles. However, in memory T cells and B cells there is no methylation. This allows us to predict the transcription of NF–kappa B in different cell types
How is Bisulphite sequencing carried out?
- Treat genomic DNA with bisulphite. Bisulphite deaminates unmethylated cytosine to uracil. If methylated, there is no effect with bisulphite treatment and leaves the cytosine in tact. Where there is methylation, the cytosine is protected and will become an uracil.
- Use PCR to amplify these changes – during PCR uracil will be replaced with thymine.
- Can use this to measure the amounts of methylation in tissues – how many cells in complex tissues show high methylation across the genome
Why is Bisulphite sequencing the main technique used?
Because it gives a full account of methylation across the genome in different tissues and is less vulnerable to error as does not use antibodies which could vary in quality
Give an example of how Bisulphite sequencing can be used?
E.g. Hox cluster genes
- How the methylation patterns of the hox cluster changes during development
- Can see that there is low methylation in sperm and high in eggs. After fertilisation, the methylation is very high. This persists to the 256-cell stage (the maternal epigenetic phenotype).
- At the sphere stage and zygotic gene transcription occurs, the methylation decreases dramatically
What is Chromatin immunoprecipitation (ChiP)?
- Chromatin Immunoprecipitation (ChIP) requires an antibody that is specific for the histone modification or protein whose genomic distribution is of interest
- Uses the same approach as MeDip to instead carry out analysis of histone modifications
What is the chromocentre in drosophila?
Heterochromatin in Drosophila polytene chromosomes is gathered together around the chromocentre, which comprises salivary gland heterochromatin from each drosophila chromosome that is rich in the silent marker H3K9me2 and deficient in the active euchromatin marker H3K4me2
What has ChiP analysis showed about histone methylation in the chromocentre?
Can map the distribution of H3K4 and H3K9 methylation. Regions within the chromocentre, it is abundant in H3K9 methylation but at the boarders there is more H3K4 methylation. In the body of heterochromatin there is no H3K4 methylation
What is position effect variegation?
Position effect variegation is the variable silencing of a gene that is understood to be active in a particular cell type but becomes silent when the gene gets relocated by chromosomal relocation
Who discovered the position effect variegation phenomenon?
Muller 1930
How was the position effect variegation phenomenon discovered?
- Observed an unusual phenotype in which the eye was variegating with some patches of red and some white.
- Red pigment is encoded by gene called white. If mutated and silenced then the eye is white
- Inversion of polytene chromosomes so that one break-point is adjacent to the Wt and the other adjacent to the heterochromatin. This results in the Wt being translocated to the heterochromatin from euchromatin on the other chromosome resulting in its silencing. The eye therefore appears white. This is called position effect variegation
How can position effect variegation be used to screen for genes that regulate the properties of heterochromatin?
Can look for mutations that restore the eye phenotype or completely wipe out wt. This allows identification of Su(Var) (suppressors of variegation) and E(Var)(enhancers of variegation)
What Su(Var) was discovered through drosophila eye pigment screening?
Su(Var)3-9
- Encodes a histone H3K9 methyltransferase which defines the chromocentre. When it is mutated the white allele is no longer in heterochromatin
- If take Wt and mutant Su(Var)3-9 genes and run a western blot and probe with anti-H3K9me2. Can see that the Su(Var) mutations have no H3K9 methylation whereas the Wt does
What is Heterochromatin Protein 1 (HP1)?
Recognizes Su(VAR)3-9 –dependent H3K9-methylation
Give evidence that HP1 binds H3K9 and not H3K4?
- Create a biochemical column on which have peptides from Histone H3 which contain methylated lysine 9 or 4 and add radiolabelled HP1. Find that HP1 will bind to lysine 9 column and not lysine 4
- Can conclude that HP1 specifically binds to H3K9
What are heterochromatin and euchromatin?
Heterochromatin - Region of chromosomes that is densely packed - can’t be transcribed
Euchromatin - Region of chromosomes that is loosely packed - can be transcribed
Give evidence that HP1a is essential for the for restricted binding of Su(Var)3-9 to heterochromatin
If HP1a is not present then then Su(Var)3-9 is no longer primarily associated with heterochromatin but is also found along the chromosome arms in the euchromatin and causes ectopic H3K9 methylation
What did Riddle et al, 2011 show about HP1 and Su(Var)3-9 distribution?
- Compared distribution of HP1, Su(Var)3-9 and H3K9 methylation in chromatin. Can see that they are all colocalised to the same region of chromatin to each other (transcriptionally silenced)
- Activation markers (H3K4) and RNA polymerase II are excluded from this location and instead emerge at the boundaries of the heterochromatic region
How does HP1 form heterochromatin?
(Machida et al, 2018)
- HP1 exists as dimers
HP1 binds to methylated H3K9 residues in nearby nucleosomes using their chromodomain.
- This pulls two nucleosomes together and compacts them.
- This stops RNA polymerase II action and stops transcription and forms heterochromatin.
What is meant by chromatin accessibility?
The accessibility of chromatin to incoming factors such as DNA binding proteins
What do changes in chromatin accessibility result in?
Changes in chromatin accessibility are accompanied by dynamic changes in patterns of histone modifications, DNA binding protein interactions and transcriptional activity of genes within chromatin
What is DNAse1?
DNAse1 are non-specific endonucleases and will fragment the DNA, at DNAse hypersensitive sites, if they are relatively free and accessible
What is DNAse1 hypersensitive site mapping?
Uses DNAse1 to identify regions of DNA in chromosomes that are relatively accessible.
Outline DNAse1 hypersensitive site mapping
- Permeablise set of cells and isolate the nuclei so that they can take up DNAse1 which will fragment free DNA that it finds. It won’t be able to access DNA that is bound tightly to the nucleosome.
- Extract the fragmented DNA and digest with specific restriction enzymes for gene of interest. The two ends of specific DNA sequence has a BamH1 restriction enzyme cleavage site at each end
- Use DNA probe to detect the restriction fragment and run Sothern blot. The band will then be detected at a certain position on the blot.
- If the DNAse1 endonuclease has fragmented this gene fragment at a DNAse1 hypersensitive site then the band will now give a smaller reading. This occurs if accessible to DNAse1
Give an example of DNAse1 hypersensitive site mapping
- Can do this in different cell types to see the accessibility of the same gene in different cell types.
- For example, in fibroblasts, the beta globin cluster is not accessible as only the original band appears on the southern blot but in erythrocytes the beta globin cluster is accessible as a band of a lower molecular weight appears.
- This is because the transcription activator responsible for activating beta globin transcription is erythroid-specific and is not present in fibroblasts
What is the locus control region (LCR)?
An enhancer region that direct the developmental and tissue specific transcription of human globin genes
How is the LCR been able to be investigated using DNAse1 hypersensitive site mapping?
It is rich in DNAse hypersensitive sites e.g. HS4.
How was the LCR discovered?
DNAse1 hypersensitive site mapping
- In erythrocytes, there is a truncated BamH1 site which identifies a specific region in the chromatin near the beta globin gene that is sensitive to DNAse1 (accessible).
What did Grosveld et al, 1987 to investigate the role of LCR?
Took LCR DNA region and produced transgenic animals
- Do a northern blot to study the levels of transcription in different transgenic lines.
What did Grosveld et al, 1987 discover about the role of LCR?
The human b-globin LCR is therefore a super enhancer, conferring position independent, transgene copy number dependant transcriptional activity to multiple, linked transcription units
What is meant by LCR being position independent?
Usually, transgenes are position dependant as if they insert in a heterochromatin rich region of the chromosome the transgene will be silenced. However, the LCS acts as an enhancer that is so powerful that it is capable of overcoming the silencing power of heterochromatin so is therefore position independent
What is meant by LCR being transgenic copy number dependant?
If measure the number of copies of the transgene in the genome and relate it to the expression of the transgene then you see a high correlation between these number
If LCR is a powerful enhancer, why in its home location (erythrocytes) does it only regulate beta global genes?
Due to the presence of boundary elements which focus its action on beta globin genes
What is Micrococcal nuclease?
Micrococcal nuclease is a smaller enzyme then DNAse1 and can access regions of chromosomes that DNAse 1 may find difficult to access
How did Riddle et al, 2011 use micrococcal nuclease to decent heterochromatin in drosophila?
- Made a transgenic fly where the white gene has been added to a euchromatic location meaning that the eye is red and one where the white gene has been added to a heterochromatic region of chromatin so the eye is white (gene is inactive)
- Isolated the nuclei from the eye and digest with Micrococcal nuclease and do a southern blot to identify the white sequences on the transgene.
- The transgene from the euchromatic region shows a smear on the southern blot showing that the enzyme is breaking the fragment down into smaller pieces.
- In the heterochromatic region, the southern blot reveals a ladder where each band is one nucleosome length of DNA separate from each other. The Micrococcal nuclease is getting in between each nucleosome and can be used to detect heterochromatin
What is ATAC sequencing used for?
To look at the chromatin accessibility across the genome and compare between different cell types
How does ATAC sequencing work?
- It uses a transposon in bacteria that move from one location to another and wherever they insert themselves they create a duplicated sequence either side of the transposable element
- Can inject these transposons with the sequences attached into developing embryos or chromatin and they will look for accessible chromatin and insert. This will mark all the available chromatin with these tags. The DNA can then be extracted and sequenced and map the location of the tags. This will map all of the accessible chromatin across the genome
Give an example of ATAC sequencing?
Liu et al, 2017
- Chromatin accessibility in early zebrafish embryogenesis from 64 cell stage to post zygotic gene activation (ZGA) to look at the period of when gene transcription begins
- The ATAC sequencing shows peaks of ATAC sequencing accessibility that increase as gene transcription begins. The genome starts to open up as development increases
- DNA methylation changes accompany the increase in accessibility revealed by ATAC sequencing
How can we discovery which DNA sequences are close together in the 3-dimensional nucleus?
Chromatin conformation capture (3C)
How is Chromatin conformation capture (3C) carried out?
- Take chromatin from isolate nuclei and cross link the DNA to chromosomal components using formaldehyde which is a non-specific cross linker that will create bridges between the DNA and proteins.
- Then use nuclease digestion to fragment the DNA and will be left with doublets which are short pairs of DNA sequences held together by proteins.
- These are regions of the DNA that were close to one another in the nucleus. Can make a library of these sequences and can map back to the reference genome. Then ask which sequence is associated with another sequence in every experiment – pair wise comparison of sequence content
- Can use this information to create a 3D model of what a region should look like
What did 3C reveal about the structure of the beta global network
The model produced showed that there is a loop where the transcribed region is and at the neck of this region exists the locus control region. The stable the neck of the loop subsequently contacts each of the genes in the cluster in turn
Give evidence for the 3D model of beta globin
- There are peaks of high contact frequency that indicate that when that fragment was sequences so was the other sequences. This showed that sequences within the LCR there are sequences in close contact to a DNA sequence (3’ HS1).
What is CTCF?
There is a specific zinc finger transcription factor called CTCF that binds to HS5 and 3’ HS1 and defines the neck of the loop in the beta globin structure.
- It is a major organising factor for 3D structure
How can we look at the proximity of DNA sequenced across the whole genome?
2D heat map of pairwise contact between different regions of the locus
- The diagonal line in the middle shows that the blue sequences are always found with the blue sequences. Can use this to compare to look for regions that sometimes interact
- Can use this to characterise the pair wise interactions across a whole chromosome
- Look at diagram
What did Dixon et al, 2016 do?
Used 2D heat mapping to define interactions of a cluster of olfactory receptor genes in the mouse
What are Topologically Associating Domains (TADs)?
- Higher order organisational units of chromatin function within the nucleus.
- TADs identify the extent of a chromatin domain containing transcriptionally co-regulated genes. TAD ends identify boundary elements that insulate genes within TADs from the effects adjacent elements
How are chromatin loops made?
Hand-cuff model
- Two ends of a TAD are brought together in 3D space by CTCF proteins which bind to each boundary via the DNA motifs ad recruit cohesin.
- This encloses the promotor and enhancers of genes to insulate its effects
- This is supported as the cohesin complex generally co-localises with CTCF throughout the mammalian genome
- However, a problem is that the number of CTCF binding sites are much more abundant than the number of TAD boundaries. Therefore, why are TADs not more abundant?
See diagram
What did E.B Lewis (20th century) discover?
His studies lead to the identification of the genes within the Antennapedia and Bithorax complex which make up the genes within the Hox clusters.
- These genes are expressed in a nested fashion – some in the anterior/posterior. The domains overlap.
- There is a collinearity between the structural organisation of these complexes of these genes and their expression domains
What are sex combs?
Sex combs are a small appendage on the foreleg and used in mating
How did E.B Lewis discover polycomb genes?
- Scr mutants do not have these or reduced sex combs.
- Lewis found some other genes which were unlinked to the homeotic complex which gave the opposite phenotype. It resulted in sex combs in the first three legs rather than just the first pair.
- He mapped the mutations and they were elsewhere – not Hox gene complex
- This family became known as the Polycomb complex
What is the phenotype of a polycomb mutant fly?
The segmental character of each of the larval segments were transformed. They usually have a distinct pattern (denticle patterns) in the thoracic segments and abdominal. However, in the Polycomb mutants each segment resembles the most posterior segment (8th).
How can the phenotype of a polycomb mutant fly be explained?
The explanation of this was that each segment now expresses many Hox genes. Usually there are fewer Hox genes expressed the more anterior of the larva - global derepression of the Hox gene
What is the function of polycomb genes in flies usually?
To restrict the expression of the homeotic cluster anteriorly
How are polycomb proteins clustered?
Into two complexes
- PRC1
- PCR2
What is the function of the Polycomb complexes?
These complexes are involved in the establishment of transcriptionally repressed chromatin. When these complexes can’t work (due to one gene mutation) then the system falls apart and it results in derepression of the Hox gene cluster
Give evidence that hox genes are evolutionary conserved?
There is collinearity between Hox gene organisation and the expression domains in drosophila, mice and human embryos showing that they are evolutionary conserved
What is the function of Hox genes in vertebrates?
Pattern the vertebral column
- There are five domains of the vertebral column
What is the function of vertebrate polycomb genes?
Polycomb genes are involved in setting the expression of Hox genes in the vertebral column and spinal cord
What happens if mutate vertebrate polycomb genes?
If mutate vertebrate Polycomb genes then it leads to posterior transformations of vertebrate elements
Give evidence for the involvement of polycomb genes in vertebrates
Carried out study on two closely related homologues of drosophila gene polyhomeatic called Phc1 and Phc2 in the mouse.
- The cervical vertebrae in the vertebral column of both a Phc1 and Phc2 mutant mouse now has ribs coming out of the cervical column.
- The final cervical vertebrae has the characteristics of a thoracic one. There is also one less of pair of ribs in Phc1 mutants.
- The final segment of the vertebral column has been converted to a more posterior which do not have ribs
What makes up PRC2?
The PRC2 includes Ecz and the enhancer of zeste proteins
What makes up PRC1?
PCR1 contains Polycomb protein at its core
What are the vertebrate and C. Elegant orthologues of the enhancer of zeste?
It has an extended region of sequence similarity at the end of the C terminus including C. elegans genes such as MES2 and the vertebrate orthologue Enx1
What does the enhancer of zeste share homology to?
Has homology to Trithorax and a sequence from Su(var)3-9 (encodes a methyl transferase specific for lysine 9 methylation
What is the domain with high homology to Su(var)3-9 and trithorax in the enhancer of zeste?
STET
- Encodes the histone methyltransferase activity of all of these enzymes
What do the STET domains of Su(var)3-9, trithorax and the enhancer of zeste methylate?
The enhancer of zeste (in vertebrates Enx1) methylates Histone H3lysine27. Su(Var) does lysine 9 where as Trithorax methylates lysine 4
What did Cao et al, 2002 investigate?
The relationship between the enhancer of zeste and histone methylation
How did Cao et al, 2002 show if there was a protein present in a fraction that had methyl transferse activity?
- Drosophila cells were cultured and prepared a protein extract
- Separated the proteins in the extract into 100 different fractions and collected
- Incubated a sample of each fractions with H-labelled S-adenosyl methionine (H-SAM) and a purified histone H3 and then a ran a western blot with the incubated extracts. If the extracts contained a protein with histone methyltransferase activity then the purified histone H3 would be methylated with a radiolabelled methyl group from H-SAM.
- After incubation, the samples were electrophoresed by SDS-PAGE and auto radiographed to identify the fractions of the cell extract that were responsible for transferring methyl groups from H-SAM to histone H3.
- Some fractions (20-53) were able to transfer the methyl group to histone H3 showing they contain these proteins
How did Cao et al, 2002 compare the methylation ability of the fractions to the expression of the enhancer of zeste?
- Electrophoresed the samples from each fragment to create a western blot
- Then probed the western blot with antibodies for Polycomb proteins and then used a secondary antibody that was conjugated to Alkaline phosphatase (AP)
- Exposed the blot to autoradiography to detect the primary antibodies bound to their protein target
- Can see that the enhancer of zeste is colocalised to the methylated activity (the same fractions have methylation ability and that protein)
What did Cao et al, 2002 conclude?
- There is a correlation between the presence of enhancer of zeste and the methylating activity
- These two experiments suggest that PRC2 contains a histone methyltransferase activity
What did Bender et al, 2004 investigate?
if Mes2 (c. elegant orthologue of enhancer of zeste) is required for trimethylation of lysine 27
How did Bender et al, 2004 investigate the role of Mes2?
- Germ cells stained for nuclei and for trimethyl H3K27 (methylation mark)
- The fluorescence antibody for DNA was labelled with RFP and the methylation mark labelled with GFP. Can see that there is Colocalisation that appeared yellow signal. Indicates that the trimethyllysine is present where DNA is in the embryo
- In mutant of Mes2, there is no Colocalisation showing and appeared red showing that there is limited methylation mark being detected when there is no Mes2 present
What can Bender et al, 2004 conclude about Mes2?
Mes2 is required for trimethylation on lysine 27
What is Esc?
Esc is a beta propeller protein that produces an aromatic cage that the trimethylated lysine27 fits in.
How does PRC2 work?
- The enhancer of zeste is the catalytic core of the complex to create the trimethylation and Esc recognises the mark. This pair of activity allows chromatin to be progressively methylated.
- Enhancer of zeste methylates histone, the Esc then binds to this methylation mark and positions enhancer of zeste in the correct location to methylate the next histone H3 in the next nucleosome
- The enhancer of zeste protein is inactive without ESC showing the need for the whole PRC2 complex
How does PRC1 work?
- Recognises the trimethyl mark through the chromodomain
- Methylation on lysine 27 from PRC2, the Polycomb protein in PCR1 then sits in between these methylations and creates condensed chromatin
What are PREs?
Cis-regulatory elements called PREs (Polycomb response elements) which recruit PRCs to establish a domain of silenced chromatin around the Hox cluster.
Does PREs affect the transcription of adjacent genes?
Yes
- Transgenically label with LacZ reporter under the control of Ubx promotor which has a PRE next to it
- Compare transgene activity in wild type and Polycomb mutant backgrounds
- See expression in anterior domains but this restriction is lost in mutants that lack Polycomb function and instead appears everywhere. PcG binding to PREs therefore affects transcription of adjacent genes.
How do PREs interact with PRCs?
PREs bind a DNA binding component of the Polycomb family and through a series of interactions it recruits PRC2 which methylates and PRC1 which reads the methylations and condenses the chromatin
What are trithorax genes?
- Animals that have mutated Trithorax have reduced sex combs but are not linked to the Hox genes
- Promote Hox gene expression
How was the relationship between trithorax genes and polycomb proteins shown?-
- In the absence of Trithorax function, Polycomb is not needed. This was shown in a double mutant of Trithorax and Polycomb and the resultant fly looked wild type – normal sex comb expression.
- There is also wild type expression of Hox genes in these mutants but in the Polycomb mutants there is ectopic expression of Hox genes in the imaginal discs.
- Polycomb proteins must be blocking the activity of Trithorax in regions where Hox gene expression must be prevented
What is the relationship between trithorax and polycomb proteins?
Functionally antagonistic
What recognise H3K4 methylation by trithorax?
PHD fingers
How does sex determination lead to an imbalance of gene product?
Females and hermaphrodites have two X chromosomes whereas males have one X and one Y. In simpler organisms, males only have one X (X0). This creates an imbalance in the number of chromosomes and therefore gene expression
What are autosomes?
Not the sex chromosomes
What are Monosomies?
When only one chromosome form the pair is present
- embryonic lethal
What are trisomes?
When there is three chromosomes in a pair instead of two
- Also embryonic lethal with the exception of Down syndrome (trisomy 21)
What is dosage compensation?
The mechanism to equalize gene expression from X-chromosome in males and females (hermaphrodites) to stop lethality
What strategy do drosophila use for dosage compensation?
Make the only X-chromosome in males twice more active.
What strategy do mammals use for dosage compensation?
Make one X-chromosome in females (hermaphrodites) totally inactive.
What strategy do C. elegans use for dosage compensation?
Make both X-chromosomes in females (hermaphrodites) twice less active.
What is Sxl?
Dosage compensation factor in drosophila
- expressed in a sex specific manner
What are the promoters for Sxl?
Sxl has two promotors: SxlPe – the establishment promotor and SxlPm – the maintenance promotor
Explain what happens in dosage compensation of female drosophila
- Expression of Sxl splicing factor from SxlPe (establishment promoter) is activated early in development. Feedbacks to maintain its own expression.
- The present Sxl protein then promotes splicing and binds to PolyU in RNA so that translation terminating exon 3 is excluded. This results in production of functional Sxl protein which maintains its own expression and prevents translation of MSL2 protein by binding its 5’-UTR and 3’-UTR of its RNA.
What is MSL2?
Male-Specific Lethal – 2
What is the MSL complex?
- A group of proteins which bind together and is essential for dosage compensation and viability.
- It consists of MSL1 which is a scaffold protein, MSL2 which is a ubiquitin ligase, MSL3 which binds trimethylated lysine 36 on histone 3 (mark of active genes), MLE which is RNA DNA helicase and MOF which is acetylates lysine 16 on histone 4. roX are two long non-coding RNAs which are required to bring the complex together.
- Present on bodies of active genes on the X chromosomes in males
What are Polytene chromosomes?
The chromosomes found in salivary glands of drosophila larvae. This is very active tissue and produces a lot of protein. To allow this it undergoes multiple rounds of endoreplication without mitosis and sister chromatid separation. This allows them to be seen in light microscopy
Why were Polytene chromosomes useful when investigating MSL complex?
The condensed and non-condensed chromatin are easily see due to the dark and light bands. Can use antibody staining to see where proteins are localised – are they associated with condensed chromatin or active chromatin. Using this technique, can see that MSL3 is localised to the active X chromosome in males.
Give evidence for the involvement of MSL3 in the spreading of MSL complex
When MSL3 protein is not present, the complex forms and fails to spread. It localised in 150 distinct sites on the X chromosome instead of the whole chromosomes
How did Alekseyenko et al, 2008 investigate the recruitment of MSL complex to the X chromosome?
- Used MSL2 chromatin immunoprecipitation to see what MSL complex bound to
- Found that there was a motif enriched in the precipitated DNA suggesting that this is what the MSL complex bound to. When put this motif on other chromosomes it too recruited MSL complex showing it is sufficient for MSL recruitment
- Named this motif MRE (MSL recognition element). This is enriched in X chromosomes in the drosophila and includes known recruitment sites of roX and roX2 genes
Outline dosage compensation in male drosophila
- If there is no Sxl protein maintained from the SxlPe promotor then the terminating exon 3 is not spliced out leading to premature truncation of the protein.
- This means that the prevention of MSL2 translation does not occur so MSL2 is expressed and forms the MSL complex
How does the MSL complex spread along the X chromosome in male drosophila?
This is achieved by MSL3 binding trimethylated lysine 36 on Histone 3. roX also helps this an unknown mechanism
How does the MSL complex exert its action in male drosophila?
Increases gene activities by two-fold.
- MOF acetylates H4K16, which weakens a repressive internucleosomal structure to open the chromosome: DNA is more accessible for transcription
- Topoisomerase II is recruited by MSL to relax torsional stress of X-linked genes making more accessible
- MSL2 ubiquitinates H2B (only shown in vitro so far) and facilitates methylation H3K4 and H3K79 (human orthologue). These modifications important for transcription elongation
Give an example of when random X inactivation can be seen in mammals
X inactivation is a random event and is then inherited by all progeny of the cells. In female cats, there are patches of fur colour as the colour determinate gene is X linked so the X has been inactivated in different cells and then passed on to progeny.
What is Xic?
Xic is a single cis-acting master switch locus essential for silencing of X chromosome in cis (silences chromosomes where it is active) ensuring initiation of random inactivation.
What are Xist and Tsix?
Xist and Tsix encode non-coding RNAs which are complementary to each other, and the expression of one inactivates the other.
How is the expression of Xist and Tsix controlled?
Rnf12 encodes a ubiquitin E3 ligase and is a known Xist activator. It ubiquitinates and thus leads to degradation of the protein Rex1 which is found at promoter regions of both Xist and Tsix. This thought to simultaneously activate Xist and inactivate Tsix.
Outline the stochastic model of mama X chromosome inactivation
- Autosomes produce a signal which results in Tsix expression whereas the X chromosomes express X linked Rnf12 from Xic which promotes Xist expression. These two proteins compete against each other
- Stochastically, there is a probability that when there is a lot of Rnf12 produced (both X chromosomes producing it) then it will bind to Xist promotor and lead to inactivation of the one X chromosome.
- This will lead to the reduction of Rnf12 as only one X chromosome producing it.
- This means that the relative expression of Tsix from autosome will be higher than the Rnf12 so will reinforce the expression of Tsix in the other X chromosome.
- This stabilises the active X chromosome and cannot express Xist
Because X activation occurs stochastically what can occur in real life?
There are some cases where both X chromosomes remain inactive or become inactive in some cells. This results in the cells eventually dying or outcompeted by cells with one active chromosome
How is the X inactivation spread across the entire X chromosome and maintained?
- Xist encodes non-coding RNA which associates with inactivated X-chromosome and associates with mediating proteins, e.g. hnRNPU/SAFA and YY1.
- Spreading must involve booster elements that are enriched on X-chromosome as XIC translocation to autosomes results in poor spreading unlike on the X chromosome
- These could be L1 long-interspersed repeats (LINE-1). They are enriched on X, and there is a burst of LINE-1 expression from inactivated chromosome at the time of inactivation.
- Locally produced short RNAs might also facilitate spreading.
How is thought that the inactivated X chromosome represses gene expression?
- Inactivated X-chromosome has organization similar fashion to constitutive heterochromatin: depletion of active chromatin marks (acetylation, H3K4me2/me3), and enrichment of inactive chromatin marks (H3K27me3, H3K9me3, and methylated DNA including on housekeeping genes).
- It also recruits PRC2 (places H3K27me3 and H2AK119u1) which is Xist-dependent,
How is X chromosome inactivation ensured that it is passed on to the cells progeny?
Protein, Xist RNA, and chromatin marks are the same on interphase and metaphase chromosomes: maintaining the same chromosome inactivated once inactivation was established. This contributes to inheritance to ensure that the same X chromosome remains inactive
Is X inactivation a continuous process?
Yes
- Some events happen much later than other e.g. DNA methylation. It is a continuous process and doesn’t all occur at once
What are the sex differences in C. elegans?
Males
- X0
Hermaphrodites (no females)
- XX
What signalling elements are released from the X chromosome in C. elegans?
XSEs are signalling elements from the X chromosome
- sex-1 (signal element on X)
- fox-1 (feminizing gene on X)
- ceh-39
- sex-2
What signalling elements are released from autosomes in C. elegans?
ASE is the signalling element from the autosome
- sea-1
Outline dosage compensation in C. elegans hermaphrodites
- Four genes on XSEs cooperatively repress expression of xol-1.
- sea-1 on ASE encodes a transcription factor, which activates xol-1 expression.
- In hermaphrodites there is twice more products of XSEs, thus xol-1 is repressed.
- XOL-1 protein is a kinase and inhibits SDC-2, which is then present only in hermaphrodites.
- SDC-2 is a key regulator of Dosage Compensation Complex (DCC) assembly.
How does the Dosage Compensation Complex (DCC) lead to gene repression in C. elegans?
- All the genes in DCC are maternally supplied, and form a complex like condensin.
- The condensin complex is conserved in all eukaryotes and is essential for proper chromosome compaction and segregation during mitosis and meiosis.
- Because of the similarity between these complexes, it is also involved in partial condensation of the X chromosome therefore reducing its activity
How is DCC recruited to the X chromosome in C. elegans?
There are X chromosomal regions which recruit DCC suggesting containing DCC recruitment sites
- Csankovszki et al., 2004: Translocation of X-chromosome regions on autosomes. Was sufficient for DCC recruitment
- Further mapping using ChIP identified smaller regions, called rex sites, enriched in a 12-bp DNA sequence motif, which are necessary (though not sufficient) for DCC recruitment on X chromosome
How does DCC spread along the X chromosome in C. elegans?
It is not known how DCC spreads from initial recruitment sites. It accumulates along X chromosome, especially at the promoters of actively transcribed genes. DCC spreading is not dependent on any particular property of X-linked DNA sequences
How much does the DCC complex reduce the activity of both X chromosomes in C. elegan hermaphrodites?
By about 2-fold
Give evidence for DCC causing reduced X chromosome activity in C. elegans?
In XX DCC mutants 40% of expressed genes show an increase in transcript levels
How is it thought that DCC causes reduced gene expression in c. elegans?
Histone 4 lysine 20 methylation, associated with gene repression, is enriched on X chromosome in hermaphrodites and is dependent on functional DCC, leading to reduction of histone 4 lysine 16 acetylation on X chromosome. Histone 4 lysine 20 methylation is greatly increased on condensed chromosomes during mitosis suggesting it affects the chromosomal structure
Does DCC have any autosomal targets?
There is one autosomal target of DCC – the her-1 gene, repression of which promotes hermaphrodites sexual development
- However, DCC recruitment to her-1 differs from that to X-chromosome: it requires SDC-3 instead of SDC-2 and uses a different DNA sequence motif.
What is the general principle of dosage compensation across species?
- Express dosage compensation factors in sex-specific manner
- Recruitment to the X-chromosome
- Spreading along X-chromosome
- Modifying chromatin to alter gene transcription
What did Csankowszki et al, 2004 identify?
A 793 base pair fragment that functions in vivo as an X recognition element to recruit DCC
- The idea of there being a single recruitment site was eliminated by analysis of mutant hermaphrodites. DCC localised at both truncated X chromosomes showing there must be multiple recognition elements
- Found a 4.5kb fragment that strongly recruits DCC and can distinguish the X chromosome from autosomes
What are through to be mammalian PREs?
Hypomethalyated CGIs (where the C and G content exceeds 50%) represent PRE like sequences that can recruit Pcg and TrxG
What happens when PcG complexes bind to CGIs?
These PcG bound CGIs correspond to repressed promotors and inhibition of transcription induces recruitment of PcG proteins to newly silenced CGIs
What is the chromatin sampling model?
Chromatin sampling model says that PcG proteins weakly interact with all potential binding sites but their stable binding is blocked by active transcription or the presence of activating TFs
Why was it though that long non coding RNAs can also regulate the recruitment of PcG complexes?
Long non coding RNAs also regulate recruitment of PcG complexes. Xist (long non coding RNAs) target PcG to the inactive chromosome. This lead to research in other long non coding RNAs (e.g. HOTAIR which associates with PRC2) that could target PcG to HOX
How have PcG complexes been linked to cancers?
PcG can play an oncogenic role by modulating cell proliferation and senescence:
- PcG can cooperate with c-MYC to generate mouse lymphomas via direct silecning of the CDKN2a.
- Also found tht elevated enhancer of zeste in prostate tumours correlates with poor prognosis
- Missense mutation in EZhZ stops mono or di methylation of H3K27 but enhances trimethylation. This hypermethylation of H3K27 can act as a driver of many human cancers and can initate cell invasio and metastasis
How much of the genome encodes for proteins?
2% of genome encodes for proteins
What are the two types of non coding RNA?
Short (22-28 nt): miRNA, siRNA, piRNA;
Long (70nt – 118 kb): lncRNA, snRNA, tRNA, rRNA.
What is the function of short non coding RNAs?
Silencing expression (RNA interference) - RNAi is a natural cellular process that silences gene expression and plays important roles in gene regulation and innate defence against invading viruses.
What are the three types of short non coding RNA?
SiRNA - cleaves mRNA miRNA - inhibits mRNA translation piRNA - cleaves products of transposons in germ cells
How do siRNA and miRNA lead to gene silencing?
- miRNAs are transcribed as longer primary pri-miRNAs which can include other miRNAs and even protein encoding exons, in addition to spacer sequences.
- Pri-miRNAs are cleaved by the RNase III enzyme Drosha to generate a 60nt pre-miRNA that then exits the cytoplasm
- They are then processed by DICER to create two mature miRNAs that are differentially assembled into RISC
- siRNA would then assembles with Argonaute (AGO2) protein and other polypeptides to form the RISC complex. AGO2 has endonuclease function and then cleaves the complementary mRNA molecule to the siRNA and silences its expression.
- However, miRNA is not perfectly complementary to the mRNA so instead prevents translation of the mRNA through the GW182 component of the RISC complex. The exact mechanism that this occur by are still unclear.
Outline the differences between siRNA and miRNA?
- They differ in length and structure. siRNA are shorter consisting of 30-100nt with a 2bt 3’ overhang whereas miRNA have 70-100nt with interspersed mismatches and hairpin structure
- siRNA are complementary to mRNA whereas miRNA are only partially complementary to mRNA typically the 3’ UTR
- siRNA can only target one mRNA whereas miRNA can target over 100
- siRNA cleaves the target and miRNA transcriptionally represses or degrades the mRNA (rare as requires good complementarity)
What developmental process are miRNA involved in?
miRNA is involved in many developmental processes including metabolism, cell proliferation, apoptosis, neuronal cell fate, ect
How does the amount of processes miRNA is involved make it susceptible to cause diseases?
If the developmental processes are misregulated they would therefore be involved in multiple diseases e.g. cancer, cardiovascular disease, skin disease ect.
What is Fragile X syndrome?
- FXS is a genetic disorder linked to expansion of CGG trinucleotide repeats on X chromosome, which causes suppression of the fragile X mental retardation 1 (FXMR1) gene, and loss of fragile X mental retardation protein (FMRP). FMRP regulates neuronal connectivity and plasticity
- One of the major causes of autism and mental retardation in humans
How did Jin et al, 2004 show that miRNA is inclined with fragile X protein?
Immunoprecipitation of fragile X syndrome protein. Shows that drosophila and human protein precipitates with both miRNAs and miRNA precursors
Why is the drosophila eye good to investigate changing phenotypes?
Drosophila eye consist of ommatidia with each having 8 photoreceptors. This regular pattern in the eye is easy to see when not normal so can look for mutations
How did Jin et al, 2004 show that FMR1 (fragile X protein) and Ago1 (involved in miRNA silencing) act in the same pathway?
- Overexpression of drosophila Fmr1 protein lead to severe effects in retinal development. The same overexpression with also insertion of P element (drosophila natural transposon) which reduces Ago1 and the eye phenotype was normal.
- The reduction of Ago1 therefore rescues the phenotype of overexpression of Fmr1.
- They therefore interact within the same pathway.
- The rescue was shown to be due to reducing the amount of Ago1 by removing the insertion, using a transposon, to see if the Fm1 would lead to the severe phenotype. This lead to the restoration of Ago1 and overexpression of Fmr1 and the disordered eye phenotype (control)
What is the current model of Fragile X syndrome protein action?
Pre-mRNA precursor binds to RISC complex and is processed. It also associates to Fragile X syndrome protein and in a phosphorylation dependant manner helps with the binding and silencing of mRNA and gene translation.
Why does the current model of Fragile X syndrome protein action allow for a quick switch between mRNA expression?
When it FMR1 is phosphorylated, it is bound to mRNA targets and when it dephosphorylates it is unbound and can be immediately translated again.
- This provides a quick switch between mRNA expression without the need for change in gene expression and waiting for other processes
What are long non coding RNAs?
They can be very long (200nt-thousands nt)
Due to their length, they can have much more diverse functions than just gene silencing.
What are the functions of long non coding RNAs?
- Gene regulation in cis. Either by Transcription through a promoter blocks its function: transcriptional interference or recruiting histone-modifying enzymes in cis;
- Scaffolding: Provide scaffold to recruit proteins and organise functional complexes
- Decoy
- Silencing through complementation (similar to miRNA)
Why are long non coding RNAs used as a scaffold instead of proteins?
It is very cost efficient
- RNA scaffolds can be very long: a typical RNA “arm” of 50 nt extends for 13 nm, whereas a 50 aa alpha helix extends for 7.5 nm (and require 150 nt to be encoded).
- This is longer and uses less nucleotides and doesn’t need protein production
More versatile
- A 100 nt RNA could easily bind multiple proteins, whereas a 100 aa domain might bind a single protein partner.
