Epigenetics MOOC Sept 2015 Flashcards
LEC 1.2 MITOTIC HERITABILITY OF EPIGENETIC MARKS
Epigenetic control is important throughout development. Identical twins are genetically identical – sometimes epigenetically different? Identical twins can have different appearances (e.g. 30% discordant for height) Sometimes, genetically identical twins both carry a disease gene, but only one has the disease phenotype.
Definitions of epigenetics
Use of the term epigenetics and its definition has changed throughout history.
Conrad Waddington, 1942 – study of epigenesis; how genotypes give rise to phenotypes in development
Robin Holliday, 1990 - temporal and spatial control of gene activity during development of complex organisms.
Our current definition (similar to Art Riggs et al, 1996):
Epigenetics is the study of mitotically heritable changes in gene expression that occur without changes in DNA sequence.
Other current definitions do not include the necessity for the epigenetic changes to be mitotically heritable.
Mitotic heritability of epigenetic state helps to maintain cell identity. Heritability of epigenetic state ensures:
• the same sets of genes are expressed in daughter cells
• tissue homogeneity
Heritability is countered by periods when epigenetic marks are removed.
• Epigenetic reprogramming in germ cells and early development
• Active remodelling of epigenetic marks during differentiation.
LEC 1.3 CHROMATIN AND THE NUCLEOSOME
DNA is compacted into chromatin
DNA exists wrapped around histone proteins
• DNA + histones = chromatin
Formation of chromatin enables compaction of the DNA
• ~2 metres of DNA is packaged into the nucleus ~10 μm in diameter
Packaging of DNA into a compressed form causes problems of accessibility for transcription and DNA replication (and DNA repair)
• Tightness of chromatin compaction negatively correlates with transcriptional activity
• Tightly packed chromatin = DNA is less accessible to the transcriptional machinery (e.g. RNA Polymerase, transcription factors)
• Loosely packed chromatin = DNA is more accessible and transcription could ensue more readily
The nucleosome = DNA + histones
The nucleosome is the smallest unit of chromatin
• Nucleosome = 146 bp of DNA wrapped around a histone octamer (2 x H2A, 2 x H2B, 2 x H3, 2 x H4) linked by exterior histone H1
The nucleosome = DNA + histones
• DNA makes 1 ¾ turns around the histone
octamer
- Histones are positively charged (lysine and arginine rich)
- Positively charged histones bind to negatively charged DNA
- N-terminal histone tails protrude from the octamer
LEC 1.4 CHROMATIN COMPACTION
DNA packaging into chromatin: nucleosomes
DNA packaged around core histones (H2A, H2B, H3 and H4) to form nucleosomes, beads on a string.
DNA packaging into chromatin: 30 nm fibre
Nucleosomal chromatin is further packaged into 30nm fibre, dependent on histone H1 interactions.
DNA packaging into chromatin: interphase chromosome
30 nm fibre can attach to scaffold proteins, to further condense the chromosome.
This is how the chromosomes are found during interphase of the cell cycle i.e. when the cell is not dividing.
DNA packaging into chromatin: metaphase chromosome
By the addition of more scaffold proteins, the final metaphase chromosome is produced. This is required for segregation of chromosomes during cell division.
Heterochromatin versus euchromatin
Euchromatin – open chromatin, less stained.
Heterochromatin – closed chromatin, densely stained
• Facultative – can differ by cell type or time e.g. Tissue specific genes, inactive X chromosome
• Constitutive – same in all cell types; performs a structural role (centromeres, telomeres, portions of sex
chromosomes, mainly Y)
Heterochromatin has several functions
- Gene silencing
- Structural integrity of the genome
Different epigenetic marks are associated with euchromatin and heterochromatin
LEC 1.5 DNA METHYLATION AT CpG ISLANDS
SPECIFIC EPIGENETIC MODIFICATIONS
1. DNA methylation
Methylation of 5’ group of cytosines within CpG dinucleotides
2. Post-translational histone modifications
Methylation, ubiquitination, phosphorylation, sumoylation, acetylation of residues in the
N-terminal tails of histones
3. Chromatin remodelling
ATP dependent chromatin remodelling complexes shift nucleosomes
4. Histone variants
Histones with varying stabilities or specialist domains that alter the function of the nucleosome
5. Noncoding RNAs
piRNAs and other siRNAs that can direct epigenetic machinery
Long noncoding RNAs – may direct epigenetic enzymes to sites in the genome
DNA METHYLATION
Almost exclusively occurs at CpG dinucleotides in mammals
– Symmetrical, so able to be maintained through cell division
DNA methylation - how does it occur?
DNA methylation is laid down by de novo methyltransferases, DNMT3a and DNMT3b in mammals
DNA methylation – a stable epigenetic mark
DNA methylation is maintained by DNMT1.
Hemi-methylated DNA is the substrate for DNMT1.
DNA methylation at CpG islands
• CpGs clustered into CpG islands, often at promoters of genes
• CpG islands tend to be protected from methylation
• Methylation at CpG island = silencing of gene expression
– Small subset of CpG islands that are dynamically methylated between cell types, most retain unmethylated status
– Mainly studied for the inactive X chromosome
X inactivation – demonstrates mitotic heritability
of DNA methylation
X inactivation is an epigenetic dosage compensation mechanism in mammals, so that males and females have the same dose of genes on the X chromosome.
Random X inactivation occurs at gastrulation in the embryo, then this epigenetic state is mitotically inherited by all daughter cells. Inactive X chromosome shows DNA methylation of CpG islands.
How does DNA methylation lead to silencing?
-
meCpG in a CpG island is associated with the formation of a repressive chromatin structure (1o mechanism)
- meCpG can be bound by methylated CpG binding proteins e.g. MeCP1 & 2. MeCP proteins have a DNA binding domain and transcriptional repression domain. MeCP proteins can recruit other factors that condense the chromatin
-
meCpG can prohibit transcription factor binding, and alter gene expression (2o mechanism, probably for rare transcription factors,
when CpG-poor promoters).
LEC 1.6 DNA METHYLATION AT INTERGENIC REGIONS
DNA methylation – where does it occur?
- CpG islands – usually unmethylated
- Intergenic regions – usually methylated
- Repetitive elements – usually methylated
DNA methylation is mutagenic
• CpGs under-represented in the genome
– as methylated C is prone to deamination to T
What is the function of DNA methylation at
intergenic regions?
- Maintain genomic integrity?
- Dnmt1 null cells display genomic instability
- Silence cryptic transcription start sites or cryptic splice sites
- silencing of repeats to prevent transposition
- mutation of the repeats (meC to T) to prevent transposition
- silencing of repeats, so avoid transcriptional interference from strong promoters
- methylation of repeats may prevent illegitimate recombination
• Genome defense model (Prof. Timothy Bestor)
– DNA methylation is mutagenic, so must also be a benefit i.e. protect the genome from transposable elements
DNA methylation and cancer
• In cancer, historically the earliest epigenetic aberration found was a genomewide lack of methylation,
- hypomethylation at intergenic regions/ repeats, and genomic instability
• Feature found in all cancers ever studied, more consistently than genetic mutations!
DNA demethylation
- DNA methylation is mitotically heritable, and originally thought to be irremovable, except by failure to maintain methylation by DNMT1.
- DNA demethylation shown to occur in early development, in primordial germ cell development and at later specific stages of differentiation.
- Around year 2000, shown this could happen without DNA replication, so could also be an active process.
- Passive DNA demethylation
- Dilution of DNA methylation with every cell division, when DNMT1 is not expressed or not in the nucleus
- Active DNA demethylation
- Not simple removal of methyl group, as C-C bond is very difficult to remove
- Enzymatic removal via intermediates, using multiple different systems
- TET proteins main players, plus AID
Summary of DNA methylation
- Occurs at CpG dinucleotides
- Associated with gene silencing when found at promoters
- Helps to maintain genomic stability
- Laid down by DNA methyltransferases
- Mitotically heritable, due to features of DNMT1
- Can be removed passively, or actively which involves TET proteins
- Essential for viability, as DNMT knockouts die in utero
Heritability of DNA methylation
Epigenetics is the study of mitotically heritable changes in gene expression that occur without changes in DNA sequence.
DNA methylation is mitotically heritable because:
1. DNMT1 recognises hemi-methylated DNA and
restores methylation on both strands
- TET proteins involved in active demethylation
are only expressed at very restricted times in
development
LEC 2.1 INTRO TO HISTONE TAIL MODIFICATIONS
Post-translational histone modifications
Methylation, ubiquitination, phosphorylation, sumoylation, acetylation of residues in the N-terminal tails of histones
– Chemical modification of histone
– Acetylation and methylation are best characterised
– Ubiquitination, phosphorylation, sumoylation, ADP-ribsolyation, citrullination
Histone tail modifications
- Most modifications occur on the N-terminal tails that protrude from the nucleosome, accessible to other chromatin proteins
- >50 sites can be modified, some with more than one type of tag e.g. Ac or Me
- Predominantly on the tails of H3 and H4, fewer on tails of H2A and H2B
Combinatorial Histone Modifications
- Many different sites able to be modified -> a large number of combinations
- The functional outcome of the various combinations is called the “histone code”
- In theory, we may be able to map and understand the histone code, and infer outcome based on the histone marks
LEC 2.2 HISTONE ACETYLATION AND METHYLATION
HISTONE ACETYLATION
- Acetylation is correlated with gene activity (universally) - reduces positive charge of histones, neutralises positive lysines, decreases attraction between histones (+) and DNA (-), hence an electrostatic explanation
- Acetylated lysines act as docking sites for other proteins, e.g. bromodomain proteins, that themselves open the chromatin (chromatin remodellers) or recruit other proteins that do so.
- Histones are acetylated by histone acetyltransferases (HAT or KAT, since lysine is acetylated)
- Acetyl groups are removed by histone deacetylases
- HAT (KAT) – 18 different genes in mouse
- HDACs – 18 different genes in mouse
- Histone acetylation is arguably not an epigenetic modification, rather just a chromatin modification
- due to the rapid acetylation/ deacetylation dynamics (circadian rhythm)
- lack of mechanism for mitotic heritability
HISTONE METHYLATION
- Histone methylation does not alter the charge of the histone
- Histone methylation can correlate either with transcriptional activity OR with inactivity
- Mono, di and tri-methylation exist
- Three of the of best characterised histone methylation marks are H3K4me, H3K9me and H3K27me
H3K4me
- Associated with gene activity
- Found around promoter of active genes, so just in the region immediately neighbouring the transcription start site (right out on the tips of those tails of histone H3 because the residue is number three from the outside working their way in).
H3K9me
- Marks transcriptionally inactive regions, usually constitutive heterochromatin, broadly over the whole gene
- Laid down by specific lysine methyltransferases, KMTs, with specific activity for H3K9
- Removed by lysine demethylases, KDMs, with specific activity for H3K9me
- So if you have aberrant methylation at H3K9 or malfunctioning proteins that normally bind H3K9me, do you get genomic instability?
H3K27me
- transcriptionally inactive regions, more commonly at facultative heterochromatin, broadly over the whole gene - hence is involved in tissue-specific regulation of gene expression
- Laid down by specific lysine methyltransferases, KMTs (e.g. Ezh2, part of polycomb repressive complex 2, PRC2), with specificity for H3K27
- Removed by lysine demethylases KDMs, with specificity for H3K27me3
How do histone modifications influence chromatin structure?
- General rule: modified histone tails are “read” by other chromatin proteins; they act as docking sites for other epigenetic factors. Interacting proteins can have the ability to alter chromatin packaging (chromatin remodellers) OR recruit chromatin remodellers
-
Methylated lysine can be bound by several different domain types.
- Chromodomains
- MBT domains
- PHD domains
- Tudor domains
- Phosphorylated serine can be bound by the 14, 3, 3 domain.
- Different chromodomain proteins recognise methylated lysines in different contexts and with different functional outcomes. 3 examples of chromodomain proteins:
-
CHD1
- ATP-dependent chromatin remodeller
- CHD1’s chromodomain has high affinity for H3K4me
-
HP1 (heterochromatin protein 1)
- essential heterochromatin protein
- binds H3K9me3
- can recruit DNMT1
- can recruit HMT to maintain and spread the H3K9me3 mark
- DNMT1 can recruit HDAC
-
CBX2
- binds H3K27 methylation
- is part of another polycomb repressive complex, PRC1 (not PRC2)
- has it’s own enzymatic activity that lays down histone H2A Lysine 119 ubiquitination (H2AK119ub), another epigenetic mark
-
CHD1
Don’t understand the hierarchy of epigenetic modifications….
LEC 2.3 CHROMATIN REMODELLING
CHROMATIN REMODELLING
ATP-dependent epigenetic factors which use the energy from ATP hydrolysis to move the nucleosomes.
They do all the work:
- Bring about changes in chromatin compaction
- Make more densely or more sparsely packed - can either be activators or repressors
- Nucleosome eviction/ disassembly, e.g. around transcription start sites - so can completely remove nucleosomes, making DNA maximally accessible to trancription machinery (also uses histone chaperone proteins)
- Histone variant deposition
- Nucleosome turnover – role in stability of epigenetic marks (clearly, if the nucleosomes can be turned over, they can be refreshed or
renewed without relying on changes in the
cell cycle and this could alter mitotic heritability)
How? Disrupt DNA-histone interactions electrostatically, allowing movement of nucleosomes.
Three main types of multi-protein complex, each with specific binding domains and ATPases. Each of them have adifferent histone tail modification that they recognise:
-
SWI-SNF (SWItch/Sucrose Non Fermentable): contains a bromodomain, hence binds regions that are acetylated and therefore actively being
transcribed and in addition to this has of course the ATPase domain. - ISWI (Imitation SWI): contains a SANT domain; we don’t yet know exactly what a SANT domain recognises, except that it appears to recognise a modified histone tail. And, of course, also has the ATPase subunit
-
CHD (Chromo domain and Helicase-like Domain): Chromo domain recognises methylated histone
tail. And remember methylated histones can be
found in different contexts - heterochromatin or euchromatin. In addition to this chromo domain, they also contain a helicase domain, and helicases are normally involved in unwinding DNA at the site of transcription or the site of DNA replication.
Role of chromatin remodellers in development and disease:
- Almost all are essential for viability in mammals (knockout studies)
- Tissue-specific deletion demonstrates important roles in differentiation, when gene expression changes must be rapid
Interplay between chromatin remodellers and histone modifiers:
- Chromatin remodelling complexes recognise histone modifications (modified histone tail, ac or me)
- Some complexes have the capability to both modify histones and act as chromatin remodellers
- NURD – contains a HDAC protein to deacetylate histones and Chd3/4 protein for nucleosome remodelling
- ORDER and hierarchy?
- Does the chromatin remodelling happen first, and then a change in histone mark?
Or because the chromatin remodelling factors
recognise modified histones. Do the modifications to the histone tails happen first or do they happen simultaneously? It seems that probably they happen either simultaneously or the histone marks come first. - Histone modification and nucleosome repositioning may be sequential or simultaneous – likely varies according to circumstance.
- Does the chromatin remodelling happen first, and then a change in histone mark?
Open questions:
- Mitotic heritability
- Interplay between epigenetic marks
- Mechanism of silencing for various marks or combinations of marks
- What are the factors that make these marks, remove them or recognise them?
- How are the particular epigenetic factors or complexes recruited to specific sites?
LEC 2.5 HISTONE VARIANTS
Histones with varying stabilities or specialist domains that alter the function of the nucleosome
Histones with varying stabilities or specialist domains that alter the function of the nucleosome.
Different variants exist for histones H2A, H3 and H1.
Each variant has specific properties that differ from the canonical histones, useful for different functions e.g.
- Increased stability vs decreased stability
- Amino acids that can be modified such as Serine (can be phosphorylated), not found in canonical histones
- Others we don’t yet understand
Histone variant deposition
How are histone variants deposited?
- Using ATP-dependent chromatin remodelling proteins
- Can be replication dependent or independent i.e. does or doesn’t rely on cell division
Histone variants - variant H3
Centromeric histone variants – e.g. CENP-A (different names in other species)
- Centromere specific (centromere is the primary constriction of a metaphase chromosome)
- CENP-A binds at the centromere, and this is the same region there is a lot of repetitive DNA.
Used to be thought that it’s the repetitive DNA which defines the centromere - not the case.
Can form a centromere somewhere else. If you have a truncation which gets rid of the portion of the chromosome that would normally contain
the centromere, the remaining part sometimes will be able to form a neo-centromere, and this neo-centromere does not form necessarily where there’s repetitive DNA but rather is simply
defined by the CENP-A protein. Hence CENP-A really has some kind of a structural role. It does change the packaging of the DNA in the region, but it’s having a structural role in maintaining genomic stability.
Histone variants – variant H2A.X
H2A.X – involved in DNA repair
- Universal histone variant, highly conserved
- C terminal motif differs from canonical H3
- Has Serine at position 139, which can be phosphorylated
- H2A.X-ph, called γ-H2A.X, localised in double strand breaks (DSB), and is involved in DNA repair
- S139 phosphorylated by kinases at DSB
- γ-H2A.X recruits DNA repair proteins, including epigenetic factors that alter chromatin state at DSB
- Phosphatase cleaves phosphate group after repair is complete
- NB: this is a histone variant whose incorporation must be replication-independent, as you would not want a cell with damaged DNA to be transcribing or copying that DNA
Histone variants – variant macroH2A
MacroH2A – inactive X chromosome
- Found only in vertebrates
- Contains large 200 amino acid C terminal “macro” domain
- Enriched on the inactive X chromosome, chromosome made up of facultative heterochromatin
Histone variants summary
Many different variants, 3 we discussed
- Variants possess particular domains or amino acids that enable specific features
- Histone variants are known that are involved in structural aspects of the chromosome, DNA repair and transcriptional silencing or activation
LEC 2.7 NONCODING RNAs - microRNAs
piRNAs and other siRNAs that can direct epigenetic machinery
Long noncoding RNAs – may direct epigenetic enzymes to sites in the genome
Non-coding RNAs
Many classes of small, mid-sized and long noncoding RNAs, only some of which we will cover
- MicroRNAs (miRNAs) – post-transcriptional gene silencing - that is, they’re involved in halting
translation or integrating the messenger RNA so that you’ll get less protein product.
But they don’t go back and alter the
epigenetic state of the chromosome, unlike piRNAs and lncRNAs - Piwi-interacting RNAs (piRNAs) – control transposable elements and direct DNA methylation at transposable elements
- Long non-coding RNAs (lncRNAs) – appear to direct epigenetic machinery and establish different epigenetic states
miRNAs – post-transcriptional gene silencing
- Discovered in C. elegans and plants
- >1,000 miRNA genes in mammals
- Each mature miRNA (19-24bp) may target many genes
- Drosha and Dicer dependent
- Repression of translation or mRNA cleavage
- Involved in development, differentiation, cancer and disease
When microRNA genes are transcribed, pri-miRNA is produced. Pri-miRNA has a stem loop structure, and the stem loop structures are recognised by the Drosha enzyme which will then cut off that stem loop and have
it exported from the nucleus. When it’s exported from the nucleus it will be bound by a second enzyme called
Dicer. And once Dicer has processed these three pre-microRNAs now, then we have
a miR duplex. Or indeed a small interfering RNA duplex
or an siRNA duplex. These double-stranded RNAs are then loaded into the RISC complex, which is a complex of proteins. At this point there are two possible outcomes:
1) if there are some mismatches between the
sequence that’s found in this MicroRNA, and a messenger RNA, then it can bind with this mismatch and this actually leads to translational repression. So it will decrease the likelihood that the messenger RNA will actually be translated into its protein product. And this is what mostly happens in animal cells. So this is the primary function, the endogenous function of MicroRNAs.
2) Whereas, in plants, what primarily happens is that you get mRNA cleavage. So the microRNA is incorporated
into the RISC complex, however, this time, if there’s perfect homology between the microRNA and the messenger RNA, the target is cleaved -> no protein can be produced. So in the lab, iff we design a microRNA to have perfect identity with a particular gene of interest, then it tends to go through this type of
mechanism. .
Using miRNAs for research purposes
- Commonly used for experimental knockdown/ RNAi in many labs around the world
- Which genes are involved in epigenetic modification of the genome, in different scenarios? We use microRNAs engineered against each of the ~1000 epigenetic modifiers to reduce the expression of each, and test their roles in different cell scenarios.
