Lecture 8 - Epigenetics
PHD (plant homeodomain) fingers
- Zinc fingers that specifically can read both unmodified and methylated lysines. - The discovery of modules that can specifically read unmodified lysines represents an expansion of the histone code.
Binding of methylated lysines by Royal-superfamily domains
Common fold, but different domains have different specificities.
Mutually exclusive binding of histones and RNA polymerase to promoters
For transcription to start, histone proteins must be displaced.
Chromatin remodelling
• Large remodelling complexes displace the histone octamer from chromatin, using energy provided by ATP. • Such remodelling allows binding of transcription factors and the RNA polymerase. • Several different types of complexes.
Role of HP-1 in heterochromatin formation
• The chromodomain of HP-1 binds specifically to histone H3 trimethylated at Lys9 (H3K9me3). • Additional HP-1 molecules are recruited through interaction between the HP-1 molecules ("self-aggregation").
Histone modifications
• The flexible tails of histones are heavily post-translationally modified. • Primarily lysines, arginines, and serines are modifed. • The modifications include acetylation, methylation, phosphorylation and ubiquitination.
The histone code concept
• The histone code: It has been proposed that the combination of different modifications at histone tails represents a code that, together with the DNA sequence and methylation status, determines chromatin structure and transcriptional activity. • This hypothesis has gained increased support from the discovery (2004) that histone lysine methylation is reversible, and that many histone demethylases exist. • Many histone modifications ("marks") appear to have specific "meanings": see Figure.
Reversal of cytosine methylation in plants
• The proteins ROS1 (Repressor of silencing) and Demeter have both been shown to be active 5-methylcytosine DNA glycosylases. • Both these proteins have been shown to prevent (or reverse) gene inactivation in plants.
Epigenetics classic definition
"Heritable changes in phenotype or gene expression caused by mechanisms other than changes in the underlying DNA sequence". Includes: Prion proteins Excludes: Transient (occurring within one cell cycle) changes in chromatin structure. Problem: The chromatin alterations that are responsible for heritable changes, also occur transiently within the cell.
Epigenetics alternative definition (Adrian Bird)
"The structural adaptation of chromosomal regions so as to register, signal or perpetuate altered activity states". Excludes: Prion proteins Includes: Transient changes in chromatin structure. This definition better covers the current, practical use of the word "epigenetics".
Epigenetic inheritance - an example
- The protein "Agouti" regulates fur colour of mice. - Mice with identical sequence of the Agouti gene can have different fur colour. - This is due to epigenetic modifications which determine how much is produced of the Agouti protein. - These epigenetic modifications are inherited maternally (from the mother).
Binding of acetylated lysine by bromodomains
Different bromodomains have different specificities.
X chromosome dosage compensation in different animals
Different mechanisms, but similar expression from male and female X chromosomes is achieved.
DNA cytosine methyltransferases in mammalian cells
Dnmt1: Maintenance methylation Dnmt3A/B: De novo methylation
Epigenetic modifications of chromatin
Epigenetic modifications of chromatin encompass DNA methylation and various chemical modifications on the histone proteins (primarily their tails). Epigenetic modifications have been proposed to constitute an epigenetic "code" that governs chromatin state and gene expression.
Interplay between chromatin modifying enzymes
Mechanism 1: • A lysine specific demethylase KDM1B is required for imprinting of genes in mouse oocytes. • KDM1B removes (activating) methyl marks at H3K4. • Unmethylated H3K4 is recognized by DNMT3L. • The "de novo" methyltransferas DNMT3A is recruited to the histone tails together with DNMT3L, leading to DNA methylation. Mechanism 2: • Methylated H3K9 recruits HP1 to chromatin. • The DNA methyltransferase DNMT1 is then recruited by binding to HP1, leading to DNA methylation.
How does DNA methylation repress gene-expression?
Two mechanisms: - Methylation directly inhibits the association of transcription factors with DNA. - Many proteins have methyl-CpG-binding domains (MBDs), and such proteins may recruit additional proteins e. g. histone deacetylases to the methylated DNA.
What causes the effects of histone modifications?
Two possibilities: 1. The modification directly alters the chromatin structure. 2. The modification creates (or removes) a binding site for an effector protein. Both appear to be true (!): 1. Phosphorylation and acetylation appear to attenuate the attraction between positively charged (Lys- and Arg-rich) histone tails and negatively charged DNA. 2. Several protein domains that specifically recognize modified (and unmodified) residues in histones have been discovered.
Histone recognising domains (modules)
- Numerous histone recognising domains (modules) exist. - Proteins may contain more than one "reader" domain and thusly are targeted to specific combinations of modifications.
Reversal of 5-meC methylation in mammalian cells - a classic example
- Rapid and active demethylation of sperm DNA upon fertilisation. - Demethylation of oocyte DNA involves slow, passive process (dilution by replication without remethylation).
Lysine demethylation by JmjC-containing proteins
- Reversal of methylation at H3-K9, H3K27, and H3-K36. - Reverses both mono-, di-, and trimethylated lysines. - Identical to the AlkB mechanism for DNA/RNA repair. - Large family of several proteins with different specificities.
Active DNA demethylation in mammals
• Many different types of enzymes have been implicated: - Tet proteins: Hydroxylation of 5-meC (to 5hmC, 5fC and 5caC). - Deaminases (AID or APOBEC proteins): Deamination of 5meC to T, or 5hmC to 5hmU. - DNA glycosylases (TDG or SMUG1): Excision of 5meC, T, 5caC or 5hmU. • Several different models proposed.
Protein complexes involved in modifying chromatin
• Many proteins that modify chromatin are part of large complexes. • Polycomb (PcG) and Trithorax (Trx) complexes were first discovered in Drosophila (as mutants). • Polycomb complexes: - Repressive effect on transcription - Have H3K27 methylase activity (repressive mark) - Bind to specific sequences, Polycomb response elements, (PREs) - Polycomb (Pc-G) complexes maintain chromatin in a repressed state • Trithorax complexes: - Activating effect on transcription - Have H3K4 methylase activity (activating mark) - Can also bind to PREs • Relative activity of Polycomb vs Tritorax complexes may determine transcriptional activity.
Histone demethylation
- Histone methylation was for a long time thought to be irreversible, since the methyl groups on histones showed a turnover time similar to that of the histone proteins themselves. - However, this view has changed dramatically due to the discovery (2004-2006) of two different classes of demethylases: • Amine oxidases (LSD1) • Oxidative demethylases (JmjC-containing proteins)
Breakthrough in 2009: Discovery of the new epigenetic modification 5-hydroxymethylcytosine (5hmC)
- A new enzyme, TET1, was found to hydroxylate 5-meC to 5-hmC. - Two similar enzymes, TET2 and TET3 exist. - 5hmC was found to be abundant in the brain, especially in Purkinje neurons. - 5-meC and 5hmC indistinguishable by the most popular methods for studying DNA methylation. - 5hmC: "The sixth DNA base". Questions: - Is 5-hmC a new epigenetic mark? - Is 5-hmC an intermediate in the demethylation of 5-meC.? - Involvement of 5meC hydroxylation in apparent demethylation in the zygote. - Further oxidation by Tet enzymes add to the complexity
Epigenetic modifications can be influenced by the environment
- Monozygotic twins (identical DNA sequence) are quite simililar with respect to epigenetic modifications early in life, but the differences increase with age - Many examples that monozygotic twins do not develop identical heritable diseases, e. g. schizofrenia. This may be due to differences in epigenetic modifications. - Comparison of monozygotic twins with respect toDNA methylation: Much larger differences for older (50 years) than for young (3 years) twins (see figure). Yellow color: Small difference between twins in pair. Green/red color: Large difference
Oxidation of 5hmC by Tet enzymes
1. Tet proteins (Tet1, Tet2, Tet3) can further oxidize 5hmC into 5-formylcytosine (5fC) and 5-carboxycytosine (5caC). 2. 5fC and 5caC are present in mammalian genomic DNA. (especially in ES cells; E14) 3. Thymine DNA glycosylase (TDG) can excise 5caC from DNA.
N6-methyladenosine (6mA) - a new DNA modification
• 2015: - Three studies showed that 6mA is present in the genomes of fruitfly (Drosophila), worms (C. elegans) and green algae (chlamydomonas). - Responsible methyltransferases and demethylases were identified. - 6mA also found in vertebrate genomes. - 6mA associated with transcriptional start sites in Chlamydomonas (but not in vertebrates).
Recruitment of remodelling complexes to chromatin
• A sequence-specific factor (activator or repressor) binds to DNA. • The remodelling complex is recruited through binding to this factor, leading to octamer displacement. • Possbly a hit-and-run mechanism; the factor leaves after the complex has been recruited.
Lysine demethylation by amine oxidation
- Reported that LSD1 (lysine specific demethylase 1) demethylates lysine 4 of histone H3 (K4-H3). - It was the first true histone demethylase to be discovered! - LSD1 is a two-step mechanism. LSD1 has dual specificityas it can act both as corepressor and coactivator: • When LSD1 is part of the Co-REST complex: Methylation at H3K4 (an activating mark) is removed, leading to transcriptional repression. • When LSD1 is involved in androgen receptor (AR) signalling: Methylation at H3K9 (a repressive mark) is removed, leading to transcriptional activation.
Epigenetic's - some interesting questions/problems
1. Epigenetic modifications can be inherited 2. Epigenetic modifications are influenced by environment and life-style This gives some interesting questions/problems: - Do we inherit from our parents not only their genes, but also effects of the lifes they have lived (life-style, traumas, diseases) ? - Many inheritable diseases are only partially heritable, maybe are epigenetic modifications determining whether disease develops or not? - Monozygotic twins have previously been considered to have identical genetic material. This should probably be reconsidered (differences in inheritable, epigenetic modifications)
Histone modifications - some main points
• A number of different enzymes introduce specific marks (methylation, phosphorylation, acetylation, etc.) to the histone tails. • Other enzymes can remove the same marks (opportunity for regulation). • Many domains ("readers") exist that recognise and bind to specific histone modifications. • Many of the enzymes that add or remove the modifications also contain reader domains (the presence of one modification may lead to the introduction of another; "crosstalk" between modifications).
Histone acetylation
• Acetylation of histone proteins is performed by specific histone acetylases (HATs). • Acetylation of histones in chromatin leads to transcriptional activation. • Acetylation of histones is also important for assembly of nucleosomes. • Acetylation of histones can be reversed by specific histone deacetylases (HDACs). • Deacetylation of histones leads to transcriptional repression.
Position effect variegation
• An epigenetic effect; Genetically identical cells have a different phenotype. • In Drosophila: - An active white locus gives red eye color, while an inactive locus gives white color. - If this locus is placed close to a heterochromatic region, the degree of inactivation may vary between individual cells, due to different spreading of the heterochromatin. - The result is a position effect variegation; patches of both red and white eye color. • Heterochromatin = orange • Euchromatin = green
X chromosome inactivation in mammals
• Background for discovery (Lyon, 1961): - Female mice that are heterozygous for an X-linked coat-color mutation show a variegated phenotype, i. e. only some areas have the mutant coat color. • Explanation: - One X chromosome is inactivated at random early in development, in a small population of precursor cells. - Cells in which the mutated X chromosome have been inactivated will give rise to spots of wild-type color. - Conversely, cells in which the wild-type X-chromosome has been inactivated will give rise to spots of mutant color. • The mammalian inactive X chromosome - a prime example of facultative heterochromatin. - Two identical sequences where one is transcriptionally active and the other is heterochromatinised.
Novel tools allow studies of the chromatin landscape on a genome-wide scale
• Binding sites for DNA-binding proteins (e.g. transcription factors) • Histone modifications • (Nucleosome positioning) • The ENCODE project has generated extensive data on chromatin landscapes
Histone acetylation and coactivators
• Coactivators: Proteins that associate with transcriptional activators (hormone receptors, transcription factors), and contribute to transcriptional activation. • Many such coactivators, e. g CBP/p300 and PCAF, have histone acetylase activity. • Conversely: Enzymes with the opposing activity, histone deacetylases, are often associated with repressors.
Maintainence (perpetuation) methylation
• DNA methylation patterns must be actively maintained not to be diluted out. • Maintainence (perpetuation) methylation propagates methylation patterns as DNA replicates. • Lack of maintenance methylation leads to loss of methylation by dilution.
Several mechanisms contribute to DNA methylation patterns.
• De novo methylation: - When a previously unmethylated site becomes methylated. • Maintenance (perpetuation) methylation: - Mechanism responsible for maintaining methylation patterns as cells divide; the hemi-methylated DNA resulting from DNA replication is converted to fully methylated DNA. • DNA demethylation: -DNA is also thought to become actively demethylated (mechanisms somewhat obscure/disputed).
Epigenomic studies of histone lysine methylation
• Discovery of histone demethylases (and the dynamic nature of histone lysine methylation) has been parallelled by the development of technologies for studying methylation genome wide (e.g. ChIP-Seq). • Many studies how on the epigenome changes during various processes (e.g. development, stress, cancer etc.). • Example: Epigenomic changes during development of embryonic stem (ES) cells into neural precursor cells (NPCs).
How are epigenetic marks transmitted at cell division?
• Example on acetylation. • "Old", modified histones are distributed randomly at replication, and mixed with newly synthesised histones. • The modification status of the old histones is, somehow, transmitted to the new histones (the mechanism is still unknown).
X chromosome dosage compensation
• Females have two X chromosomes, wheras males have only one. • A similar gene expression level from each of the X chromosomes would give a much higher expression of X-linked genes in females. • Mechanisms exist to prevent the different "gene dosage" between males and females to result in different expression levels; dosage compensation. • Different animals have very different mechansims for achieving this.
Imprinted genes
• For some genes, only the allele (copy) which has been inherited from one parent will be expressed. • This phenomenon is due to different methylation of the two alleles. • Some genes (e.g H19) are inactive when inherited from the father, others (e. g. Igf2) are active when inherited from the mother. Imprinted genes - recent knowledge: • Far more genes than previously thought are imprinted, or at least show an expression bias, depending on the origin of the gene (paternal or maternal). • Imprinting can be highly tissue specific. Example: Mouse that only had one copy (maternal or paternal) of the Grb10 gene were studied. - Maternal copy expressed throughout entire body, paternal copy expressed in brain. - The mice that lacked the paternal copy were "socially dominant". - The mice that lacked the maternal copy had a higher body weight than normal. - Conclusion: An imprinted gene may be expressed in very different parts of the body, and may give rise to different traits, depending on the parental origin.
Active cytosine demethylation in mammals
• Has during the last 10-15 years been of the hottest, as well as most competitive and controversial, topics in biology. • Several processes have been described, where active DNA demethylation appears to be involved. • Several articles have been published in very high impact journals during the last two decades, describing different mechanisms for demethylation, but many of these studies have been problematic.
Histone modifying enzymes
• High degree of complexity. • Several different enzymes exist that perform the same function. • The same enzyme is likely to have different names in different organisms. • One enzyme may have several, alternative names in different organisms. • Solution: see figure. - K-Demethylases (KDMs; Formerly Lysine Demethylases) - K-Acetyltransferases (KATs; Formerly Acetyltransferases) - K-Methyltransferases (KMTs; Formerly Lysine Methyltransferases)
DNA methylation (5-methylcytosine) in gene regulation
• Important mediator of gene regulation in vertebrates and flowering plants. • Methylation is associated with repression of transcription. • DNA methylation is an important step in the formation of heterochromatin. • DNA methylation is important in mediating imprinting, i. e. the silencing of one of the two alleles of a gene. • In mammals: Methylation at CG dinucleotides, referred to as CpG.
"Conflicting" (bivalent) chromatin marks
• In embryonic stem cells, the promoters of some genes have both repressive (H3K27me) and activating (H3K9Ac, H3K4me) marks. • Such bivalent modifications found on genes encoding highly conserved, tissue specific transcription factors. • These genes are "poised" for transcription (both "brake" and "gas pedal" on).
Reversal of DNA methylation
• Many epigenetics marks (e. g. histone methylation, acetylation, and phosphorylation) are reversible by an active mechanism. • Active vs. passive mechanism: - Passive: New methylations are not provided after replication, leading to net reduction in modification level. - Active: A specific enzyme removes modification • What about DNA methylation? Active reversal? • Vertebrates: Reversal appears to occur, but the mechanisms involved still remain somewhat elusive. • Plants (Arabidopsis): Enzymes capable of demethylation have been characterized, and they have been shown to mediate gene activation.
The chemistry of histone modifications
• Methylation of lysines and arginines: Charge remains unaltered (positive). • Acetylation of lysine: Charge altered (from positive to neutral), may decrease interaction of histone tails with DNA (negative charge). • Phosphorylation of serine: Charge altered (from neutral to negative).
Histone methylation
• Occurs at Lys and Arg residues. • Can both give activation and repression, depending on the position in the histone tail. • Typical activating mark: Methylation of Lys in position 4 of histone H3 (H3K4). • Typical repressive marks: Methylation at H3K9 and H3K27. • Introduced by specific methyltransferases. • Several specialized enzymes are involved in histone methylation. • Most methyltransferases involved in lysine methylation of histones have a SETdomain (named after the founding members Su(var3-9), E(z), Trx). • There are three types of methylated Lys and Arg (see figure).
Histone phosphorylation
• Occurs on H1 at mitosis. • Occurs at Ser10 in H3 on connection with gene activation. • Upon double strand breaks in DNA, the histone variant H2AX is phosphorylated, and associates with the breaks (previous lecture). • Example: The JIL-1 kinase in flies phosphorylates H3S10, and disruption of JIL-1 causes chromatin condensation, suggesting that this phosphorylation is required to keep the chromosome in an open conformation.
CpG islands
• Often found in the promoter region of housekeeping genes in vertebrates. • High frequency of CpGs (relative to rest of the genome). • 0.4 - 3 kb long, rich in G + C (>55 %). • CpG islands display a low level of CpG methylation (compared to rest of genome). • The dinucleotide CG is underrepresented in the human genome, why? - C is prone to deamination, giving U. - U in genome is easily removed by uracil glycosylase. - Deamination of meC will give T, a normal base, and mutations may form.
X chromosome inactivation in mammals - mechanism
• One locus on the X chromosome, the Xic (X-inactivation center), is sufficient to give inactivation. • The Xic expresses a non-coding RNA, Xist. • Prior to inactivation both copies of X express Xist, which is rapidly degraded. • At the time of inactivation, an antisense RNA, Tsix, is expressed from the future active X-chromosome, leading to repression of Xist expression from this chromosome. • Xist from the future inactive X-chromosome is stabilized, and covers the chromosome. • X chromosome inactivation by Xist is followed by additional chromatin modifications: - Histone deacetylation, ubiquitination,(de)methylation - DNA methylation
Types of chromatin remodelling
• Sliding -Alters the position of the DNA sequence on the nucleosomal surface • Space adjustment -Alters the spacing between nucleosomes • Displacement of histone octamer -One ore more histone octamers completely removed from DNA
Suppressors and enhancers of position effect variegation
• Su(var) (suppressors of variegation) mutants are defective for proteins involved in propagating the heterochromatic state, i.e. histone deacetylases and methylases. • E(var) (enhancers of variegation) mutants are defect in proteins needed to activate transcription, i.e. histone acetylases and components of chromatin remodeling complexes(SWI/SNF). Relationship between position effect variegation and proteins acting on chromatin: • Su(var) 3-9 affects gene encoding histone methyl transferase SUV39H1, which is responsible for methylation at H3K9. • Su(var) 2-5 affects heterochromatin protein 1 (HP-1) which binds to H3K9me.
