Epigenetics

Give three mechanisms of epigenetics.

-DNA methylation -Histone modifications -Non-coding RNA

What is a chromatosome?

A nucleosome plus the H1 histone.

What are the products of H19 and IGF2 expression?

H19: a long non-coding RNA involved in downregulation of body weight and cell proliferation. IGF2: insulin-like growth factor 2, a growth-promoting hormone involved in gestation.

Where does ubiquitination occur?

Histones H2A and H2B only.

How does histone acetylation alter gene expression?

LOOSENING OF DNA -Acetylation of the histone tails reduces their negative charge -This weakens the interaction of the negatively-charged histone tails with positively-charged DNA -DNA is loosened around the histone -This probably allows the DNA to be more accessible to transcription factors BINDING OF REGULATORY PROTEINS -Regulatory proteins bind to acetylated histones through bromodomains -These proteins only recognise a short sequence of four amino acids which includes acetylated lysine -Bromodomain binding is associated with transcriptionally active chromatin

How do effector proteins bind to methylated histones? Describe this feature.

They bind to methylated lysine residues via chromodomains. The chromodomain is a highly conserved motif that is around 60 amino acids in length.

Why do epigenetic changes during differentiation have such relevance in modern science?

They can be used to monitor reprogramming of cells to form iPSCs.

What are some patterns of histone methylation?

-Methylation occurs on histones H3 and H4 only -Specific lysine and arginine residues are commonly methylated on H3 and H4 -Residues can be mono-, bi-, or tri-methylated

How does histone methylation have an effect on gene expression?

-Methylation results in the recruitment of effector proteins to chromatin -These remodel the chromatin, which can lead to increased or decreased transcriptional activity

How does histone modification change throughout differentiation? How can this be utilised during reprogramming of cell fate?

-Throughout differentiation, levels of H3 and H4 acetylation decrease -Throughout differentiation, levels of H3K9 and H3K27 methylation increase

What is a nucleosome?

A structural unit of a eukaryotic chromosome, consisting of a complex of eight histone proteins around which DNA is coiled twice.

What is X-inactivation? Why does it occur?

Inactivation of one of the copies of the X chromosome in female mammals. The copy that is inactivated is chosen on a random basis. This occurs to prevent females from having twice as much X-chromosome gene product as males.

What is histone phosphorylation indicative of? Give examples.

It can indicate active or inactive chromatin. H2A phosphorylation is commonly associated with gene silencing, whereas phosphorylation of H2B is commonly associated with transcriptional activation.

What is histone methylation indicative of? How does this compare to DNA methylation?

It can indicate active or inactive chromatin. DNA methylation, on the other hand, is typical of transcriptional inactivity.

How does global transcription change throughout differentiation?

It decreases.

How does chromatin condensation change throughout differentiation?

It increases.

What is the catalytic domain of histone methyltransferases?

SET domain.

How does X-inactivation occur during differentiation?

X-inactivation occurs at the multipotent stage.

Name three pieces of evidence for the existence of epigenetic effects.

-Importance of methyltransferases -Role of environmental factors -Inheritance of epigenetic traits

What is a feature of IGF2 and H19 expression?

Both are highly expressed during foetal and early postnatal development.

In which histone proteins does acetylation most commonly occur?

H3 and H4, although it can also occur in H2A and H2B.

What is a feature of enzymes that carry out methylation and demethylation?

They are often specific to a certain residue at a certain position.

Give seven examples of histone modifcation. What are the most and least common of these?

-Acetylation: addition of an acetyl group donated from acetyl CoA (common) -Methylation: addition of a methyl group (common) -Phosphorylation: addition of a phosphate group -Ubiquitination: addition of a ubiquitin molecule -SUMOylation: addition of a small ubiquitin-like modifier protein (H2A and H2B only; uncommon) -Biotinylation: addition of biotin (uncommon) -ADP-polyribosylation: addition of ADP-ribose polymers (uncommon)

How does Polycomb activity change throughout differentiation?

-At the pluripotent stage, genes involved in differentiation are repressed by Polycomb proteins so as to avoid further differentiation -At the multipotent stage, fewer genes are repressed by Polycomb proteins such that only lineage-specific differentiation genes are repressed by Polycomb proteins, so as to prevent differentiation into a single lineage -At the unipotent stage, Polycomb proteins dissociated from lineage-specific genes, preventing their silencing

How does DNA methylation change throughout differentiation?

-At the totipotent stage, there is global DNA demethylation -At the pluripotent stage, there is some promoter methylation but only enough to induce a hypomethylated state -At the multipotent and unipotent stages and promoters become hypermethylated

Outline how maintenance methylation occurs.

-Before replication, DNA is fully methylated at CpG sites -During replication, daughter DNA strands are synthesised which currently lack methylation at CpG sites, leading to the creation of hemimethylated DNA (methylated on only one strand) -The methyltransferase recognises hemimethylated DNA and binds to it, moving along the DNA and methylating at CpG sites until the daughter strand is also fully methylated

How does X-inactivation occur?

-Chromatin is packaged into heterochromatin which is very dense and inaccessible to transcription factors -Inactivation of the chromosome occurs through the upregulation of the ncRNA Xist which spreads around the X-chromosome, coating it in order to silence genes -Xist recruits Polycomb complexes which modify histones on the inactivated X chromosome in order to facilitate heterochromatin formation -Histone modifications are made on both X-chromosomes in order to upregulate transcription on the active chromosome (Xa) and downregulate transcription on the inactive chromosome (Xi) -Xa is more heavily methylated than Xi; although hypermethylation is typically associated with reduced expression, in this case the hypermethylation occurs in gene bodies where it is believed that hypermethylation could actually stimulate transcriptional elongation

Name the two ways in which DNA can be methylated.

-De novo methylation: establishment of methylation patterns after fertilisation due to erasure of methylation -Maintenance methylation: perpetuation of methylation patterns that are already present after DNA replication

Outline how the flowering process in Arabidopsis is controlled by histone deacetylase.

-Flowering must occur at the correct time for optimal conditions (e.g. temperature, insect populations) -Arabidopsis has 'memory' of cold weather due to histone deacetylase activity -Flowering locus C (FLC) is a gene encoding a protein which represses flowering -Acetylation of the histones associated with this gene increases its transcriptional activity; the gene is expressed and flowering is thus repressed -Flowering locus D (FLD) encodes a histone deacetylase -After a period of cold weather where a subsequent period of warm weather is anticipated, the FLD gene is activated -The histone deacetylase removes acetyl groups from the FLC histones, reducing transcriptional activity of FLC -Expression of FLC is reduced; flowering is no longer suppressed

Describe a recently postulated theory relating to histone modification.

-In contrast to the typically transient nature of modifications, it is believed that there may be some modifications that are passed down to future generations -The mechanism for transmission of these inherited modifications is not yet clear -Current indications are that histones are randomly distributed to daughter strands during DNA replication -It is possible that these histones are used as a template to maintain modifications on new histones

What could be some possible purposes of histone phosphorylation?

-Mediate recruitment of proteins -Mediate release of effector proteins

Give examples of commonly phosphorylated residues (positions not needed).

-Serine and threonine on H2A, H2B, H3, H4 -Histidine on H4

How do Polycomb complexes bring about X-inactivation?

-The PRC2 complex carries out di- and tri-methylation of H3K27 via its EZH1 and EZH2 subunits -The PRC1 complex carries out monoubiquitination of H2AK119 via its RING1A and RING1B ubiquitin ligase subunits -Both modifications lead to chromatin compaction -It is believed that the PRC1 complex may also play an additional role in chromatin compaction independent of its ubiquitin ligase activity

What are some caveats for the experiments carried out on coat colour?

-The effects of methylation on coat colour are only seen if the agouti gene is transferred through the paternal line; different research has shown no effects when the gene is transferred through the maternal line -The actual density of CpG methylation does not appear to increase in mothers fed the methyl-rich diet compared to the mothers fed the methyl-poor diet, despite the phenotypic changes -The transcriptional promoter at the agouti gene is incompletely methylated regardless of whether the gene is activated or silenced -Methylation could perhaps be acting indirectly through histone modifications

How have the effects of methylation on flowering of Arabidopsis been experimentally demonstrated?

-The flowering of Arabidopsis in relation to patterns of trimethylation on H3K27 (H3K27me3) on the FLC gene was tracked using ChIP -Plants were kept in a cold environment for periods of 0, 2 and 4 weeks -It was found that the longer the plant is kept in the cold, the more H3K27me3 there is compared to non-methylated H3K27 in the nucleation region of the FLC gene -Plants kept in a cold environment for 4, 6 and 8 weeks were then investigated; no great increase in methylation was seen across this period (methylation was approaching 100% saturation after 4 weeks) but there was no decrease either -Plants kept in a cold environment for each time period were placed into a warm environment for 7 days and ChIP experiments were then carried out -There was little change in trimethylation at H3K27 in the nucleation region after 7 days in the warm environment, but further along the FLC gene in the distal region there was an increase in H3K27me3 -A larger increase in H3K27me3 in the distal region corresponded to a greater length of time spent in the cold period; it thus appears that H3K27me3 in the nucleation region provides some epigenetic memory that influences H3K27me3 in the distal region -This indicates that Arabidopsis is able to monitor the length of the cold period through H3K27 trimethylation in the nucleation region of the FLC gene, and is able to monitor the length of the warm period through H3K27 methylation in the distal region of the FLC gene -It is proposed that the changes in methylation act as a switch to alter FLC expression

How is the Kcnq1 locus imprinted?

-The locus consists of a gene for a long non-coding RNA (ncRNA) as well as 8-10 protein coding genes -The paternal ncRNA gene promoter is hypomethylated, allowing expression of the gene -The ncRNA represses the adjacent protein-coding genes -The maternal ncRNA gene promoter is hypermethylated, repressing gene expression -The ncRNA is not transcribed and thus there is no repression of the protein-coding genes; they are thus expressed

List some processes that histone phosphorylation is associated with.

-Transcriptional regulation -Apoptosis -Cell cycle progression -DNA repair -Chromosome condensation -Developmental gene regulation

Outline a breakthrough that has been made in "chromosome therapy" for the prevention of Down's syndrome.

-Trisomy for chromosome 21, the cause for Down's syndrome, causes imbalance of gene expression -This can be corrected by silencing the extra chromosome through altered expression of Xist -The Xist gene, along with an inducible promoter, was integrated into the additional copy of chromosome 21 in Down's syndrome iPSCs using genomic editing techniques -This results in normal expression of the Xist ncRNA, resulting in execution of the silencing processes involved in X-inactivation

Name and describe the amino acid composition of the eight proteins that make up the nucleosome core.

-Two H2A histones: slightly lysine-rich -Two H2B histones: slightly lysine-rich -Two H3 histones: arginine-rich -Two H4 histones: arginine-rich

Describe three mechanisms for gene silencing.

-Unmethylated DNA forms a more open conformation than methylated DNA; the conformation formed by methylated DNA is less accessible for non-histone proteins such as transcription factors -Methyl groups themselves can physically impede the binding of transcription factors -Methyl-CpG-binding proteins preferentially bind to methylated DNA through their methyl-CpG-binding domains (MBDs); this inhibits transcription (A combination of all three of these mechanisms tends to occur at any one time.)

What is imprinting?

A form of epigenetic gene regulation that results in expression of a single allele in a parent-of-origin-specific manner. If an allele inherited from one parent is imprinted, it is silenced and only the allele inherited from the other parent is expressed.

Name the enzymes that carry out acetylation and deacetylation, giving examples of families.

Acetylation: histone acetyltransferases. Families: MYST, GNAT, p300. Deacetylation: histone deacetylases. Families: classes I-IV.

Which histone proteins are capable of being phosphorylated?

All of them, including H1.

What is a feature of the tertiary structure of the nucleosome proteins? What is a consequence of this?

Although the proteins are globular, their N and C termini protrude from the protein. Consequence: allows modification of the N and C termini.

Give examples of two diseases that arise as a result of imprinting. What do the two diseases have in common in terms of imprinting?

BECKWITH-WEIDEMANN SYNDROME (BWS) -There are many causes of BWS -However, one cause is abnormal DNA methylation, resulting in altered regulation of gene imprinting -The genes H19 and IGF2 are both affected by BWS - both of these genes are typically imprinted -Over half of BWS cases also have defective imprinting at the Kcnq locus, which is adjacent to the H19/IGF2 locus -BWS is characterised by excessive growth (in particular an excessively large tongue) and an increased risk of childhood cancer SILVER-RUSSELL SYNDROME -Silver-Russell syndrome is a form of dwarfism, often with asymmetrical growth -The exact cause of Silver-Russell syndrome is unknown but it is known that it involves hypomethylation of H19 and IGF2, both of which are typically imprinted -Silver-Russell syndrome is also associated with maternal uniparental disomy on chromosome 7 - this is an imprinting error whereby the offspring receives two maternal copies and no paternal copies of chromosome 7

Describe two techniques that are used in experiments into epigenetics.

BISULPHITE SEQUENCING/PCR -This capitalises on the different ways in which cytosine and 5-methylcytosine respond to bisulphite treatment -Bisulphite treatment converts cytosine to uracil but does not affect methylated cytosine -This allows tracking of whether cytosines were methylated or not through PCR -The PCR results will show methylated cytosine as C, and normal cytosine as U CHROMATIN IMMUNOPRECIPITATION (ChIP) -This is use to detect whether a particular protein binds to a particular DNA sequence -Chromatin sequences are detected through use of a specific antibody -Chromatin is treated with formaldehyde, cross-linking protein and DNA so that they remain bound to one another -Cross-linked chromatin is then extracted and sheared to a manageable size -An antibody specific to the chromatin protein in question is added, resulting in precipitation of the antibody-chromatin complexes -DNA is purified from the immunoprecipitate and the sequence of interest is detected through a variety of methods including Southern blotting and PCR; if the sequence is present this indicates that the specific DNA sequence does bind to the specific protein

Give examples of how ubiquitination can be implicated in cancer.

BRCA1 GENE -The tumour suppressor gene BRCA1 contains a ubiquitin ligase domain -Females with a mutated BRCA1 gene are likely to express ubiquitin ligase with reduced activity, resulting in reduced histone ubiquitination -Females with a mutation in the BRCA1 gene have an 87% risk of breast cancer and a 44% risk of ovarian cancer respectively PROSTATE AND BREAST CANCER -Monoubiquitinated H2A is dramatically downregulated in prostate cancer -Monoubiquitinated H2B is dramatically downregulated in breast cancer

How has the role of environmental factors in producing epigenetic effects been highlighted experimentally?

COAT COLOUR INVESTIGATIONS -The effects of diet on coat colour have been investigated in mice -Mice have a gene for coat colour: B = black, b = brown -They also have a gene for the presence of an agouti (yellow) band: A = agouti (due to transient expression of the agouti G protein), a = non-agouti -AA creates pure yellow mice but these do not survive to birth in the wild; insertion of a transposon allows them to be created in the lab -AA mice can have different coat colours, from yellow to 'psuedo-agouti' (i.e. lab-created agouti), depending on the extent of DNA methylation -One mother is fed a methyl-rich diet, and one mother is fed a methyl-poor diet, which affects DNA methylation in offspring -Offspring of mothers who were fed a methyl-poor diet tended to have a more yellow coat colour, and offspring of mothers who were fed a methyl-rich diet tended to have a more psuedo-agouti coat colour -High levels of DNA methylation leads to coat colour tending towards psuedo-agouti, and low levels of DNA methylation leads to coat colour tending towards yellow LICKING/GROOMING BEHAVIOUR -Different mothers show different attitudes to licking and grooming their offspring (this is important after birth as it warms the offspring up) -Mothers who are dedicated to licking and grooming (high LG) tend to produce confident offspring which show reduced hormonal responses to stress -Mothers who are more indifferent about licking and grooming (low LG) tend to produce fearful offspring with an increased hormonal response to stress -High LG mothers produce high LG offspring, and vice versa -Some genes related to stress, including the gene for the oestrogen receptor, show altered different methylation patterns between high-LG and low-LG offspring -If timid offspring from a low-LG mother are cross-fostered by a high-LG mother, there is a phenotypic change as the timid mice become more confident

What are some patterns of CpG appearance?

CpG sites are underrepresented in the genome, so there are fewer sites than would be expected if the genome were completely random. CpG 'islands' are often found near transcription start sites.

Describe a process that differs between cytosine and methylated cytosine. What are the consequences of this?

DEAMINATION When deaminated, unmethylated cytosine becomes uracil. 5-methyl-cytosine becomes thymine. Uracil doesn't appear in DNA so repair mechanisms are able to detect the presence of uracil and thus remove it with a great deal of ease. Thymine does occur in DNA, and although a mismatch occurs as a result of deamination this is not as easy to detect by repair mechanisms as the presence of uracil. Thus the mismatch may be missed and this will result in a base substitution (and thus a permanent mutation) on both strands in one of the daughter DNA molecules after replication.

Name and describe the enzymes involved in DNA methylation.

DNA METHYLTRANSFERASES (DNMTs) Dnmt1 -Maintenance methyltransferase -Ubiquitously expressed in mammals Dmnt3a and 3b -De novo methyltransferase -Developmentally regulated Dnmt2 -tRNA methyltransferase -Methylates cytosine on tRNA

Why do histones not end up being degraded as a result of ubiquitination?

Degradation occurs as a result of polyubiquitination. Histones only undergo monoubiquitination.

How has the importance of methyltransferases been highlighted experimentally?

Dmnt1 MUTATIONS -Mouse homozygous mutants for Dmnt1 die 10.5 days into gestation (development period between conception and birth; in mice 10.5 days is around halfway through pregnancy) -Homozygous mutants are observed to have a number of abnormal features e.g. smaller than wt, smaller yolk sac than wt, presence of an abnormal structure near the tail Dmnt3a MUTATIONS -Mouse homozygous mutants for Dmnt3a mostly appear normal at birth -However, they suffer great retardation in growth and only survive until around 3 or 4 weeks after birth Dmnt3b MUTATIONS -Mouse homozygous mutants for Dmnt3b die during gestation -Mutants are observed to have a number of abnormal features e.g. growth impairment, rostral neural tube defects Dmnt3a/b MUTATIONS -Mouse double homozygous mutants for Dmnt3a and Dmnt3b die during early development and have a huge range of defects

How are H19 and IGF2 imprinted? What do the two genes have in common? How does this differ to Kcnq1?

H19 is paternally imprinted, resulting in expression of the maternal allele and repression of the paternal allele. IGF2 is maternally imprinted, resulting in expression of the paternal allele and repression of the maternal allele. The genes are at the same locus and show highly similar expression patterns. This indicates that the two genes share regulatory elements. Silencing of both genes occurs from the same element, H19 imprinting control element (H19ICE), located just upstream of the H19 promoter. Kcnq1 is independently regulated.

What are some patterns of H3 and H4 modification?

H3 and H4 tend to carry more modifications on their N termini compared to other proteins. H3 in fact appears to carry no modification at all on the C terminus. H3 unusually appears to have modification within the globular protein (methylation of lysine at residue 79).

Give an example of histone phosphorylation and two processes it is associated with. What is interesting about this?

H3 serine phosphorylation is associated with gene activation and mitosis. In terms of chromatin structure, gene activation (very open structure) and mitosis (supercoiled structure) are essentially opposites of one another. Different effects are generated by phosphorylation at different residue positions.

Which of the histone proteins carries the most modification?

H3.

Describe two models for an example of the mode of action of histone methylation.

H3K9 -H3K9 (i.e. lysine-9 residue on histone H3) is dimethylated by G9a methyltransferase -H3K9 is then recognised by the adaptor protein HP1, which recognises dimethylation -This in turn results in recruitment of DNA (cytosine-5)-methyltransferase 1 (DNMT1) which methylates CpG sites on DNA near the histone -This results in gene inactivation -DNMT1 is able to interact with G9a and a positive feedback loop is generated, with the complex moving down the DNA to perpetuate further inactivation H3K4 -Methylation of H3K4 results in inactivation of the NuRD complex and of SUV39 -The NuRD complex is involved in chromatin remodelling and histone deacetylation -SUV39 is a histone lysine methyltransferase -A number of other residue modifications) are capable of inhibiting the activity of SUV39 (e.g. H3K9 acetylation, H3S10 phosphorylation) -Inactivation of these proteins affects histone modification at other sites

Give an example of a histone residue that can have differential functions depending on its modification.

H3K9 -Has a repressive function when methylated -Has an activatory function when acetylated

In general, how can methylation be associated with gene expression?

Hypermethylation is associated with inactive genes. Hypomethylation is associated with active genes.

What are the families of methyl-CpG binding proteins? Give examples. What is the primary difference between these families?

METHYL-CpG-BINDING DOMAIN PROTEINS -Some bind to methylated DNA (e.g. MBD1, MBD2) -Some bind to DNA that has been altered in some other way, but not to methylated DNA (e.g. MBD3 binds to hydroxymethylated DNA) SET AND RING FINGER ASSOCIATED PROTEINS (SRAs) -e.g. UHFR1 and UHFR2 ZINC FINGER -e.g. Kaiso DIFFERENCE: they all recognise methylated DNA in different ways

Which enzymes carry out histone methylation and demethylation?

METHYLATION: -Histone methyltransferase (HMT) -Histone lysine methyltransferases (HKMTs) DEMETHYLATION: -Histone demethylases

What occurs during DNA methylation?

Methyl groups are added to CpG sites on the DNA.

Give some patterns of acetylation in the histone proteins?

Occurs on specific histones at specific residues. Frequently occurs at Lys residues.

Name the four factors that can be added to a cell culture in order to reprogram cells. What are these referred to as?

Oct3/4 Sox2 c-Myc Klf4 Referred to as Yamanaka factors.

What is polycomb repression? How was it discovered?

Polycomb repression is wide-ranging repression of genes by the polycomb group of protein complexes, transcription factors that are master regulators of gene expression. -Discovered through research into Drosophila developmental (HOX) genes -It was found that polycomb proteins repress several thousands of genes involved in differentiation throughout development in Drosophila, vertebrates and plants -Specific expression programmes are also maintained throughout the lifetime of the organism by polycomb proteins

Describe an example of how X-inactivation can be seen phenotypically.

TORTOISESHELL CATS -Tortoiseshell cats are black and ginger -Tortoiseshell cats are almost always female because the gene for ginger/non-ginger coat colouring is on the X chromosome -Female cats can thus have one gene for ginger fur and one gene for non-ginger (black) fur -Because X-inactivation occurs randomly, some cells can have one X chromosome inactivated and other cells can have the other X-chromosome inactivated -Thus some cells involved in producing coat colour will produce a ginger coat and some will produce black, resulting in the tortoiseshell phenotype -This is not possible in males because they inherit only one X chromosome and can thus only have either ginger or black fur

What is dosage compensation? Give an example.

The processes by which organisms equalise the expression of genes between equal biological sexes so that those with additional copies of a particular chromosome do not have higher amounts of gene product than those with reduced copies. EXAMPLE: X-inactivation

What is epigenetics?

The study of changes in organisms caused by modification of gene expression rather than alteration of the genetic code itself.

Which enzymes are involved in ubiquitination of histones, and in reversing the process?

Ubiquitination: ubiquitin ligase. Deubiquitination: deubiquitinating peptidases (DUBs)

What does acetylation typically indicate?

Upregulation of transcription.

How has it been determined experimentally that bromodomains are associated with transcriptionally active chromatin?

Utilising ChIP to identify bromodomains generally produces transcriptionally active genes.

To what extent is DNA methylation conserved?

Widely, but not completely. It appears to have been lost in worms, Drosophila and some yeast but has been found in a wide range of prokaryotes, bacteria and archaea.