Epigenetic mechanisms

1. Epigenetic mechanisms

2. Definition of epigenetics • Coined by Conrad Waddington to describe changes in gene expression during development refers to a change in gene expression without a change in the sequence of the gene or modifications in gene expression brought about by heritable, but potentially reversible, changes in DNA methylation and/or chromatin structure

3. Examples of epigenetic modifications observed in genome • Position effects • Paramutation • Transvection • Protein conformation • Imprinting • X-inactivation • Histone modification • DNA methylation

4. Position effects • Change in the level of gene expression brought about by a change in the position of the gene relative to its normal environment • Chromosomal rearrangement may separate promoter and transcription unit from an essential distant regulatory element • Leading to reduction or absence of expression • Rearrangement may dissociate transcription unit from an element that serves to silence expression • Leading to inappropriate activation of the gene • Rearrangement may place gene with an enhancer from a second gene • Leading to inappropriate expression of the gene

5. Examples of disorders caused by position effects mechanisms • Burkitts lymphoma -translocation event that places the cMYC gene under the control of an immunoglobulin enhancer • Holoprosencephaly – caused by mutations in/deletions in the sonic hedgehog (SHH) gene. Can also be due to chromosomal rearrangements that involve translocations 15-25kb upstream of gene • Aniridia – caused by mutations in/deletions of the PAX6 gene. Can also be due to chromosomal rearrangements that involve breakpoints downstream of gene • suggests that the rearrangement separates the PAX6 gene from an essential regulatory element

6. Position effects • Proximity of genes to centromeres, telomeres or heterochromatic regions can suppress expression • Facioscapularhumeral muscular dystrophy (FSHD) • Maps to 4q35 (close to telomere) • Shown to be associated with deletion of copies of a repeat unit (D4Z4) from the subtelomeric region • 95% patients with FSHD have contraction of repeat units (1-10 vs control up to 100)

7. FSHD 4qA 4qB D4Z4 repeat blocks Allelic variants 4qA/B – equal in population But FSHD allele is always of 4qA type 6.2kb beta satellite repeat upstream genes • Actual disease mechanism is unclear – no specific gene identified as causative • but considered that the D4Z4 units form boundary between the heterochromatic • and euchromatic region • upon contraction the local chromatin relaxes and allows transcription of • genes normally under repression, • or • upon contraction, the boundary between regions is abolished allowing • heterochromatinisation and subsequent downregulation of genes in region

8. Paramutation • A meiotically heritable change in expression of one allele invoked by another allele • Initially observed and extensively studied in maize and other plants (pigment genes) but recent evidence suggests it may be more widespread • Usually results in a decrease in expression • Paramutagenic alleles – have ability to induce change in allelic partner • Paramutable alleles – can be induced to alter by paramutagenic allele • Neutral alleles – can neither change or invoke change • Possible human example – the insulin minisatellite which is associated with protection against diabetes • Paternally inherited class I alleles do not predispose to disease when have been allelic to class III allele in father.

9. Transvection • Another trans sensing mechanism ie involves the interaction of homologous alleles • Observed in Drosophila eg spreading of DNA methylation through successive generations • Modifications can occur across homologs • No known examples in human genome

10. Protein conformation • Observed in prions • infectious agents implicated in scrapie, BSE and Creuzfeldt-Jakob disease • have little or no associated DNA • consist primarily of single protein (PrP) which is host encoded and is present at low levels in disease free individuals • Exists as two stable conformers (wild type and abnormal infectious type) • Infectious nature of disease believed to derive from capacity of abnormal protein (PrPsc) to interact with wild type protein and convert it to abnormal type • Observed in yeast • Similar scenario where protein conformer alters a second • Results in inheritance of altered form in daughter cells to produce new strain with different metabolic phenotype

11. Imprinting • Most autosomal alleles expressed from both alleles – maternal and paternal origin • Subset of genes are “imprinted” ie expressed from only the mother’s or father’s allele • Alleles identical in each case but regulated differently • Involves several different mechanisms • Differential DNA methylation • Allele specific RNA transcription • Anti sense transcripts • Histone modification • Differences in replication timing • Example of disorders involving imprinting • Prader Willi syndrome • Angelman syndrome • Beckwith Wiedemann • Discussed later

12. X- inactivation • Method of ensuring dosage compensation • In mammals dosage is equalised between sexes by inactivating 1 X chromosome in female cells • Requirement for counting the X chromosomes • Choosing which to inactivate • Inactivation process • Occurs under control of X inactivation centre (XIC) • Region of ~1Mb which contains several control elements and at least 4 genes • Two of these genes are • Xist – encodes a large non coding RNA • Tsix – encodes an RNA which is synthesised antisense to Xist; regulates activity of Xist

13. X- inactivation • Both Xs are expressing Xist and Tsix at low levels prior to X inactivation • Prior to inactivation the two X chromosomes “communicate” at the XIC - decision is made as to which chromosome will be inactivated • Selected inactive chromosome adopts a heterochromatic form which upregulates the expression of Xist - coats the inactive X chromosome • Process also involves methylation of the inactive X chromosome and modification of the histones • Modification allows silencing of most of the X chromosome genes and thereby dosage compensation

14. DNA methylation and histone modification • One of major mechanism in epigenetics is chromatin modification • Usually brought about by methylation and histone modfication • Key players in determining chromatin state and subsequent transcriptional activity of genes of interest

15. CpG dinculeotide • Dinucletide repeat CpG accounts for approx 1% genome • CpGs generally under represented in human genome, but occur close to expected frequency in small regions (~1kb) called CpG islands • Approx 45,000 such islands – most reside within or near promoters or 1st exon of gene Expected CpG frequency

16. CpG islands Have “open” chromatin structure • Contains nucleosomes enriched in acetylated histone (decreased affinity for DNA and each other) • Allows access for transcription factors etc Associated with gene transcription

17. DNA methylation Occurs at 5’ position of cytosine within the CpG dinucleotide Approx 70% CpGs are methylated methylated CpGislands are associated with transcriptional silencing

18. DNA methylation • Most CpG dinucleotides occur in the “bulk” DNA (non coding) – these are hypermethylated • Reside within parasitic DNA elements/retrotransposons such as endogenous retroviruses, L1 elements, Alu elements • Proposed that methylation has arisen as a genome defense mechanism to limit spread of these elements thru’ genome • Suggested that expansion of genome methylation occurred at onset of vertebrate evolution and was accompanied by an increased dependence upon methylated DNA mediated silencing as a means of transcriptional control

19. How does methylation occur • De novo methylation occurs via group of enzymes known as de novo methyl transferases (DNMTs • Include DNMT1, DNMT3a, DNMT3b • Highly expressed in embryonic cells • Highly specific programme of methylation to include repetitive DNA/parasitic DNA, etc • Changes with development of cell types • Accessory factors also required • ds RNA may direct methylation; ~ Xist known to trigger X inactivation prior to methylation • Considered that transcriptional repression may be trigger for methylation rather than effect

20. Repressor models • Methyl domain binding proteins are known to be involved in methylation process • Recruit other repressor proteins • Represssion of transcription • Different models exist for different stages (of repression/development) • Examples include the Sin3A and NuRD complexes

21. Repression of transcription Open chromatin; active transcription De novo methyl transferases Methylation of CpGs Repressor complex; methyl domain binding ptns histone deactelyases Closed chromatin; transcriptional silence

22. Timing of methylation Somatic tissues sperm Trophoblast lineages zygote Methyln level egg Primordial germ cells Primordial germ cells blastocyst • Gametes are terminally differentiated – have specific methylation patterns and • are erased upon fertilistation • New pattern initiated by de novo methylation from blastocyst stage

23. Methylation in human disease • ICF- rare recessive disease • Immunodeficiency • Facial anomalies • Mental retardation and developmental delay • Chromosomal instability (1, 9 & 16) • Due to mutations in DNMT3b (de novo methyl transferase) • Specific areas of hypomethylation • Instability sites on chromosomes 1, 9 & 16 • Satellite DNAs 2 & 3 • DNA repeat D4Z4 • X chromosome • Loss of methylation results in either relilef from transcriptional silencing or an upregulation of normally silenced genes

24. Rett syndrome • X chromosome • Usually presents in females only • Normal development until 6-18months then • Gradual loss of speech, purposeful hand use • Develop microcephaly, ataxia, autism • Due to mutations in the MeCP2 gene (methyl binding domain protein) • Again hypomethylation occurs at specific sites • Target genes not yet identified (conflicting results – result of tissue specificity and system examined; UBE3A?)

25. DNA methylation in cancer • Hypermethylation of promoter regions is most well catergorised epigenetic change to occur in tumours • Seen in virtually every tumour type • Associated with inappropriate silencing • Is at least as common as disruption of TSGs eg BRCA1 – associated with familial breast cancer- evidence that 10-15% non familial cases have tumours with hypermethylation of BRCA1

26. DNA methylation in cancer • Also hypomethylation – may result in enhanced genomic instability; activation of oncogenes • Expression profile of DNMTs also observed in tumours • DNMT1, 3a, 3b all increased with progression of cancer • Expression profile varies depending on cancer type • Methylated CpG may also undergo spontaneous deamination; C>T and result in germline mutation • ~50% inactivating point mutations in TP53 are C>T mutations

27. Epigenetics in summary • Modifications in gene expression brought about by heritable, but potentially reversible, changes in DNA methylation and/or chromatin structure • Provide an “extra layer” of transcriptional control that regulates gene expression • Like the basic trancriptional unit and regulation, disruption of these mechanisms may result in human disease