1. Epigenetics: Modification of DNA and chromatin to control DNA structure and Gene Expression

2. Genetic Variation is a Change in the DNA Sequence
SNPs
Indels
Rearrangements
Mendelian Inheritance

3. X

4. Epigenetics relies on DNA methylation
Epigenetics - heritable change in phenotype without a change in DNA sequence
Organismal or cell generations
A mechanism involved in epigenetic phenotype regulation
DNA methylation is a transcription (OFF) switch
Histone methylation is a transcription (OFF) switch
Histone acetylation is a transcription (ON) switch
Will also involve other molecular mechanism

5. DNA methylation in different species
S. cerevisiae
Arabidopsis
C. elegans
Drosophila
Vertebrates
S. pombe
N. crassa
DNA
methylation — — + —
(+)‏ + +
DNA
methylation
binding
proteins — + + + + + +
Imprinting — — — —
(+)‏ + +
A. Peaston TJL

6. DNA methylation -- 5'CpG3' cytosine guanine H N O C1' CH3 DNMT
A. Peaston TJL

7. Establishment of DNA methylation7 5 10 15 20
Days post conception
Relative methylation at CpG sites
Establishment of DNA methylation
~ 70%
Yoder, Walsh & Bestor, 1997 Trends Genet

8. Establishment of DNA methylation: imprinting8
Concept – CpG islands are a special case: inactive X-chromosome is heavily CpG island methylated, whereas autosomes and active X are generally unmethylated.
Concept – in the developing germ cells of male embryos, the female-derived unmethylated alleles of imprinted genes e.g. H19 become methylated, thus the mature male will pass on the male imprint to the next generation via his germ cells whilst his somatic cells will retain both the maternal and paternal allele-specific methylation.
Similarly, in developing germ cells of female embryos, the male-derived methylated alleles of imprinted genes will become unmethylated, and the female will thus pass only female-pattern methylation of those alleles to her offspring although her somatic cells will retain both maternal and paternal allele-specific methylation. 8
Establishment of DNA methylation: imprinting 5 10 15 20
Days post conception
Relative methylation at CpG sites
H19
Imprinted
gene
Maternal allele in
male germ cells
Paternal allele in
female germ cells
Inactive X
Autosomes
& active X
CpG
islands

9. DNA methylation and cell memory
Self-complementary
CpG dinucleotides
DNA replication
Maintenance methylation
Holliday and Pugh 1975 Science
Riggs 1975 Cytogenet Cell Genet

10. Law & Jacobsen Nat Rev. Genet 2010

11. DNA demethylation Passive Active e.g. zygotic paternal genome,
IL2 promoter
A. Peaston TJL

12. DNA demethylation -- CpG islands
OFF
? CpG methylation default, islands exclude global activity ?
A. Peaston TJL

13. Law & Jacobsen Nat Rev. Genet 2010

14. DNA Demethylation and 5hydroxymethyl cytosine
5-methylcytosine oxidized to 5 hydroxymethylcytosine
Tet family (ten-eleven translocation)

15. Wu & Zhang 2011 Cell Cycle

16. How does DNA methylation inhibit transcription?
Titratable transcriptional inhibition
DNA-protein complex specific for methylated DNA
MeCP2, MBD1-4, Kaiso
Methyl-binding proteins recruit different co-repressor complexes
HDAC
Sin3A
HKMT
MeCP2 Me me
A. Peaston TJL

17. How does DNA methylation inhibit transcription?
Biochemical experiments using
artificial CpG methylation & transfection
re-expression of X-linked genes after 5-azacytidine treatment
Interference with transcription factor or other protein binding
e.g. CTCF binding
Mat
Pat
Igf2
ICE
CTCF E
H19 me
A. Peaston TJL

18. Assessing DNA methylation
Avy/a
Morgan et al, Nature Genetics 1999

19. DNA methylation at the agouti locus
Avy allele
Hypothesis
IAP OFF
LTR CpG methylated
pseudoagouti
LTR CpG unmethylated
IAP ON
yellow

20. Methylation-sensitive restriction enzymes
Avy allele
B BamH1
M Msp1
H HpaII
Isoschizomers, C/CGG
A. Peaston TJL

21. Bisulfite sequencing Locus-specific detailed single-molecule analysis
5MeCpG
CpG
bisulfite
DNA
PCR UG CG TG
Frommer et al, PNAS 1992

22. Bisulfite Sequencing Sperm 95% Oocytes 98% Zygotes 90% Blastocyst 62%
Lane et al, Genesis 2003

23. Higher throughput Pyrosequencing of clones (You still have to clone)
SAGE

24. Next Generation Sequencing
Higher throughput
Next Generation Sequencing
High throughput, non-biological cloning

25. And permutations.....
Murrell, Rakyan & Beck, Hum Mol Genet 2005

26. Chromatin and DNA methylation
Neurospora and Arabidopsis – methylation depends on H3K9me
Plants – RNAi targets chromatin modification, gene silencing & DNAme
Mouse reduced CpG methylation with :
deficiency of H3K9 methyltransferases
deficiency of EZH2 (H3K27 methyltransferase)
increased CpG methylation with :
antisense transcription through promoters
unsettled role of RNAi
Many species – link to SWI/SNF-like remodeling proteins

27. The nucleosome core. Site of action
The nucleosome core. Site of action. Tails that get modified are evident in this image.

29. Histone modifying enzymes
2929
Histone modifying enzymes often are part of large multi-subunit complexes.
Many modifying enzymes have cell context-dependent substrate-specificity often with other substrates as well as histones.
Histone modifications provide marks recognized by specific domains e.g. bromo and chromo domains .
Bromo domain recognition sites are recognized by chromatin remodelling proteins of the Trithorax group, whilst chromo domains are recognized by at least 5 related families of proteins including the polycomb family of proteins.
An additional level of complexity – several trithorax group proteins are able to perform more than one type of modificatin reaction e.g. some trithorax proteins methylate H3K4 a mark of active gene expression, as well as performing nucleosome remodelling activity.
Also, the histone modfying enzymes provide a link with cellular response to external stimuli. E.g. Mitch Smith, Hbo1 HAT (histone acetyl transferase). Under physiologic stresses that stabilize p53 (in yeast), P53 binds Hbo1 protein and represses its activity. Moreover, Hbo1 also has documented roles in transcription and DNA replication.
Histone modifying enzymes 29

30. Chromatin remodelling – Polycomb and Trithorax
3030
Example: recruiting ATP-dependent remodelling complexes such as Swi-snf group (Trithorax group) remodel chromatin by
by nucleosome octamer sliding,
altering nucleosome structure by DNA looping
or replacement of core histones with histone variants
Polycomb complexes recognize and promote repressive histone methylation and promote tight packing of nucleosomes.
Note interactions btw two systems. Illustration in next slide.
Chromatin remodelling – Polycomb and Trithorax 30

31. 3131
Concept: non-coding RNAs including for example Xist (large) and small RNAs directly transcribed fom genome or from double-stranded RNA degradation (RNA interference) also regulate epigenetic DNA modifications in ways that are only just beginning to be understood.
e.g. slide illustrates that RNAi guides histone modification during S-phase. Fission yeast Swi6 = mammalian HP1. In G2, heterochromatic repeats have high levels of Swi6 and H3K9me2 and retain cohesin. In M, phosphorylation of H3S10 causes loss of Swi6 & transcription of the repeats in early S. Also H3K9me2 levels fall during replication, but are restored during S, guided by RNAi of the repeat transcripts and accompanied by recruitment of Swi6. 31
Kloc et al Current Biology 2008

32. Epigenetic changes and phenotype
It is not just DNA methylation that leads to various pathologic phenotypes, but abnormalities of DNA methylation have certainly been linked with these pathologic processes, in some cases as cause, in many cases it is not yet clear.
3232
Epigenetic changes and phenotype
DNA methylation and mutation
Genomic instability
Cancer
Complex disease
Environmental effects on DNA methylation
A. Peaston TJL

33. Epigenetics/Epigenomics
Modification of histones
Mostly Acetylation (+) and Methylation (-)
Histone acetyl transferases
Histone deacetylases
Modification of DNA
In eukaryotes, mostly 5-methylcytosine (-)
Dnmt (DNA methyl transferases)
Removal?
A sixth base; 5-hydroxymethylcytosine

34. Epigenetics/Epigenomics
Methylated DNA recruits HDACs
Increased condensation of chromatin decreased expression
Methylated chromatin may recruit Dnmts
Transcription factors may recruit Dnmts, HDACs or HMTs or HATs depending on function
Signals can propagate along the chromosome using bi-functional enzymes (chromodomain and HMT activity)