The tangled web of epigenetics Bryan Turner Advances in Genetics: a Second Teachers’ Conference

1. The tangled web of epigenetics Bryan Turner Advances in Genetics: a Second Teachers’ Conference Nowgen Centre, Manchester 28 June 2017

2. What is epigenetics? Epigeneticsis the study of the processes by which genotype gives rise to phenotype. Waddington 1942 “The changes in gene activity during development are generally referred to as epigenetic”. Holliday 1987 The boundary between genetics and epigenetics is blurred

3. The power of epigenetics Epigenetics defines the processes by which organisms access and use the information stored in DNA Epigenetics can tell us • How we interact with our environment • How to escape the tyranny of DNA • How to cure cancer The boundary between genetics and epigenetics is blurred

4. The Power of Epigenetics Epigenetic processes generate multiple phenotypes from the same genotype “…endless forms most beautiful and most wonderful…”

5. Epigenetics is underpinned by molecular mechanisms that regulate how, when and where genes are expressed Genome function (gene expression) is regulated by the interaction between DNA sequence, histones and non-histone proteins (eg.transcription factors).

6. Epigenetics is underpinned by molecular mechanisms that regulate how, when and where genes are expressed Genome function (gene expression) is regulated by the interaction between DNA sequence, histones and non-histone proteins (eg.transcription factors). Chromatin and the nucleosome are key players in epigenetic processes

7. DNA is packaged as chromatin Nucleosomes 30nm fibre RNA, non-histone proteins Metaphase chromosome

8. An evolving understanding of the nucleosome A 1980 1974 1980 110Å 1997

9. Histones are intimately associated with DNA and inevitably influence its functions….but how? Luger et al (1997) Nature 389, 251-60

10. An evolving understanding of the nucleosome 1974 1989 1997

11. Histone tails are subject to multiple, enzyme-catalyzed post-translational modifications (PTM) S-adenosyl methionine AcetylCoA HATs HMTs Methylation (lysine, arginine) Acetylation (lysine) HDACs HDMs Kinases Phosphatases ATP Phosphorylation (serine)

12. Histone tails are subject to multiple, enzyme-catalyzed post-translational modifications (PTM) H3 H4 H2B C-term H2A C-term H3K79 H2A H2BK123 H2AK119 H3 Histone globular regions 5 2 4 36 9 23 14 18 20 Acetyl lysine 20 16 15 12 Methyl arginine 8 12 H4 5 H2B Methyl lysine 3 5 Phosphoryl serine 1 Ubiquityl lysine

13. Proteins with specific binding domains recognise specific modifications and thereby influence chromatin structure and function

14. Multiple protein binding domains recognise specific modified amino acids…a code? Musselman et al (2012) Nature Struct.Mol.Biol. 19 (12) 1718-27

15. Nucleosome signalling (A beautiful paradigm) Changes in nucleosome structure through incorporation of histone variants or the actions of modifying enzymes, can influence genome function. The nucleosome core particle carries on its surface, a variety of chemical signals in the form of modifications of specific histone amino acids. Modifications are put in place and removed by families of enzymes. Histones carrying specific modifications can be bound by proteins carrying defined binding domains. These proteins regulate the function of genomic regions to which they are bound.

16. Roles of chromatin modification histone modifications Short-term effects Long-term effects Ongoing processes Transcription, replication, repair Heritable changes Cellular memory

17. Immunostaining with modification-specific antibodies shows how H4 isoforms differing only in degree of methylation are differently distributed on metaphase chromosomes H4K20me1 + DAPI H4K20me3 + DAPI Female mouse ES cells

18. In Mouse Embryo Fibroblasts H4K20me3 is located at centromeres of autosomes, but all along the Y Y IIF H4K20me3 + DAPI Y FISH Y paint + DAPI

19. H3K4me3 on human lymphoblastoid cell chromosomes

20. The nucleosome is more than just a DNA packaging device: It is a sophisticated signalling system that regulates gene expression It also, inevitably, links genome function to environmental agents

21. Environmental agents can alter the activities of Chromatin-modifying enzymes with wide-ranging effects on cell function Proteome Trans-generational (phenotypic) change If heritable Metabolic enzymes Transcriptome Germ line Metabolome Place for a ”true code” ? Gene expression Somatic Somatic (phenotypic) change If heritable Chromatin modifying enzymes DNA histone TFs Direct Via signalling pathways Environmental agents Adapted from Turner, Phil.Trans.Roy.Soc.B 364, 3403 (2009)

22. Jean-Baptiste Lamarck (1744-1829) More than just neglect….. Lamarck is the only major figure in the history of biology whose name has become…a term of abuse. Most scientists’ contributions are fated to be outgrown, but very few authors have written works which, two centuries later, are still rejected with an indignation so intense that the sceptic may suspect something akin to an uneasy conscience. C.H.Waddington, in Evolution after Darwin, Chicago University Press, 1960 Philosophie Zoologique, 1809 The environment drives heritable phenotypic change

23. How we escape the tyranny of DNA

24. Paradigm shifts in genetics 1850-1900 : Proto-genetics Mendel, inheritance Darwin, natural selection 1900–1950 : Age of genetics gene concept, mutation, genotype-phenotype 1950–2000 : Age of DNA DNAstructure, genetic code, Selfish Gene, genomics Has DNA become the all-powerful deity of a new religion…

25. with its prophets…… ….who ask us to wonder at the marvels created by DNA in a deterministic universe

26. …and of course a patron saint Charles Darwin (1809-1882) The combination of Natural Selection and Mendelian Genetics provides an almost complete explanation for the earth’s biodiversity “The Newton of Biology” and a major beneficiary of the Age of DNA The Origin of Species by Means of Natural Selection, 1859

27. But DNA is just an inert coding device, useless without epigenetic processes to interpret its messages.

28. But DNA is just an inert coding device, useless without epigenetic processes to interpret its messages. Epigenetic processes can be manipulated and changed by environment, lifestyle, diet etc Philosophically, epigenetics is an empowering science!

29. Paradigm shifts in genetics 1850 -1900 : Proto-genetics Mendelian inheritance Darwin, natural selection 1900 –1950 : Age of genetics gene concept, mutation, genotype-phenotype 1950–2000 : Age of DNA structure, genetic code, genome sequence 2000 - : Age of epigenetics epigenetic code, epigenome, epigenetic medicine

30. Jean-Baptiste Lamarck (1744-1829) Epigenetics will free us from the tyranny of DNA Vive la révolution Philosophie Zoologique, 1809 The environment drives heritable phenotypic change

31. Epigenetic drugs to treat cancer

32. Histone acetate groups turn over rapidly in eukaryotic cells Intermediary metabolism CoA Acetyl CoA 0 2h 5h HATs Lysine Acetyl Lysine - HDACs Inhibitors Therapeutic HDAC inhibitors have chemotherapeutic value, but effectiveness is patchy. Valproate is a widely used anti-epileptic. Environmental

33. Classes of HDACi in clinical trials against various cancers • I, II, III indicates the phase of the clinical trial; App indicates • approval for clinical use in the USA (FDA, against specified cancers) • or China.

34. First interesting question Histone acetylation would be expected to cause massive (lethal) disruption of epigenetic systems Why are many normal cells so tolerant? And why are some cancers sensitive?

35. First interesting question Histone acetylation would be expected to cause massive (lethal) disruption of epigenetic systems Why are many normal cells so tolerant? And why are some cancers sensitive?

36. Second interesting question Why should human cells mount such a complex resistance response to a particular class of anti-cancer drugs?

37. HDAC inhibitors are naturally occurring (environmental) agents Short chain fatty acids (eg. butyric) are products of gut microflora and found at mM concentrations in the colon Trichostatin A and other hydroxamic acid derivatives are secreted by certain fungi and are anti-fungal antibiotics. Depsipeptide (FDA approved) is a fermentation product of Chromobacterium violaceum

38. Second interesting question Why should human cells mount such a complex resistance response to a particular class of anti-cancer drugs? The answer may lie in our evolutionary history

39. Life on earth is made of only two cell types Prokaryotes Eukaryotes Archaea Eukaryota Bacteria No cytoskeleton proteins, unique ribosomal proteins, RNAs Occasional actin, tubulin Eukaryotic homologies in ribosomal proteins, RNAs Nuclear envelope, mitochondria cytoskeleton Archaeal homologies in ribosomal proteins, RNAs

40. The first eukaryotes had to fight to survive First prokaryotes Stromatolites First animals Cambrian explosion First eukaryotes Single celled rRNA phylogeny Earth formed Multicellular fossils 4.5 4.0 3.0 2.0 1.0 0 Billions of years before present. Gya Oxygen levels

41. A microbial arms race Prokaryotes attacked (or defended themselves against) the earliest eukaryotic interlopers (predators?) by secreting toxins to disrupt their unique epigenetic systems.

42. A microbial arms race Prokaryotes defended themselves against the earliest eukaryotic predators by secreting toxins to disrupt their unique epigenetic systems. We see these today as bacterial anti-fungals (TSA, depsipeptide) T

43. A microbial arms race Prokaryotes defended themselves against the earliest eukaryotic predators by secreting toxins to disrupt their unique epigenetic systems. We see these today as bacterial anti-fungals (TSA, depsipeptide) Eukaryotes fought back, and we see this today as fungal antibiotics such as penicillin

44. A microbial arms race Prokaryotes defended themselves against the earliest eukaryotic predators by secreting toxins to disrupt their unique epigenetic systems. We see these today as bacterial anti-fungals (TSA, depsipeptide) Eukaryotes fought back, and we see this today as fungal antibiotics such as penicillin They also developed ways of defending their epigenetic systems against chemical attack, which we have detected as a survival response against HDACi

45. Naturally occurring HDACi and their target organisms * All classes of HDACiexceptdibromotyrosines are in clinical trials against various cancers (June 2016)

46. Classes of HDACi in clinical trials against various cancers • I, II, III indicates the phase of the clinical trial; App indicates • approval for clinical use in the USA (FDA, against specified cancers) • or China.

47. Human cells can mount a chromatin-based response that limits the toxic effects of HDAC inhibitors Are cancers susceptible to HDACi those in which key components of the resistance response have been lost through mutation? Can we develop drugs to target these components in cancer cells? “Seen in the light of evolution, biology is perhaps the most satisfying and inspiring science. Without that light, it becomes a pile of sundry facts…” Theodosius Dobzhansky Turner B (2011) FEBS Lett 585, 2032-40 (Epigenetics special edition)