1. MCB 140 09-28-07 1

2. 2 Shmoo Al Capp (1948) – Li’l Abner

3. MCB 140 09-28-07 3 Marsh and Rose diagram

4. MCB 140 09-28-07 4 The next two lectures, and beyond 1.Genetics and epigenetics – examples (distinction, similarities, overlap) 2.The power of the evolutionary perspective 3.A brief preview – mutations 4.A brief preview – genetic screen 5.A brief preview – suppressor genetics 6.Today – yeast, flies, humans. Wednesday – plants. Thursday night (7 pm onwards) – review session for midterm.

5. MCB 140 09-28-07 5 Amazing but true A wild-type haploid yeast cell contains THREE copies of mating type-determining genes: Copy #1: the  1 and  2 genes (silent). Copy #2: the a1 and a2 genes (also silent). Copy #3: An additional copy of genes in item 1, or of the genes in item 2, but active. Whichever genes are contained in copy #3 determines the mating type.

6. MCB 140 09-28-07 6 cen MAT HML  HMRa  a1a2  cell  activesilent In an  strain, the genetic information at MAT and at HML  is identical. The one at MAT is expressed, but the one at HML is not – it is epigenetically silenced.

7. MCB 140 09-28-07 7 A note on homework 1.Good job – I am impressed, and pleasantly surprised, both with the number of responses, and the quality of the writing. 2.Many gave the correct answer – two mating types evolved to prevent X from happening. No one, however, gave a complete answer: why is X bad? In other words, why did yeast evolve to protect themselves against X happening? What would happen to yeast if X were to happen frequently?

8. MCB 140 09-28-07 8 Loss of silencing at the silent mating type cassettes creates a “nonmater” – a haploid that is a/  and that thinks it’s a diploid. cen MAT HML  HMRa  a1a2  cell  active

9. MCB 140 09-28-07 9 Screen for silencing mutants A sample “screen”: 1.Take haploid cells. 2.Mutate them. 3.Screen for those that don’t mate. Problem: mating is so much more than proper silencing of mating type loci!!

10. MCB 140 09-28-07 10 The mating pheromone response Jeremy Thorner Thorner diagramAlso see Fig. A.13.

11. MCB 140 09-28-07 11 How to screen for silencing mutants cen MAT HML  HMRa  a1a2 a cell a1a2 activesilent Jasper Rine and Ira Herskowitz (1987) Genetics 116: 9-22.

12. MCB 140 09-28-07 12 How to screen for silencing mutants cen mata1-1 HML   a1a2 activesilent Jasper Rine and Ira Herskowitz (1987) Genetics 116: 9-22. HML   Note: mata1-1 is a special allele of the a gene – it is recessive to 

13. MCB 140 09-28-07 13 Jasper Rine and Ira Herskowitz (1987) Genetics 116: 9-22. Rine schematic mate to a cells

14. MCB 140 09-28-07 14 The data Colonies screened: 675,000 Colonies that mated to a: 295 Major complementation groups: 4 silent information regulators: SIR1, SIR2, SIR3, SIR4 Jasper Rine and Ira Herskowitz (1987) Genetics 116: 9-22.

15. MCB 140 09-28-07 15 > 1 metre < 10 -5 metres 15,000x compaction Compaction into chromatin brings the eukaryotic genome to life

16. MCB 140 09-28-07 16 “Beads on a string”?

17. MCB 140 09-28-07 17 The Nucleosome Core Particle: 8 histones, 146 bp of DNA

18. MCB 140 09-28-07 18 Histones: Conserved and Charged H.s. = Lycopersicon esculentum

19. MCB 140 09-28-07 19

20. MCB 140 09-28-07 20 “Extremely conserved histone H4 N terminus is dispensable for growth but essential for repressing the silent mating loci in yeast” (M. Grunstein) Kayne et al. (1988) Cell 55: 27-39. Fig. 3 kayne Deletion of histone tail led to no obvious effect, except the yeast stopped mating. Why? Loss of silencing at the mating type loci!

21. MCB 140 09-28-07 21 Acetylation of lysine in histone tail neutralizes its charge (1964) Covalent modification of histones as a regulatory mechanism?

22. MCB 140 09-28-07 22 “Genetic evidence for an interaction between SIR3 and histone H4 in the repression of the silent mating loci in Saccharomyces cerevisiae” Johnson et al. (1990) PNAS 87: 6286-6290. Reverse genetics: introduce point mutations in H4 tail!!

23. MCB 140 09-28-07 23 Johnson et al. (1990) PNAS 87: 6286-6290. Table 2

24. MCB 140 09-28-07 24

25. MCB 140 09-28-07 25 And 5 years later … Sir3p and Sir4p bind H3 and H4 tails Hecht et al. (1995) Cell 80: 583.

26. MCB 140 09-28-07 26 The key question How do the SIRs spread over the mating type loci genes? = how do the SIRs actually silence txn?

27. MCB 140 09-28-07 27 Roy Frye (Pitt) “Characterization of five human cDNAs with homology to the yeast SIR2 gene: Sir2-like proteins (sirtuins) metabolize NAD and may have protein ADP- ribosyltransferase activity” BBRC 260: 273 (1999). 1. Bacteria have proteins homologous to Sir2. 2. So do humans (>5). 3. The bacterial proteins are enzymes, and use NAD to ADP-ribosylate other proteins.

28. MCB 140 09-28-07 28 J. Denu: Sir2p is a NAD-dependent histone deacetylase (HDAC) Tanner et al., PNAS 97: 14178 (2000) Sir2p

29. MCB 140 09-28-07 29 Rusche L, Kirchmaier A, Rine J (2002) Mol. Biol. Cell 13: 2207.

30. MCB 140 09-28-07 30 The unique power of an evolutionary perspective on biology Caloric restriction  longevity Why? 1.Lin, S. J., Defossez, P. A. & Guarente, L. Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae. Science 289, 2126−2128 (2000) 2.Howitz KT, … Sinclair DA. Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature. 2003 Sep 11;425(6954):191-6.  resveratrol 3.Baur et al (2006). Resveratrol improves health and survival of mice on a high-calorie diet. Nature. 444(7117):337-42 4.Lagouge et al. (2006) Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1alpha. Cell. 2006 Dec 15;127(6):1109-22.

31. MCB 140 09-28-07 31 Baur et al. Nature 444: 337. Lagouge et al. Cell 127: 1109.

33.  Hermann Joseph Muller 1946 Nobel Prize in Medicine: "for the discovery of the production of mutations by means of X-ray irradiation"

34. 13.13 Genetic screen: 1.Su(var)2-5 2.Su(var)3-9

36. Su(var)3-9 = heterochromatin protein 1 (HP1)

37. Who would have thunk it? NCBI: Su(var)3-9 contains a domain (the SET domain) that is somewhat similar to, ahem, RUBISCO methyltransferase. Su(var)3-9 is a HISTONE methyltransferase.

38. Histone methylation

39. Calling David Duchovny and Gillian Anderson Su(var)3-9 was given this name because it was the 9 th gene isolated on the 3 rd chromosome in a screen for Su(var)s. It methylates lysine 9 in histone H3. This was discovered 18 years after it was named.

40. And finally HP1 preferentially BINDS histone H3 methylated on lysine 9. That’s why Su(var)3-9 determines localization of HP1 to heterochromatin (it methylates histones in heterochromatin).

43. HP1

44. ===

45. Homology (orthologs of heterochomatin proteins in fission yeast, insects, and humans)

46. Analogy Fission yeast, flies, mammals.Budding yeast.

47. 47 From egg to embryo ?

48. 48

49. Homeotic mutations (W. Bateson) “… Not that there has merely been a change, but that something has been changed into the likeness of something else.” Genetics Allele Heterozygous Homozygous

50. wt antennapedia

51. Nature, October 10, 2002 The polycomb group protein EZH2 is involved in progression of prostate cancer Varambally et al. Prostate cancer is a leading cause of cancer-related death in males and is second only to lung cancer. Although effective surgical and radiation treatments exist for clinically localized prostate cancer, metastatic prostate cancer remains essentially incurable. Here we show, through gene expression profiling, that the polycomb group protein enhancer of zeste homolog 2 (EZH2) is overexpressed in hormone-refractory, metastatic prostate cancer. … Dysregulated expression of EZH2 may be involved in the progression of prostate cancer, as well as being a marker that distinguishes indolent prostate cancer from those at risk of lethal progression.

52. Nature Genetics, Feb. ‘07 “Epigenetic stem cell signature in cancer” – Peter Laird “Polycomb-mediated methylation on Lys27 of histone H3 pre-marks genes for de novo methylation in cancer” – Howard Cedar “A stem cell–like chromatin pattern may predispose tumor suppressor genes to DNA hypermethylation and heritable silencing” – Stephen Baylin

53. 53 Nature Aug. 2007 Cancer Cell July 2007

54. Fischle, Wang, Allis COCB 2003 David Allis: “the histone code”

55. 55 Theodosius Dobzhansky “Nothing in biology makes sense except in light of evolution.”