MCB /27/06 1 E. coli = E. lephant ? F. Jacob J. Monod A. Pardee D. Hawthorne H. Douglas Y. Oshima

MCB /27/06 2 Analogy and homology as tools in genetic investigation Animal Mandibular Arch (ventral) Mandibular Arch (dorsal) Hyoid Arch (dorsal) SharkMeckel's cartilage Palatoquadrate cartilage Hyomandibular cartiliage AmphibianArticular (bone)Quadrate (bone)Stapes MammalMalleusIncusStapes

MCB /27/06 3

4 a cells produce a pheromone and  receptor  cells produce  pheromone and a receptor diploid (a/  ) cells produce none of the above

MCB /27/06 5 Shmoo Al Capp (1948) – Li’l Abner

MCB /27/06 6 Marsh and Rose diagram

MCB /27/06 7 The phenotype of a haploid yeast cell with respect to mating is determined by transcription factors An  cell produces two transcription factors, Mat  1p and Mat  2p, that ensure expression of  specific genes, including the pheromone and receptor, and repress expression of a specific genes. In an a cell, Mat  1p and Mat  2p are not expressed, and a different transcription factor is expressed, Mata1p. The  genes are off, and the a genes (pheromone and receptor) are on.

MCB /27/06 8 A.9

MCB /27/06 9 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.

MCB /27/06 10 A.11 A.12

MCB /27/06 11 “An easily understood, workable falsehood is more useful than an incomprehensible truth.”

MCB /27/06 12 cen MAT HML  HMRa  a1a2  cell  activesilent

MCB /27/06 13 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

MCB /27/06 14 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!!

MCB /27/06 15 The mating pheromone response Jeremy Thorner Thorner diagramAlso see Fig. A.13.

MCB /27/06 16 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.

MCB /27/06 17 How to screen for silencing mutants cen mata1-1 HML   a1a2 activesilent Jasper Rine and Ira Herskowitz (1987) Genetics 116: HML   Note: mata1-1 is a special allele of the a gene – it is recessive to 

MCB /27/06 18 Jasper Rine and Ira Herskowitz (1987) Genetics 116: Rine schematic mate to a cells

MCB /27/06 19 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.

MCB /27/06 20 Question What molecular mechanisms are responsible for silencing at the mating type loci?  heterochromatin formation in metazoa  prostate cancer  breast cancer  ageing  “normal” gene regulation in mammals

MCB /27/06 21 Homework

MCB /27/06 22 How can one explain the evolution of two distinct mating types in budding yeast? Surely a pathway could have just evolved for the fusion of two identical haploid cells?

MCB /27/06 23 Two mating types have evolved under selective pressure to avoid inbreeding M D1 D2 D1 D2 One evolutionary advantage of mating is the production of novel genotypic combinations via the fusion of two genomes with different life histories. x

MCB /27/06 24 Granddaughters of any given mother can switch mating type

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MCB /27/06 27 cen MAT HML  HMRa  a1a2  cell  cen MAT HML  HMRa  a1a2 a cell a1a2 activesilent

MCB /27/06 28 Epigenetic inheritance 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. Epigenetic: mitotically stable (persists through cell division) change in gene expression state that is not associated with a change in DNA sequence. Examples: X chromosome inactivation; imprinted genes; transgene silencing in gene therapy.

MCB /27/06 29 > 1 metre < metres 15,000x compaction Compaction into chromatin brings the eukaryotic genome to life

MCB /27/06 30 “Beads on a string”

MCB /27/06 31 The Nucleosome Core Particle: 8 histones, 146 bp of DNA

MCB /27/06 32 Histones: Conserved and Charged H.s. = Lycopersicon esculentum

MCB /27/06 33

MCB /27/06 34 “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: Fig. 3 kayne

MCB /27/06 35 Kayne et al. (1988) Cell 55: Fig. 6 and 7 of Kayne.

MCB /27/06 36 Kayne et al. (1988) Cell 55:

MCB /27/06 37 Acetylation of lysine in histone tail neutralizes its charge (1964)

MCB /27/06 38 “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: Reverse genetics: introduce point mutations in H4 tail!!

MCB /27/06 39 Johnson et al. (1990) PNAS 87: Table 2

MCB /27/06 40

MCB /27/06 41 And 5 years later … Sir3p and Sir4p bind H3 and H4 tails Hecht et al. (1995) Cell 80: 583.

MCB /27/06 42 Houston, we have a … Every nucleosome in the cell has an H3 and H4 tail (two of each, actually). Why do the SIRs bind only where they bind?

MCB /27/06 43 The silencers “Hawthorne deletion” (1963) and onwards: two silencers flank the mating type loci:

MCB /27/06 44 The key question How do the SIRs spread from the silencer and over the mating type loci genes? = how do the SIRs actually silence txn?

MCB /27/06 45 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.

MCB /27/06 46 J. Denu: Sir2p is a NAD-dependent histone deacetylase (HDAC) Tanner et al., PNAS 97: (2000) Sir2p

MCB /27/06 47 Rusche L, Kirchmaier A, Rine J (2002) Mol. Biol. Cell 13: 2207.

MCB /27/06 48 acetylation Histone tail acetylation promotes chromatin unfolding (somehow)

MCB /27/06 49 Next time: the genetics of heterochromatin in metazoa