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ShmooAl Capp (1948) – Li’l Abner

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Marsh and Rose diagram

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The next two lectures, and beyond

1. Genetics and epigenetics – examples (distinction, similarities, overlap)

2. The power of the evolutionary perspective3. A brief preview – mutations4. A brief preview – genetic screen5. A brief preview – suppressor genetics6. Today – yeast, flies, humans. Wednesday –

plants. Thursday night (7 pm onwards) – review session for midterm.

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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.

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cen MATHML HMRa

a1a2

cell

active silentsilent

• 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.

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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?

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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 MATHML HMRa

a1a2

cell

active activeactive

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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!!

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The mating pheromone response

Jeremy Thorner

Thorner diagramAlso see Fig. A.13.

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How to screen for silencing mutants

cen MATHML HMRa

a1a2

a cell

a1a2

active silentsilent

Jasper Rine and Ira Herskowitz (1987) Genetics 116: 9-22.

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How to screen for silencing mutants

cen mata1-1HML

a1a2

active silentsilent

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

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

Rine schematic

mate to a cells

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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.

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> 1 metre< 10-5 metres

15,000x compaction

Compaction into chromatin brings the eukaryotic genome to life

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“Beads on a string”?

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The Nucleosome Core Particle:8 histones, 146 bp of DNA

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Histones: Conserved and Charged

H.s. = Lycopersicon esculentum

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“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!

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Acetylation of lysine in histone tail neutralizes its charge (1964)

Covalent modification of histones as a regulatory mechanism?

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“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!!

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

Table 2

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And 5 years later …

Sir3p and Sir4p bind H3 and H4 tails

Hecht et al. (1995) Cell 80: 583.

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The key question

How do the SIRs spread over the mating type loci genes?

= how do the SIRs actually silence txn?

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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.

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J. Denu: Sir2p is a NAD-dependenthistone deacetylase (HDAC)

Tanner et al., PNAS 97: 14178 (2000)

Sir2p

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

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The unique power of an evolutionary perspective on biologyCaloric restriction longevityWhy?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.

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Baur et al. Nature 444: 337.Lagouge et al. Cell 127: 1109.

Hermann Joseph Muller

1946 Nobel Prize in Medicine:

"for the discovery of the production of mutations by means of X-ray irradiation"

13.13

Genetic screen:1. Su(var)2-52. Su(var)3-9

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

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.

Histone methylation

Calling David Duchovny and Gillian Anderson

• Su(var)3-9 was given this name because it was the 9th gene isolated on the 3rd chromosome in a screen for Su(var)s.

• It methylates lysine 9 in histone H3.

This was discovered 18 years after it was named.

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).

HP1 HP1

HP1 HP1 HP1 HP1 HP1 HP1HP1 HP1= = =

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

Analogy

Fission yeast, flies, mammals. Budding yeast.

47

From egg to embryo

?

48

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

wt antennapedia

Nature, October 10, 2002

The polycomb group protein EZH2 is involved in progression of prostate cancerVarambally 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.

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

Nature Aug. 2007

Cancer Cell July 2007

Fischle, Wang, Allis COCB 2003

David Allis: “the histone code”

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Theodosius Dobzhansky

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