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Shmoo Al Capp (1948) – Li’l Abner. Marsh and Rose diagram. The next two lectures, and beyond. Genetics and epigenetics – examples (distinction, similarities, overlap) The power of the evolutionary perspective A brief preview – mutations A brief preview – genetic screen - PowerPoint PPT Presentation
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1MCB 140 09-28-07
2MCB 140 09-28-07
ShmooAl Capp (1948) – Li’l Abner
3MCB 140 09-28-07
Marsh and Rose diagram
4MCB 140 09-28-07
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.
5MCB 140 09-28-07
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.
6MCB 140 09-28-07
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.
7MCB 140 09-28-07
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
9MCB 140 09-28-07
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
14MCB 140 09-28-07
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
16MCB 140 09-28-07
“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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25MCB 140 09-28-07
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?
27MCB 140 09-28-07
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.
28MCB 140 09-28-07
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.
30MCB 140 09-28-07
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.
31MCB 140 09-28-07
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
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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.”