Download a2 - Molecular and Cell Biology

MCB 140 09-28-07
1
Shmoo
Al Capp (1948) – Li’l Abner
MCB 140 09-28-07
2
Marsh and Rose diagram
MCB 140 09-28-07
3
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.
MCB 140 09-28-07
4
Amazing but true
A wild-type haploid yeast cell contains THREE
copies of mating type-determining genes:
• Copy #1: the a1 and a2 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 140 09-28-07
5
• In an a strain, the genetic information at
MAT and at HMLa is identical.
• The one at MAT is expressed, but the one
at HML is not – it is epigenetically
silenced.
a2 a1
a cell
HMLa
silent
cen
a2 a1
a2 a1
MAT
HMRa
active
silent
MCB 140 09-28-07
6
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?
MCB 140 09-28-07
7
Loss of silencing at the silent mating type
cassettes creates a “nonmater” – a haploid
that is a/a and that thinks it’s a diploid.
a2 a1
a cell
HMLa
active
cen
a2 a1
a2 a1
MAT
HMRa
active
active
MCB 140 09-28-07
8
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 140 09-28-07
9
The mating
pheromone
response
Also see Fig. A.13.
Jeremy Thorner
Thorner diagram
MCB 140 09-28-07 10
How to screen for silencing mutants
a2 a1
a cell
HMLa
silent
a2 a1
a2 a1
MAT
HMRa
active
silent
cen
Jasper Rine and Ira Herskowitz (1987) Genetics 116: 9-22.
MCB 140 09-28-07 11
How to screen for silencing mutants
a2 a1
HMLa
silent
cen
a2 a1
a2 a1
mata1-1
HMLa
active
silent
Note: mata1-1 is a special allele of the a gene – it is recessive to a
Jasper Rine and Ira Herskowitz (1987) Genetics 116: 9-22.
MCB 140 09-28-07 12
Rine schematic
mate to a cells
Jasper Rine and Ira Herskowitz (1987) Genetics 116: 9-22.
MCB 140 09-28-07 13
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 140 09-28-07 14
Compaction into chromatin brings
the eukaryotic genome to life
15,000x
compaction
< 10-5 metres
> 1 metre
MCB 140 09-28-07 15
“Beads on a string”?
MCB 140 09-28-07 16
The Nucleosome Core Particle:
8 histones, 146 bp of DNA
MCB 140 09-28-07 17
Histones: Conserved and
Charged
H.s. = Lycopersicon esculentum
MCB 140 09-28-07 18
MCB 140 09-28-07 19
“Extremely conserved histone H4 N terminus is
dispensable for growth but essential for repressing
the silent mating loci in yeast” (M. Grunstein)
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!
Kayne et al. (1988) Cell 55: 27-39.
MCB 140 09-28-07 20
Acetylation of lysine in histone tail
neutralizes its charge (1964)
Covalent modification of histones as a regulatory mechanism?
MCB 140 09-28-07 21
“Genetic evidence for an interaction between SIR3
and histone H4 in the repression of the silent
mating loci in Saccharomyces cerevisiae”
Reverse genetics: introduce point mutations in H4 tail!!
Johnson et al. (1990) PNAS 87: 6286-6290.
MCB 140 09-28-07 22
Table 2
Johnson et al. (1990) PNAS 87: 6286-6290.
MCB 140 09-28-07 23
MCB 140 09-28-07 24
And 5 years later …
Sir3p and Sir4p bind H3 and H4 tails
Hecht et al. (1995) Cell 80: 583.
MCB 140 09-28-07 25
The key question
How do the SIRs
spread over the
mating type loci
genes?
= how do the SIRs
actually silence txn?
MCB 140 09-28-07 26
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 ADPribosyltransferase 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 140 09-28-07 27
J. Denu: Sir2p is a NAD-dependent
histone deacetylase (HDAC)
Sir2p
Tanner et al., PNAS 97: 14178 (2000)
MCB 140 09-28-07 28
Rusche L, Kirchmaier A, Rine J (2002) Mol. Biol. Cell 13: 2207.
MCB 140 09-28-07 29
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.
MCB 140 09-28-07 30
Baur et al. Nature 444: 337.
Lagouge et al. Cell 127: 1109.
MCB 140 09-28-07 31
Hermann Joseph Muller
1946 Nobel Prize in Medicine:
"for the discovery of the production of
mutations by means of X-ray irradiation"
Genetic screen:
1. Su(var)2-5
2. Su(var)3-9
13.13
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
HP1=HP1
HP1
Homology
(orthologs of heterochomatin proteins
in fission yeast, insects, and humans)
Analogy
Fission yeast, flies, mammals.
Budding yeast.
From egg to embryo
?
47
48
Homeotic mutations (W. Bateson)
Genetics
Allele
Heterozygous
Homozygous
“… Not that there has merely
been a change, but that
something has been changed
into the likeness of something
else.”
wt
antennapedia
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.
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
Nature Aug. 2007
Cancer Cell July 2007
53
David Allis: “the histone code”
Fischle, Wang, Allis COCB 2003
“Nothing in biology
makes sense except
in light of evolution.”
Theodosius
Dobzhansky
55