Transcript document

Suppressor and Enhancer Screens
Suppressor and enhancer screens
1. General considerations
2. Using suppressors to delineate a signaling pathway:
vulval induction in C. elegans
3. Suppressor screens don’t always work
pha-1 suppressors
4. Using enhancers to identify new glp-1 interacting genes
Final paper: Developmental Genetic
Analysis in Xenopus
Due: December 8, 2008
No more problem sets.
Read carefully the instructions for the
assignment on the web site.
Saturation screens have limitations:
1. not all phenotypes are amenable to
saturation screening
2. sometimes it is not practical to do enough
screening to reach saturation
3. many genes involved in a process will not
appear in the screens because of
a) pleiotropy
b) redundancy
c) bad luck
Suppression:
A + suppressor --> less severe “A” phenotype
Enhancement:
A + enhancer --> more severe “A” phenotype
“synthetic” phenotype
Modes of suppression:
1. compensating mutation
2. bypass suppressor:
• Gain-of-function can suppress
mutation in positive regulator
• Loss-of-function can suppress
mutation in a negative regulator
3. indirect supressor:
• informational suppressors (e.g. tRNA
nonsense suppressors, alternative splicing, NMD)
•
Physiological suppressors
(e.g. mutations in collagen suppress glp-1)
4. intragenic suppressor
• second site compensatory mutation
• true revertant
Enhancer
Weak mutation in A - no phenotype or weak
phenotype
Mutation in B enhances A phenotype
equivalent to synthetic lethal in yeast
Can identify genes in a pathway and can
identify genes with redundant functions.
Enhancers in a pathway
Enhancers due to redundancy
Genotype
gene A(null)
gene A
x
gene B
Activity sufficient;
little or no phenotype
gene A
gene B (null)
gene B
x
Activity sufficient;
little or no phenotype
gene A
gene A(null);
x
gene B
x
Herman, R and Yochem J. (September 16, 2005), WormBook, ed. The C. elegans
Research Community, WormBook, doi/10.1895/wormbook.1.7.1, http://www.wormbook.org.
Activity reduced or
eliminated;
strong phenotype
Advantages:
1. Both enhancers and suppressors can
identify genes at any point in the
pathway.
2. Is not restricted to proteins that directly
interact.
3. Can identify genes that would not appear
in directed phenotype-based screens.
Disadvantages:
1. Noise:
• Intragenic revertants
• Indirect suppressors
2. Suppressors and enhancers may have
no phenotype alone.
3. Cloning suppressors and enhancers with
no phenotypes can be difficult.
ac
2°
P3.p
P4.p
P5.p
2°
1°
P6.p
2°
P7.p
P8.p
2°
Hyp 7
Question: What is the mechanistic basis for signaling?
What are the signals and receptors?
What are the molecular targets of the signals?
How is signaling regulated?
Suppressors in analysis of Vulval development:
Working out the components of the signaling pathway
Observation: extensive screens had identified
many genes with roles in vulval specification,
but many other genes were expected to be
undetectable in these screens. (specifically,
loci whose null phenotype would be embryonic
or larval lethality or sterility.)
How can one identify such genes?
One way is luck, the other way is to search
for extragenic suppressors.
Identification of one essential locus was by luck
Identified by a single allele with a Vul phenotype: n1045
Mapped near a known larval lethal called let-23.
n1045 failed to complement lethal alleles of let-23.
Cloning of let-23 reveals it is a receptor tyrosine kinase
(RTK).
Epistasis relationship of let-23 to lin-15
leads to a suppressor screen
let-23(n1045) -- Vul and Egl
lin-15 (n309) -- Muv (Egl+)
n309; n1045 -- Vul and Egl
n1045 is epistatic to n309 but….
another way to look at this is that a mutation in
a Vul gene suppresses the Muv phenotype of
lin-15(n309)
Aroian, R. V., and Sternberg, P. W. (1991). Multiple functions of let-23 a
Caenorhabditis elegans receptor tyrosine kinase gene required for vulval induction.
Genetics 128, 251-268.
Screen for suppressors of lin-15
(Two screens in different labs: Sternberg, Horvitz)
Mutagenize lin-15 (n309) worms (n765ts)
Screen in F2 for nonMuv, Vul animals
100,000 gametes screened (38,000)
10 extragenic suppressors
2 are recessive new let-23 Vul mutations (as
predicted)
1 is a recessive mutation in a new gene (later)
7 dominant suppressors mapped to a single locus
(5 recessive and 2 dominant suppressors in this
locus among others)
Han, M., Aroian, R. V., and Sternberg, P. W. (1990). Genetics 126, 899-913.
Beitel,G.J., Clark, S.G., and Horvitz, H.R. (1990) Nature 348:503.
Digression:
The two new let-23 alleles made it possible to
screen for additional loss-of-function alleles by a
noncomplementation screen.
let-23(sy1)/Df = viable Vul
Therefore screening for new mutations that fail
to complement the Vul phenotype of sy1 will
identify putative null alleles. (much more difficult
to screen for new lethal alleles of let-23.)
How to analyze the new suppressors:
Map them
map near genes defined by two existing mutations:
lin-34 (single Muv allele) and let-60, a larval lethal.
What is the nature of the suppressor mutations and
what is their relationship to one another?
Suppressor (Vul) x (self)
2
Vul x (self)
Vul ; Muv
2
1
1
Muv x (self)
only Muv
Suppressor is dominant; recessive lethal
Suppressor (Vul) x (self)
Dead
1
s/s; lin-15
Vul x (self)
Muv x (self)
s/+; lin-15
+; lin-15
Vul ; Muv
2
1
only Muv
s/+; lin-15 +; lin-15
Additional properties of the suppressors:
1) s/+; + still dominant Vul, recessive lethal
2) failed to complement the let-60 recessive lethals
What is the nature of the dominant suppressor mutation?
How to test?
Make deletion heterozygote:
Df (let-60)/+ --> not dominant Vul
Therefore, must be gain of function
What is the loss-of-function phenotype of the
suppressor locus?
Can isolate loss-of-function mutations by
reverting the gain-of-function allele.
F1 screen
x
dominant
gof
x
x
wild type
x
x
x
x
recessive
lof
Method 1: revert the dominant Vul
let-60 + dpy-20+ + /+ let-65 + unc-22 (balanced lethal)
Only Vul hets survive, screen for rare nonVul animals by
looking for eggs.
Result: 1 cis-revertant
Method 2: suppress the suppressor!
unc-24 let-60/dpy-20 let-3; lin-15/lin-15
Mutagenize and score for Muv animals in the F1
Result: 2 suppressors of suppressor (one cis- one trans)
Both cis-mutations mapped to let-60 region,
failed to complement recessive let-60 alleles and
are wild type as heterozygotes. (come back to trans
mutation in a bit)
Conclusion: dominant Vul mutations are in the
let-60 locus, and loss-of-function phenotype is
lethal.
What is the role of let-60 in vulval development?
How to assess? Loss of function animals die
before vulva forms!
“The biology of a given gene is often revealed using
non-null mutations, a valuable point to stress as we
approach a post-genomic sequencing project era.
Classical forward genetics will be as useful as ever
for isolating such special alleles because genetic
screens can select out relatively rare, but informative,
mutations. Of course, rare alleles can also be
misleading and need to be interpreted in the context
of a complete genetic analysis, including knowledge
of null phenotype.”
Sternberg and Han (1998) Trends in Genetics 14: 466.
Solution: use weak lof allele that allows some
survival.
let-60(s1124lf)/let-60(s1124lf) rare survivors fail to
induce vulva.
Conclusion: lof phenotype is Vul.
Because the dominant mutations produce a
phenotype identical to the recessive mutations
they are called “dominant negative” (Muller’s
antimorph)
One suppressor of the let-60 suppressor of lin-15
was unusual
It mapped to the let-60 region and it functioned as
a trans-dominant suppressor
That is: let-60(dn)/sup; lin-15 = Muv
sup/+ ---> Muv (10%) sup/sup --> Muv (90%)
This phenotype was similar to that described for
lin-34, a mutation closely linked to let-60.
lin-34/let-60(dn) = weak Muv (i.e. lin-34
suppresses let-60(dn)
lin-34/let-60(lf) ---> rescues the lethality; some let60/let-60 progeny survive
Conclusion:
lin-34 mutations are gain of function
(hypermorph) mutations in let-60. (G13E)
If excess let-60 is provided in trans it can
counteract the effect of the dominant negative
And maternal expression can rescue the
complete loss of zygotic let-60. (even let60(lf)/let-60(lf) homozygotes from lin-34/let-60
hets can survive.)
Summary of let-60 story:
let-60 can mutate in three ways
1) lof – Vul
2) dn - Vul
3) gof - Muv
Therefore let-60 plays a pivotal role in vulval induction.
What is let-60??
Han and Sternberg cloned let-60 and
found that it was a member of the
Ras family of small GTPases.
Compare to the fly eye.
RTK
Sev
LET-23
Ras
Ras1
LET-60 RAS
There was one other new gene identified as a
lin-15 suppressor. lin-45
Larval lethal, few survivors are Vul,
phenotypes are similar to other genes in the
Vul pathway.
Summary of suppressor screen:
1. two new let-23 alleles
2. identified let-60 as having an essential role
in vulval development.
3. identified an additional new gene, lin-45
SO WHAT ?????
let-23 cloned: receptor tyrosine kinase
let-60 cloned: ras
lin-45 cloned: raf
Epistasis showed relationships between these
genes and other existing lin mutants
lin-3
let-23
let-60
lin-45
lin-1
vulval fate
lin-15
AND
The let-60(gof) mutations provided a new
starting point for additional suppressor screens
Suppression of the dominant ras mutation
let-60(n1046gf) = Muv
Look for non-Muv
let-60(n1046); +/* or */* in F2 after mutagenesis
Any gene required downstream of ras will show a nonMuv phenotype.
sur-1 one of 50 or so sur mutants
Null mutation is larval lethal
Epistasis puts it downstream of raf but upstream of lin-1
= map kinase = mpk-1
Wu Y, Han M. (1994) Suppression of activated Let-60 ras protein defines a role of
Caenorhabditis elegans Sur-1 MAP kinase in vulval differentiation.Genes Dev.
8:147-59.
Not all suppressors screens are productive:
The story of pha-1 suppressors
pha-1 (pharynx abnormal) - poor differentiation of pharynx
Schnabel, H., Bauer, G., and Schnabel, R. (1991). Suppressors of the organ-specific
differentiation gene pha-1 ofCaenorhabditis elegans. Genetics 129, 69-78.
Screen for suppressors of pha-1
EMS
pha-1 (ts) x (self) 15°
15°
F1
pha-1; */+
25° (restrictive temp)
F2
All die, except pha-1 suppressors
pha-1/pha-1; */+ or */*
Results:
360,000 genomes
220 suppressors!
All recessive
Three complementation groups, sup-35, 36, 37
Suppress most but not all pha-1 alleles
Have no phenotype on their own.
Published in 1991 - no follow-up
Enhancers
Enhancers.
The basic idea is similar to finding suppressors.
1. the process in question involves the combined
action of many proteins.
2. the successful execution of the process
requires sufficient levels of all the components.
3. If levels of two components are lowered
simultaneously, that can result in strong
phenotypes not seen when only one component
is lowered.
Enhancers of sevenless:
1. Hypersensitive background
2. F1 screen
3. Enhancers in essential genes can be
recovered
4. Enhancing mutations are likely to have
recessive phenotypes and can include
null alleles
5. Provided a means to identify Ras1 as a
component of R7 fate specification in
photoreceptor development.
Screen for enhancers of glp-1
glp-1 (germline proliferation)
Suppressor screens to identify
interacting genes failed:
only intragenic revertants
and
Indirect suppressors (collagen
mutants??!!)
glp-1 enhancer screen
Sensitized background:
glp-1 (ts):
15° nearly wild-type
25° sterile
But 20° fertile (but 50% maternal effect lethal)
At 21° start to see sterility
Qiao, L., Lissemore, J., Shu, P., Smardon, A., Gelber, M. B., and Maine, E. M.
(1995). Enhancers of glp-1, a gene required for cell-signaling in Caenorhabditis
elegans, define a set of genes required for germline development. Genetics 141,
551-569.
EMS
glp-1(ts) x (self)
15°
glp-1(ts) ; */+ x (self)
20°
All should be fertile
except
1. enhancers
2. recessive zygotic steriles that have nothing to do
with glp-1
To eliminate zygotic steriles:
a) keep only those with Glp-looking germline
b) keep only those that require glp-1 for the phenotype.
Results:
Mutations are all recessive and define seven
complementation groups.
1) five alleles of lag-1 (known to function
downstream of glp-1.)
2) one allele of glp-4 (similar phenotype to
glp-1)
3) Five ego genes
four have phenotypes on their own
ego-1 - produces abnormal oocytes
ego-2 - no phenotype on its own.
ego-3 - complex defects in germ line: abberant
transition from mitosis to meiosis
ego-4 & 5- these have defects in germ line
proliferation and oogenesis, oocytes that are
produced and fertilized always die as embryos.
Conclusions:
1. all of the genes affect multiple aspects of development (like
glp-1 itself)
2. the screen can identify genes in the glp-1 pathway (e.g. lag-1)
3. the phenotypes of the ego genes are consistent with a role in
the glp-1 pathway.
4. ego-1 and ego-3 are epistatic to a glp-1(gf) allele
Overall, enhancement is a way to recover lof mutations identifying
new players in a common process.
5. ego-1 encodes an RNA-dependent RNA polymerase that has a
role in germline RNAi.
6. ego-2 encodes a Bro1 domain protein facilitates ligand
production or action. Role in secretory pathway?
Summary:
Suppressor and enhancer screens can help
to identify genes acting in a particular
process, but which also have essential roles
in other processes.