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 workpha-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) pleiotropyb) redundancyc) 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 regulator3. 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.

Genotype gene A

gene BActivity sufficient; little or no phenotype

x

gene A

gene BActivity sufficient; little or no phenotype

xgene A

gene B

Activity reduced or eliminated; strong phenotypex

x

gene A(null)

gene B (null)

gene A(null);

Enhancers in a pathway Enhancers due to redundancy

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.

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.

P3.p P4.p P5.p P6.p P7.p P8.p

ac2° 2°

2°2°

1°

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.

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).

Identification of one essential locus was by luck

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.

Epistasis relationship of let-23 to lin-15 leads to a suppressor screen

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 suppressors2 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 themmap 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?

2 1Vul x (self) Muv x (self)Vul ; Muv only Muv 2 1

Suppressor is dominant; recessive lethal

Suppressor (Vul) x (self)

Vul x (self) Muv x (self)s/+; lin-15 +; lin-15

Vul ; Muv only Muv 2 1

s/+; lin-15 +; lin-15 s/s; lin-15

Suppressor (Vul) x (self)

1Dead

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.

x

x x

dominantgof

wild type

x xx x

recessivelof

F1 screen

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 usingnon-null mutations, a valuable point to stress as weapproach 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 let-60/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 let-60(lf)/let-60(lf) homozygotes from lin-34/let-60 hets can survive.)

Summary of let-60 story:

let-60 can mutate in three ways1) lof – Vul2) dn - Vul3) 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 RasSev Ras1LET-23 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 kinaselet-60 cloned: raslin-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

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.

Suppression of the dominant ras mutation

let-60(n1046gf) = Muv

Look for non-Muvlet-60(n1046); +/* or */* in F2 after mutagenesis

Any gene required downstream of ras will show a non-Muv 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

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

pha-1 (ts) x (self) 15°

EMS

pha-1; */+

15°

25° (restrictive temp)

All die, except pha-1 suppressors

F1

F2

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 andIndirect suppressors (collagen mutants??!!)

glp-1 enhancer screen

Sensitized background:

glp-1 (ts):

15° nearly wild-type25° 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.

glp-1(ts) x (self)

glp-1(ts) ; */+ x (self)

15°

20°

All should be fertileexcept

1. enhancers2. recessive zygotic steriles that have nothing to do

with glp-1

EMS

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 genesfour 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.