Analysis of function-2

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BRCA1 follow-up Gene knockouts

Synthetic interaction

Analysis of function-2

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Final exam

- Thursday Dec 17

-Emphasis on material starting Nov. 6 (Lecture 10)

- Help hours during

fi

nals week… stay tuned

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Proto-oncogenes and tumor suppressor genes

Proto-oncogene Cell growth and proliferation Tumor suppressor gene In a diploid cell…

Cancer-promoting form… Oncogene

Expected behavior of allele

Dominant

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Cell growth and proliferation

Tumor suppressor gene

Inherited cancer susceptibility

TS+

ts

-Tumor suppressor gene

loss of heterozygosity (LOH)

Cell growth and proliferation

TS+

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-From quiz section

Molecular detection of LOH

↓ ↓ 300 200 150 100 50 Functional BRCA1 protein Siz e standar d in bp 300 200 150 100 50 Marker 1 1 2

Lanes: Healthy Cancer Healthy Cancer Healthy Cancer Healthy Cancer Marker

2 3 3 4 4

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Resolving the paradox…

Pedigrees

show dominant inheritance pa

tt

ern

Mutant allele is recessive to wild type

at

cellular level

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So we’ve mapped the gene… what next?

Gene knockout or

“knockdown” in model systems

…does the phenotype mimic the disease?

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» gene knockdown… reduce expression of the gene

» gene knockout… completely delete the gene (or a critical portion of the gene)

» “knock-in”… replace one allele (e.g., wild type) with another (e.g., a specific mutation)

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your favorite gene

URA3

What is a knockout mutation

A complete loss of function allele

Often produced by replacing a gene or

portion of a gene with a selectable marker

Knock-out mutations are engineered deliberately

…by modifying a gene with recombinant DNA technology, and replacing the wild type allele with the knockout (“KO”) allele.

Goal?

To understand what a gene normally does, find out what happens if the gene is missing

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target ORF (for example)

select for Ura+_{transformants}

Ask… with the target gene knocked out, what phenotype do these cells have? (Besides Ura+₎

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URA3

Systematic ORF deletions

The deletions are carried out in diploids. Why?

In case the gene is essential for life ura3/ura3

URA3

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meiosis URA3

meiosis …then sporulate, look at

phenotype of haploid with the knockout

How do we know 

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There are 6,000+ in yeast…

make 6,000+ different strains, each with one gene deleted

Strain #1

Strain #2

Strain #3

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Selectable marker

Unique tags or “barcodes”

Strain #6244 Strain #6243

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target protein

nucleus What can we do with a yeast deletion collection?

Lots of possibilites, eg.:

» which genes help cells survive desiccation? » understanding drug metabolism

potential anti-cancer drugs…

which genes cause increased or decreased sensitivity to the drug?

drug

? ?

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Yeast “chemical genomics” to understand drug action pool of deletion strains growth with drug added sequence ? sequence compare etc.

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Interpreting the results

If a barcode is reduced or is missing?

If a barcode is present in excess?

target protein

nucleus drug

? ?

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✕

Gene X

Finding genetic interactions can help understand function

Suppose the gene knockout only raises more questions…

Knowing a gene’s “associates” may tell us about the pathway involved

No detectable phenotype or

Phenotype we can’t understand

Gene Y

Gene Z Gene W

Look for mutations in other genes that alter the phenotype of the first mutation

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mutant ph.

Example 1. If a knockout has no phenotype

If a knock-out has wild type phenotype, does that mean . . . 1. the gene doesn’t do anything?

2. there is a second gene carrying out the function?

X Y Gene 1 Gene 2 X Y Gene 1 Gene 2 X Y Gene 1 Gene 2 wt pheno wt pheno

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Combination of two mutated genes…

Testing for functionally related genes: “Synthetic eﬀects”

• phenotype becomes stronger: • phenotype becomes weaker:

genetic enhancement genetic suppression

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Practice question

A, B, and C are independently assorting genes in yeast. Each gene is separately knocked out with URA3 in ura3/ura3 diploid cells, creating heterozygotes A/a, B/ b, and C/c (where lower case = gene that has been replaced by URA3). These heterozygotes show normal growth on complete and on –ura plates.

(i) The diploids are sporulated and one tetrad from each diploid is tested for

growth on a complete plate and then replica-plated onto a –ura plate as shown:

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(ii) One Ura+ spore from tetrad A/a was mated to a Ura+ spore from tetrad B/b. The resulting diploid (strain ‘AB’) showed normal growth on complete and on – ura plates. Many of cells of this diploid strain were sporulated and the spores tested for growth. The tetrads fell into three categories (I, II, and III) as shown below. Explain the results, giving genotypes of the spores as necessary to

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✕

Gene X

Gene Y

Gene Z Gene W

“Synthetic lethal” screens Suppressor screens

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Synthetic lethality: may be a basis for therapies

Proto-oncogene Cell growth and proliferation Tumor suppressor gene

Drug

How to treat loss of tumor suppressors?

Why not use

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Synthetic lethality: may be a basis for therapies

Cell growth and proliferation Tumor suppressor gene

Some essential function

Some other gene

Hypothesis: TS gene’s essential function is fulfilled by some other gene

Proposal: Knock out other gene → synthetic lethality → specifically kill cancer cells

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Synthetic lethality: basis for BRCA LOF anti-cancer drugs

Cell growth and proliferation BRCA1

Repair of broken DNA

PARP

Inhibitor drug

BRCA1 deficient cells No drug Rampant chromosome aberrations ↓ Cell death + drug

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✕

Gene X

Gene Y

Gene Z Gene W

“Synthetic lethal” screens

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What is a suppressor mutation?

Phenotype caused by one mutation reversed or reduced by 2nd mutation

Detects direct or indirect interactions

Example: mutation in a heterodimeric protein A B A* B* B

⃠

✔

A*

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Suppressors can reveal indirect (pathway) interactions

Rett syndrome:

» X-linked dominant

» LOF mutation in MECP2

» Females: loss of hand and language skills, autism features » Males: perinatal death

Mecp2 protein: binds to methylated CG

dinucleotides in DNA transcriptional regulation Transcription

mis-regulation Rett syndrome

?? --CG--

--GC--CH3

CH3

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A mutation in SQLE suppresses the MECP2 mutant phenotype Acetyl-CoA Squalene Cholesterol SQLE Mecp2 mutant Cholesterol elevated in brain Statins

Can statin treatment reverse the MECP2 mutant phenotype? Yes! (at least in mice)

Mecp2 Sqle*

Cholesterol

closer to normal Double mutant