Identification of a daf-3 corepressor and mechanism of cofactor switch in high temperature induced dauer

formation

L1

L2

L3

Dauer

L4

Egg

Adult

C. Elegans Life Cycle

Adverse ConditionsFavorable Conditions

Lack of Food

Dauer Inducing Factors

HeatOvercrowding

TGF-B in Dauer Formation

daf-5

daf-1 daf-4

daf-3

Dauer promoting genes

daf-7

daf-14daf-8

Pheromone

TGF-β Pathway <26°C

daf-3

TGF-β Pathway >26°C

Dauer CONSTITUTIVEDauer DEFECTIVE

daf-5

No Effect

HYPOTHESIS

daf-5

daf-1 daf-4

daf-3

Dauer promoting genes

daf-7

daf-14daf-8

Pheromone

TGF-β Pathway >26°C

coREP

daf-3

Specific Aims

Identify daf-3 corepressor in high temperature induced dauer formation

Determine the mechanism of daf-3 cofactor exchange.

Specific Aim 1: Identify the daf-3 corepressor

RNAi mediated loss of known daf-3 binding proteins in WT

Non-lethal deletion mutants loss of known daf-3 bindg proteins

DAF-C PHENOTYPE AT 27°C

VSAdult Dauer

Specific Aim 1: Identify the daf-3 corepressor

Protein extract from 20C and 27°CALTERNATE APPROACHES

Purified with daf-3 antibody20°C 27°C

Specific Aim 1: Identify the daf-3 corepressorALTERNATE APPROACHES

Two Dimensional Gel

Identification of proteins by MALDI-MS

pH

Size

daf-5daf-3

Dauer promoting genes

coREP

• Temperature dependent cofactor expression

Real-time PCR of daf-5 and the corepressor at 20°C and 27°C

20°C 27°C

daf-5daf-3

Dauer promoting genes

coREP

• Temperature dependent cofactor expression

Ectopically express daf-5 and the daf-3 corepressor with HSP-GFP transgenics

20°C (+) Pheromone27°C (-) Pheromone

•Expect dauer promoting conditions•Expression of the corepressor should result in REDUCED dauer formation •Expect dauer repressing conditions•Expression of daf-5 should result in INCREASED dauer formation

daf-5daf-3

Dauer promoting genes

coREP

Competitive binding of the corepressor

•Corepressor-RFP/daf-5-CFP/daf-3-GFP young adults at 20°C and 27°C

RFP CFPGFP

20°C27°C

daf-5daf-3

Dauer promoting genes

coREP

Changes in daf-3 structures

20°C 27°CRNA Isolation

20°C 27°C

Changes in daf-3 structures: Northern Blot

Isoform XDaf-3 Null

+

Changes in daf-3 structures: Isoform Rescue

27°C

SUPPRESSION OF THE DAF-C PHENOTYPE

Specific Aims 2:Determine the mechanism of daf-3 cofactor exchange.

• Temperature dependent cofactor expression - real-time PCR of daf-5 and the corepressor at 20°C and 27°C

- Ectopically express daf-5 and the daf-3 the corepressor with HSP-GFP transgenics

• Competitive binding of the corepressor- corepressor-RFP/daf-5-CFP/daf-3-GFP young adults at 20°C and 27°C

• Changes in daf-3 structures- Examine relative amounts of daf-3 mRNA isoforms a-c at

both 20°C and 27°C via northern blot

- Rescue the daf-d phenotype at 27°C by expression of isoform’s cDNA in the daf-3 mgD90 mutant