This study aims to identify the corepressor of daf-3 that mediates dauer formation in C. elegans in response to high temperatures, and to determine the mechanism by which daf-3 switches cofactors between repressor and activator in different temperatures. The specific aims are to first identify the daf-3 corepressor using RNAi, deletion mutants, protein extracts, and mass spectrometry. The second aim is to study the temperature dependent expression of daf-5 and the corepressor, their competitive binding to daf-3, and changes in daf-3 mRNA isoforms, to understand how daf-3 switches cofactors between repressor and activator in response to temperature

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

2. L1
L2
L3
Dauer
L4
Egg
Adult
C. Elegans Life Cycle
Adverse ConditionsFavorable Conditions

3. Lack of Food
Dauer Inducing Factors
Heat
Overcrowding

4. 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

5. 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

6. Specific Aims
 Identify daf-3 corepressor in high temperature
induced dauer formation
Determine the mechanism of daf-3 cofactor exchange.

7. 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
VS
Adult Dauer

8. Specific Aim 1: Identify the daf-3 corepressor
 Protein extract from 20C and 27°C
ALTERNATE APPROACHES
 Purified with daf-3 antibody
20°C 27°C

9. Specific Aim 1: Identify the daf-3 corepressor
ALTERNATE APPROACHES
Two Dimensional Gel
Identification of proteins by MALDI-MS
pH
Size

10. 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

11. 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

12. 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

13. daf-5daf-3
Dauer promoting genes
coREP
Changes in daf-3 structures
20°C 27°C
RNA Isolation

14. 20°C 27°C
Changes in daf-3 structures: Northern Blot

15. Isoform X
Daf-3 Null
+
Changes in daf-3 structures: Isoform Rescue
27°C
SUPPRESSION OF THE DAF-C PHENOTYPE

16. 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