The transcription factor DAF-16 regulates a plethora of phenotypes in C. elegans, including longevity, stress tolerance and pathogen resistance. WormBase (WS212) predicts seven alternative isoforms of DAF-16, but little is known about the differential expression or regulation of these isoforms.
We set out to analyse the expression of the DAF-16 isoforms using quantitative real-time PCR (qRT-PCR). We were able to design isoform-specific primer sets for six of the seven isoforms; the seventh (R13H8.1e.1) is identical to R13H8.1e.2 with the exception of one missing exon and therefore cannot unambiguously be identified. We isolated RNA from mixed stage populations grown at 20°C on OP50, checked for genomic DNA contamination and linearly converted RNA to cDNA using reverse transcriptase. Isoform specific PCR on this cDNA pool detected isoforms R13H8.1a, R13H8.1b, R13H8.1c and R13H8.1d. Additional PCR investigations and cDNA sequencing demonstrated that isoforms R13H8.1e.2 and R13H8.1f are expressed but, under the conditions we tested, both isoforms retain introns currently annotated as being spliced out. R13H8.1e.2 retains the intron in the 5’UTR, whilst R13H8.1f retains the intron between exons one and two. We have submitted a revision of this annotation to WormBase for inclusion in WS215.
Using these splice form-specific primer sets, we determined the expression level of each of the six isoforms relative to the housekeeping gene gpd-3 under resting (20°C) and heat-shock (37°C for three hours immediately prior to RNA extraction) conditions (Table 1). Under both conditions, isoform R13H8.1b is by far the most strongly expressed and is the only isoform to significantly increase its expression upon heat-shock. This isoform was also the only variant to show increased expression in response to heavy metal stress (7mM CuCl2) or pathogen challenge (Staphylococcus aureus exposure). Thus, the dominant DAF-16 form likely to be present in wildtype C. elegansis R13H8.1b. Determining the role(s) of the alternative isoforms of this key transcription factor will be of great relevance for our understanding of longevity and stress response in C. elegans.
| Isoform | Length (bp) | Fold expression relative to gpd-3 | |
|---|---|---|---|
| 20°C | 37°C | ||
| R13H8.1a | 2575 | 1 | 0.44 |
| R13H8.1b | 3021 | 18621.20 | 274998.94 |
| R13H8.1c | 3035 | 0.39 | 0.31 |
| R13H8.1d | 1616 | 1.68 | 0.96 |
| R13H8.1e.1 | 1128 | Cannot be discriminated fromR13H8.1e.2 | |
| R13H8.1e.2* | 1027 | 0.18 | 0.10 |
| R13H8.1f* | 2852 | 0.03 | 0.11 |
Table 1. Expression of daf-16 isoforms at 20°C and following 3hr heat-shock at 37°C. Expression levels were normalised to gpd-3 and then presented as fold-expression relative to R13H8.1a under resting (20°C) conditions. *Note that isoforms R13H8.1e.2 and R13H8.1f retain introns currently annotated as spliced out - see text for details.
