Figure 2. Regulation of cyp-35B1 expression by
daf-16 and
hsf-1 in
daf-2(
e1370). A. RT-PCR analysis of endogenous cyp-35B1 and
sod-3 mRNA levels in
daf-2(
e1370) adults under control conditions or under
daf-16 or
hsf-1 RNAi. Results are average from three independent experiments. T-test was used to determine significance. cyp-35B1 mRNA was significantly reduced by
daf-16 RNAi and
hsf-1 RNAi (p = 0.003 and 0.046, respectively).
sod-3 mRNA was significantly reduced by
daf-16 RNAi (p,0.001) but not by
hsf-1 RNAi (p = 0.55). B. Expression of cyp- 35B1:gfp transcriptional reporter was low or undetectable in wildtype non-dauer larvae (i) and adults (iii), but was expressed in the intestine of dauer larvae (ii) and
daf-2(
e1370) adults (iv). In
daf-2(
e1370) adults, cyp- 35B1:GFP expression was abrogated by
daf-16 RNAi (v) and substantially reduced by
hsf-1 RNAi (vi). The spot of GFP fluorescence in the head reflects the transformation marker,
gcy-7:GFP, expressed in the ASE/L neuron. Bars, 100 mm. In some lines,
hsf-1 RNAi resulted in increased or broader intestinal cyp-35B1:GFP expression. Since this phenotype was not recapitulated by the endogenous cyp-35B1 mRNA (panel A), we attribute this observation to mysregulation of the cyp-35B1:gfp transgene under conditions of reduced
hsf-1 activity.