Figure 2.
aakg-4 transcription is directly regulated by DAF-16/FoxO. A, B)
aakg-4 but not
aakg-5 mRNA levels are increased in
daf-2 animals in a
daf-16 dependent manner. p,0.05, compared to N2, p,0.05, compared to
daf-2. Mean values from 4 trials. Error bars, standard deviation. C) DAF-16 binds to the promoter of
aakg-4 but not
aakg-5.
aakg-4 peak 2 is positioned within 1 Kb of the transcriptional start site and contains two DAF-16 binding elements (Figure S5). One representative experiment is shown which contained 3 immuno-precipitation replicates from the same chromatin preparation (error bars show the standard deviation between them). The horizontal dotted line shows the % input from a negative control region (39 of gene R11A5.4) that does not bind DAF-16 [22]. Statistical analysis of additional trials is presented in Table S5. A western blot showing the specificity of the DAF-16 antibody is shown in Figure S4. D) Confocal microscopy shows Paakg-4::gfp to be broadly expressed in C. elegans. Images show Paakg-4::gfp expression pattern in 1 day old hermaphrodites. (i) Whole worm expression pattern. (ii) Paakg-4::gfp is seen in tail sensory organs: phasmid sheath (PhaSh), socket cells (PhaSc) and neurons (PhaN), as well as in epithelial rectal cells (RectE), anal-depressor muscle (ADM), pre-anal ganglion rectal neurons (RectN), body wall muscles (BWM), posterior intestine (Int) and dorsal cord neuronal processes (DC). (iii) Paakg-4::gfp is expressed in vulval (VlvM), uterine muscles (UtM) and ventral cord processes (VC). (iv) Head expression ly localizes to the ring ganglia (RingN) plus 6 pharyngeal neurons (PhxN). (v) It is also seen in amphid sensory organs including sheath (AmphSh), socket cells (AmphSc) and neurons (AmphN). Table S10 compares expression of Paakg-4::gfp with other AMPK subunits. E) Quantification of GFP fluorescence in worms expressing the Paakg-4::gfp reporter shows that fluorescence increased in
daf-2 animals dependent on
daf-16. The same was also true for a second set of strains generated from a different extrachromosomal array. Means from 3 independent trials shown. Error bars, standard error. Animals contained the wuEx256 transgene array. p,0.01 compared to N2, p,0.001 compared to
daf-2. F) gfp mRNA was increased in
daf-2 animals in a
daf-16-dependent fashion. p,0.05 compared to N2, p,0.01 compared to
daf-2. Means from 3 independent trials shown. Error bars, standard deviation. Prior to transgene quantification animals were maintained at 15uC and then shifted to 25uC for 24 hr at the L4 stage.