NHR-49 and HLH-30 upregulate icl-1 expression upon fasting by turning-on transcription in more nuclei, more alleles, and increasing burst duration. (A) Confocal images showing the transcriptional kinetics of icl-1 upon fasting in WT, nhr-49, and hlh-30 mutant animals. L4 stage animals were imaged for 90 min. All images are maximum intensity projections from z-stacks. Arrowheads: nascent RNAs. Scale bars: 5 μm. (B) Quantification of the tissue-wide transcription activity is defined by the percentage of nuclei with at least one RNA spots. n = 7 animals for WT, n = 10 animals for nhr-49(zac598) and n = 10 animals for hlh-30(tm1978) were measured, including the ones shown in A. (C) Heatmaps showing the nuclear-wide transcription activity of icl-1 in different strains shown in B, which is defined by the total intensity of spots and the number of active alleles per nucleus. n = 222 nuclei for WT, n = 276 nuclei for nhr-49(zac598) and n = 227 nuclei for hlh-30(tm1978) were quantified. (D) Allele-wide transcriptional kinetics of icl-1 in the strains showed in A. n = 50 alleles for WT, n = 38 alleles for nhr-49(zac598) and n = 70 alleles for hlh-30(tm1978) were quantified. (E) Representative quantifications to show the transcriptional dynamics of a single allele during fasting as shown in D. (F) Quantification of the percentage of total burst time (left) or total pause time (right) in different strains as shown in D. n represents the number of alleles. (G) Quantifications of single burst durations (left) or single pause durations (right) in different mutants as shown in D, n represents the number of bursts or pauses. (H) Quantification of single burst amplitude defined by the maximum total intensity of spots in a complete burst in D. n represents the number of bursts. All values are displayed as mean ± SEM. ns: not significant. ****P < 0.0001 (one-way ANOVA with the Tukey correction).
Figure 7.

NHR-49 and HLH-30 upregulate icl-1 expression upon fasting by turning-on transcription in more nuclei, more alleles, and increasing burst duration. (A) Confocal images showing the transcriptional kinetics of icl-1 upon fasting in WT, nhr-49, and hlh-30 mutant animals. L4 stage animals were imaged for 90 min. All images are maximum intensity projections from z-stacks. Arrowheads: nascent RNAs. Scale bars: 5 μm. (B) Quantification of the tissue-wide transcription activity is defined by the percentage of nuclei with at least one RNA spots. n = 7 animals for WT, n = 10 animals for nhr-49(zac598) and n = 10 animals for hlh-30(tm1978) were measured, including the ones shown in A. (C) Heatmaps showing the nuclear-wide transcription activity of icl-1 in different strains shown in B, which is defined by the total intensity of spots and the number of active alleles per nucleus. n = 222 nuclei for WT, n = 276 nuclei for nhr-49(zac598) and n = 227 nuclei for hlh-30(tm1978) were quantified. (D) Allele-wide transcriptional kinetics of icl-1 in the strains showed in A. n = 50 alleles for WT, n = 38 alleles for nhr-49(zac598) and n = 70 alleles for hlh-30(tm1978) were quantified. (E) Representative quantifications to show the transcriptional dynamics of a single allele during fasting as shown in D. (F) Quantification of the percentage of total burst time (left) or total pause time (right) in different strains as shown in D. n represents the number of alleles. (G) Quantifications of single burst durations (left) or single pause durations (right) in different mutants as shown in D, n represents the number of bursts or pauses. (H) Quantification of single burst amplitude defined by the maximum total intensity of spots in a complete burst in D. n represents the number of bursts. All values are displayed as mean ± SEM. ns: not significant. ****P < 0.0001 (one-way ANOVA with the Tukey correction).

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