Regulation of Caenorhabditis elegans HLH-30 subcellular localization dynamics: Evidence for a redox-dependent mechanism

Abstract

Basic Helix-Loop-Helix (bHLH) transcription factors TFEB/TFE3 and HLH-30 are key regulators of autophagy induction and lysosomal biogenesis in mammals and C. elegans, respectively. While much is known about the regulation of TFEB/TFE3, how HLH-30 subcellular dynamics and transactivation are modulated are yet poorly understood. Thus, elucidating the regulation of C. elegans HLH-30 will provide evolutionary insight into the mechanisms governing the function of bHLH transcription factor family. We report here that HLH-30 is retained in the cytoplasm mainly through its conserved Ser201 residue and that HLH-30 physically interacts with the 14-3-3 protein FTT-2 in this location. The FoxO transcription factor DAF-16 is not required for HLH-30 nuclear translocation upon stress, despite that both proteins partner to form a complex that coordinately regulates several organismal responses. Similar as described for DAF-16, the importin IMB-2 assists HLH-30 nuclear translocation, but constitutive HLH-30 nuclear localization is not sufficient to trigger its distinctive transcriptional response. Furthermore, we identify FTT-2 as the target of diethyl maleate (DEM), a GSH depletor that causes a transient nuclear translocation of HLH-30. Together, our work demonstrates that the regulation of TFEB/TFE3 and HLH-30 family members is evolutionarily conserved and that, in addition to a direct redox regulation through its conserved single cysteine residue, HLH-30 can also be indirectly regulated by a redox-dependent mechanism, probably through FTT-2 oxidation.

Keywords: 14-3-3 proteins; DAF-16; Diethyl maleate; HLH-30; Redox.

Figure 1.. Ser201 is the main residue regulating C. elegans HLH-30 subcellular localization.

a) Protein sequence homology between human TFEB and TFE3 and C. elegans HLH-30 (from the 14-3-3 binding motif to the Leucine zipper motif), using the Clustal Omega Program [28]. C. elegans Ser201 and Ser283 residues and their equivalent residues in TFEB/TFE3 are boxed in red. Identical residues are shadowed in black and marked with an asterisk while similar residues are shadowed in grey and marked with a black dot. Mammalian cysteine residues are drawn in yellow and C. elegans cysteine in blue. Numbers on the left indicate amino acid residue. b) Representative fluorescence micrographs of ARPE-19 cells expressing HLH-30-FLAG. Scale bar 10 μm. The graph shows the quantification of HLH-30-FLAG nuclear labeling of two independent experiments with >300 cells per assay. **** p<0.0001 by two-tailed unpaired t-test. c) Representative fluorescence micrographs of C. elegans expressing HLH-30::GFP endogenous reporter. Cytoplasmic, diffuse labeling; Weak nuclear, nuclear labeling of some cells in the posterior region of the animal (arrows) and not nuclear labeling in the head area; Strong nuclear, nuclear labeling both in the posterior and head regions of the animal. Scale bar 50 μm. The graph shows the quantification of HLH-30::GFP subcellular localization of three independent experiments with >50 animals per genotype and assay. **** p<0.0001 by 2way ANOVA with Bonferroni’s multiple comparisons test. Error bars are S.E.M. d) HLH-30 subcellular localization upon stress treatments of three independent experiments with >50 animals per genotype and assay. **** p<0.0001 by 2way ANOVA with Bonferroni’s multiple comparisons test. Error bars are S.E.M.

Figure 2.. C. elegans HLH-30 subcellular localization is regulated by 14-3-3 proteins and independently of DAF-16.

a) Synergistic effect of ftt-2 and par-5 RNAi downregulation on the subcellular localization of the HLH-30::GFP reporter. b) ftt-2(n4426) mutation induces the nuclear translocation of the HLH-30::GFP reporter. c) Similar effect of ftt-2 and par-5 RNAi downregulation on the subcellular localization of the DAF-16::GFP reporter. d) daf-16(mu86) mutation does not impair HLH-30::GFP subcellular localization under basal or stress conditions. e) hlh-30(tm1978) mutation does not impair DAF-16::GFP localization under basal or stress conditions. f) Expression of GFP1–9 together with endogenous tagging of ftt-2 and hlh-30 with GFP10 and GFP11, respectively, produces fluorescence in multiple neurons. Tagging of only ftt-2 or hlh-30 does not produce fluorescence, whereas the combination of FTT-2::GFP10 and DAF-16::GFP11 leads to faint cytoplasmic signal. Scale bar 10 μm. All graphs show the quantification of three independent experiments with >50 animals per genotype and assay. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001 by 2way ANOVA with Bonferroni’s multiple comparisons test. DEM, diethyl maleate. Error bars are S.E.M.

Figure 3.. HLH-30 S201A substitution has a minor effect on expression of HLH-30 regulated genes in C. elegans.

Wild type (WT) and hlh-30(S201A) animals were infected with S. aureus for 4 h, fasted for 4 h or kept on E. coli as control. qRT-PCR for each gene was performed in triplicate, except for hlh-30 which was performed in duplicate, using snb-1 as internal control. Graphs represent the primary data and the heatmap of the linear row-normalized data is depicted for comparison. ns, not significant, ** p<0.01 (WT vs S201A under fasting conditions) by two-tailed unpaired t-test. Error bars are S.E.M.

Figure 4.. ftt-2 and par-5 gene downregulation mildly regulates expression of HLH-30-regulated genes in C. elegans.

Wild type animals were subjected to feeding RNAi directed against ftt-2 and par-5; empty vector was used as control. Following RNAi, animals were infected with S. aureus for 4 h, fasted for 4 h, or kept on E. coli as control. qRT-PCR for each gene, as indicated, was performed in duplicate using snb-1 as internal control. Graphs represent the primary data and the heatmap of the linear row-normalized data is depicted for comparison. ns, not significant, * p<0.1, ** p< 0.01, *** p< 0.001 by two-tailed unpaired t-test. Error bars are S.E.M.

Figure 5.. In vivo role of C. elegans HLH-30 Ser201 residue.

a) Survival of hlh-30 mutants to infection by Staphylococcus aureus. Data are the mean ± S.E.M. of three independent experiments initiated with 90 animals per strain. ****p<0.0001 by 2way ANOVA compared to wild type control. b) Synthetic developmental delay phenotype of eat-2(ad1116) worms carrying mutations in the hlh-30 gene. Data are the mean ± S.E.M. of three independent experiments with at least 25 animals per assay. ns, non-significant; ****p<0.0001 by 1way ANOVA with multiple comparison test. c) Effect of hlh-30 mutations on the dauer formation phenotype of daf-2(e1370) worms. Data are the mean ± S.E.M. of four independent experiments. ns, non-significant; ***p<0.001 by 1way ANOVA with multiple comparison test.

Figure 6.. DEM regulates C. elegans HLH-30 subcellular localization through its interaction with FTT-2.

a) DEM but not other stresses cause a transient nuclear localization of the HLH-30::GFP reporter. b) XPO-1 is not a primary target of DEM to regulate HLH-30::GFP subcellular dynamics. c,d) Importin IMB-2 assists HLH-30 nuclear translocation but it is not a DEM target. e) Ser201 residue is needed for transient HLH-30::GFP nuclear localization upon DEM treatment. f) ftt-2 RNAi downregulation abolishes HLH-30::GFP reporter cytoplasmic retrotranslocation upon DEM treatment. All graphs show the quantification of three independent experiments with >50 worms per genotype and assay. ** p<0.01; *** p<0.001; **** p<0.0001 by 2way ANOVA with Bonferroni’s multiple comparisons test. DEM, diethyl maleate. Error bars are S.E.M.

Figure 7.. FTT-2 residue Cys191 regulates transient nuclear localization of HLH-30 upon DEM treatment.

a) Genomic organization of ftt-2 locus. The positions of common FTT-2 Cys96 and isoform specific Cys191 residues are shown in black. The position of Cys167 residue specific for isoform c is shown in red. b) Mutation of ftt-2 Cys96 and/or Cys191 residues does not alter HLH-30 subcellular location in a wild type background. c) Downregulation of par-5 expression delays the cytoplasmic retrotranslocation of HLH-30 when ftt-2 Cys191, but not Cys96, is mutated. All graphs show the quantification of three independent experiments with >50 worms per genotype and assay. ** p<0.01; *** p<0.001; **** p<0.0001 by 2way ANOVA with Bonferroni’s multiple comparisons test. DEM, diethyl maleate. Error bars are S.E.M.