pgen.1008508.g004.tif (573.43 kB)

MDT-15 and HIZR-1 are co-dependent for metal responsive gene induction and physically bind in cadmium and zinc-enhanced fashion.

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posted on 2019-12-09, 18:31 authored by Naomi Shomer, Alexandre Zacharie Kadhim, Jennifer Margaret Grants, Xuanjin Cheng, Deema Alhusari, Forum Bhanshali, Amy Fong-Yuk Poon, Michelle Ying Ya Lee, Anik Muhuri, Jung In Park, James Shih, Dongyeop Lee, Seung-Jae V. Lee, Francis Christopher Lynn, Stefan Taubert

[A-B] qRT-PCR analysis of zinc and cadmium-responsive genes. The graphs show fold-inductions of mRNAs normalized to the average of wild-type control(RNAi) worms. Error bars: SEM. Statistical analysis: * p < 0.05, ** p < 0.01, unpaired Student’s t-test for indicated comparisons. [A] Comparison of wild-type and hizr-1(am285) gf worms grown on control or mdt-15 RNAi (n = 5 independent repeats). [B] Comparison of wild-type and mdt-15(yh8) gf worms grown on control, hizr-1, or nhr-49 RNAi (n = 3–4 independent repeats). [C-E] Protein-protein interaction analysis using the Y2H system. Graphs show average interaction strength (arbitrary units, A.U.). Error bars: standard deviation. [C] Interaction between MDT-15 and HIZR-1, with Empty Vector (EV)–HIZR-1 and MDT-15–EV as negative controls, and MDT-15–SKN-1c as a positive control (n = 3 independent repeats). Statistical analysis: n.s. = not significant; ** p < 0.01, *** p < 0.001, **** p < 0.0001, One-way ANOVA, multiple comparisons, Tukey correction. All comparisons for “MDT-15 + EV” unless indicated by lines. Expression of HIZR-1 prey fusion proteins is shown in panel A of S1 Fig. [D] Average interaction strength between MDT-15 and HIZR-1; MDT-15 and NHR-64; and MDT-15 and NHR-49, all with no treatment, or treated with 200μM zinc, 5μM cadmium, or 200μM manganese (n = 3 independent repeats). Statistical analysis: n.s. = not significant; **** p < 0.0001, One-way ANOVA, multiple comparisons, Sidak correction; all comparisons to the pertinent “no supplement” control. Expression of HIZR-1, NHR-64, and NHR-49 prey fusion proteins is shown in panel B of S1 Fig. [E] Interaction between MDT-15 bait and HIZR-1 prey without zinc supplementation and with 1, 5, 10, 25, or 50μM supplementary zinc (n = 3 independent repeats). Statistical analysis: n.s. = not significant; *** p < 0.001, **** p < 0.0001, One-way ANOVA, multiple comparisons, Dunnett correction; all comparisons to “0μM zinc”. Expression of HIZR-1 prey fusion proteins is shown in panel C of S1 Fig. [F] The diagram shows the MDT-15 deletions analyzed for HIZR-1 binding in [G]. Numbers indicate amino acids; the N-terminal, NHR-binding KIX-domain (“KIX”) and the C-terminal autoactivation domain (“CT”) are shaded gray. [G-H] Protein-protein interaction analysis using the Y2H system. Graphs show average interaction strength (arbitrary units, A.U.). Error bars: standard deviation. [G] Interaction between empty vector (EV) or MDT-15 mutant (MDT-15ΔCT, MDT-15-KIX, or MDT-15ΔKIXΔCT) baits and a full-length WT HIZR-1 prey (n = 3 independent repeats). Statistical analysis: n.s. = not significant, **** p < 0.0001, One-way ANOVA, multiple comparisons, Sidak correction; comparisons are to “MDT-15ΔCT” unless indicated by lines. Expression of HIZR-1 prey fusion proteins is shown in panel D of S1 Fig. [H] Interaction between HIZR-1 and MDT-15 or HIZR-1-D270N and MDT-15 with and without zinc treatment (n = 4 independent repeats). Statistical analysis: n.s. = not significant, ** p < 0.01, *** p < 0.001, One-way ANOVA, multiple comparisons, Sidak correction; comparisons to “HIZR-1” unless indicated by lines. Expression of HIZR-1 prey fusion proteins is shown in panel E of S1 Fig.