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. 2018 Apr 17;7:e35037. doi: 10.7554/eLife.35037

Figure 2. A forward genetic screen identifies C. elegans isy-1 as a causal regulator of asp-17::GFP expression.

(A) Schematic of the C. elegans ISY-1 protein showing the domain structure predicted by SMART (top) (http://smart.embl-heidelberg.de) and a multiple sequence alignment of ISY-1 homologues from major metazoans showing the conservation of the glutamate residue substituted to lysine by the dma50 mutation isolated from EMS screens. (B) Nomarski and fluorescence images showing the phenotype of intestinal asp-17p::GFP in wild type and dma50 mutants. (C) Fluorescence images showing the distribution of ISY-1::GFP driven by the endogenous isy-1 promoter. Arrows indicate neuronal, hypodermal and intestinal nuclei. (D) Nomarski and fluorescence images showing intestinal asp-17p::GFP in a transgenic strain expressing RNAi against isy-1 specifically in intestine. (E) Schematic of the C. elegans isy-1 gene with mCherry tagged at the C-terminus (top); Nomarski and fluorescence images showing rescue of dma50 by various transgenes (below). (F) Quantification of fluorescence intensities showing rescue of dma50 in asp-17p::GFP activation. N ≥ 3 independent biological replicates; *** indicates p<0.001. Scale bar: 20 µm.

Figure 2.

Figure 2—figure supplement 1. EMS screens identified isy-1 as a regulator of asp-17.

Figure 2—figure supplement 1.

(A) Fluorescence images showing up-regulation of asp-17p::GFP but not the co-injection marker unc-54p::mCherry by CW. (B) Schematic illustrating the workflow of EMS mutagenesis to isolate mutants with abnormal asp-17p::GFP expression. (C) Table showing the mutants isolated from the screen, including dma50 that defines the gene F53B7.3, named as isy-1. (D) Chromosome genetic maps showing DraI SNP sites and the mapped interval for dma50. (E) Table listing candidate genes with mutations identified by WGS, with two genes highlighted in yellow within the mapped interval. (F) fluorescence images showing that RNAi of isy-1 phenocopies dma50. N ≥ 3 independent biological replicates; *** indicates p<0.001. Scale bar: 20 µm.

Figure 2—figure supplement 2. ISY-1 is a C. elegans member of evolutionarily conserved protein family and its level is not regulated by cold-warming.

Figure 2—figure supplement 2.

(A) Multiple sequence alignment (Cluster Omega, visualized by Jalview) of ISY-1 family members in major metazoan species. (B) Cladogram of ISY family proteins. (C) Sanger sequencing confirmation of isy-1(dma50). (D) RNA-seq measurements of isy-1 and asp-17 levels under CW conditions and in isy-1(dma50) mutants. (E) Western blot of isy-1p::ISY-1::mCherry showing no regulation by CW. N ≥ 3 independent biological replicates; *** indicates p<0.001.