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. 2016 Sep 7;5:e15477. doi: 10.7554/eLife.15477

Figure 4. The H3K27 demethylase UTX-1 is required for GeCo enhancement.

(A) UTX-1 is critical for GeCo enhancement. Candidate Notch-activated genes, selected from Supplementary file 2 with available RNAi clones, were assayed for a role in GeCo in glp-1(ar202) animals, expressing CHE-1–induced nGFP and treated with lin-53 RNAi. While the additional depletion of utx-1 had the strongest impact on GeCo+ and GeCo, the depletion of C07G1.6 and aldo-1 had a weaker effect. Representative fluorescence micrographs are below the quantification chart. White dashed line outline the animal body, yellow lines outline gonadal areas with GeCo. P-values for GeCo+ were calculated using Student's t-test: p1=0,000013; p2=0,026; p3=0,021; p4>0,1. At least 250 animals were scored per condition. Error bars represent SEM. nGFP = gcy-5::gfp. Scale bars = 10 μm. (B) As in A, but RNAi was performed against jmjd-1.2 (H3K9/27me2 demethylase); jmjd-3.1, jmjd-3.2, and jmjd-3.3, (H3K27me2/3 demethylases); and jmjd-2 (H3K9/36 demethylase). Only RNAi against jmjd-1.2 suppresses GeCo+, though to a lesser degree compared to utx-1 RNAi. Representative fluorescence micrographs are below the quantification chart. P-values for GeCo+ were calculated using Student's t-test: p1=0,0042; p2=0,035; p3>0,2. At least 190 animals were scored per condition. Error bars represent SEM. Scale bars = 10 μm.

DOI: http://dx.doi.org/10.7554/eLife.15477.016

Figure 4—source data 1. Quantification of GeCo upon double RNAi against lin-53 and Notch-activated genes.
(A) Quantification of GeCo+ upon RNAi against lin-53 and Notch-activated genes. (B) Quantification of GeCo+ upon RNAi against lin-53 and Histone demethylases. Figure 4—figure supplement 1A source data: Quantification of GeCo+ upon RNAi against lin-53 and utx-1 with and without rrf-1 (pk1417) background. More details can be found in the corresponding figure legends.
DOI: http://dx.doi.org/10.7554/eLife.15477.017
elife-15477-fig4-data1.xlsx (65.3KB, xlsx)
DOI: 10.7554/eLife.15477.017

Figure 4.

Figure 4—figure supplement 1. UTX-1 is required for the GeCo+ enhancement upon the depletion of PRC2.

Figure 4—figure supplement 1.

(A) Adult glp-1(ar202) animals treated with lin-53 or mes-3 RNAi were additionally subjected to either control or utx-1 RNAi. Depletion of utx-1 strongly suppressed the GeCo+ phenotype. Suppression upon utx-1 co-depletion with lin-53 is also detectable in the rrf-1(pk1417) background which is permissive for RNAi in the germline but not in the somatic gonad and the DTC. Right: quantification: n = 715 were scored for lin-53; utx-1 RNAi; n = 500 for mes-3; utx-1 RNAi and n = 270 were scored for lin-53; utx-1 RNAi in rrf-1(pk1417). P-values were calculated using Student's t-test: p1=0,0588; p2=0,0042; p3=0,2454; p4=0,01713; p5=0,40479; p6=0,00271. Error bars represent SEM. (B) anti-HA antibody staining for the 3xHA-tagged CHE-1 protein, which is being induced after heat-shock treatment in the different genetic backgrounds: glp-1(ar202); otIs305 (hspprom::che-1::3xHA) ntIs1 (gcy-5prom::gfp) treated with our without RNAi against lin-53 and utx-1. As additional controls the strain otIs305 (hspprom::che-1::3xHA) ntIs1 (gcy-5prom::gfp) with or without lin-53 RNAi and heat shock treatment was used. No obvious changes in the induction of CHE-1::3xHA in the germlines of the different genetic backgrounds can be detected. Scale bars = 1 μm.
DOI: http://dx.doi.org/10.7554/eLife.15477.018