Skip to main content
. 2016 Sep 7;5:e15477. doi: 10.7554/eLife.15477

Figure 5. UTX-1 is regulated by GLP-1Notch and PRC2.

(A–C) Expression of utx-1 is regulated by PRC2 and GLP-1Notch. Top: dissected gonads expressing a GFP reporter, driven from the utx-1 promoter (putx-1::GFP, fused to histone 2B for nuclear localization to facilitate quantification), subjected to the indicated RNAi (A), crossed into the indicated genetic background (B) or carrying the indicated mutations in the reporter gene (C). a, b, and c indicate gonadal regions containing germ cells in mitosis (a), and leptotene/zygotene (b) or pachytene (c) stages of meiosis. Below: the corresponding GFP quantifications. The diagrams show GFP intensities relative to the control (indicated by green arrows) in regions a-c. Results are represented as average changes in the GFP intensity (relative to mock RNAi-ed or untreated animals). The error bars represent SEM. The numbers of analyzed gonads were as follows: n = 44 for wild-type reporter; n = 36 for glp-1(ar202); n = 55 for wild-type reporter on control RNAi; n = 48 for mes-2 (RNAi); n = 15 for mes-3 (RNAi); n = 29 for mes-6 (RNAi), and n = 20 for the LAG-1 binding sites deleted reporter. (A) The putx-1::GFP reporter is repressed by PRC2. In all mes-depleted gonads, the reporter was de-repressed in proliferating cells (a) as well as in more proximal gonadal regions (b-c). (B) The putx-1::GFP reporter is upregulated by GLP-1Notch. In the gain-of-function glp-1(ar202) mutant, the reporter was strongly derepressed in the proliferating cells in the distal-most gonad (a). Its expression was also increased in the more proximal regions (b-c), which, in this mutant background, contain proliferating cells instead of meiotic cells. (C) putx-1::GFP expression depends on the predicted LAG-1/CSL binding sites in the promoter. Upon deletion of putative LAG-1/CSL binding sites, the reporter expression was abolished. The changes in GFP intensities were highly significant (p-values were measured by independent t-tests) p1=4.85–13, p2=1.38–20, p3=1.18–7. (D) LAG-1 binds the utx-1 promoter. Lysates of animals expressing FLAG-tagged LAG-1 (strain wgIs591; lag-1::TY1::EGFP::3xFLAG), either in wild-type or glp-1(ar202) background, were subjected to ChIP-qPCR analysis of the indicated genes. Negative controls and additional tested genes are shown in Figure 5—figure supplement 3. The qPCR amplicons were tested in at least three independent experiments. The results are shown as fold enrichment in anti-FLAG IP compared to IP with unspecific antibody. The 3’UTR of lst-1 serves as a negative control. Interestingly, LAG-1 binding in the glp-1(ar202) gain-of-function background is stronger to the utx-1 promoter than to the reported GLP-1Notch targets lst-1 and sygl-1. The asterisk indicates a p-value < 0.05 (Students t-test). Error bars represent SEM.

DOI: http://dx.doi.org/10.7554/eLife.15477.019

Figure 5.

Figure 5—figure supplement 1. The functional utx-1 transgene is expressed in the same pattern as a GFP reporter coupled to the utx-1 promoter.

Figure 5—figure supplement 1.

Shown is an adult with an outlined gonad. The full-length GFP-tagged functional UTX-1 (rrrSi189) is repressed in the distal, proliferative part. Nuclei entering meiosis and developing oocytes express the fusion protein progressively stronger. This expression pattern is identical with the pattern seen in the utx-1 promoter reporter strains (rrrSi185, rrrSi281).
DOI: http://dx.doi.org/10.7554/eLife.15477.020
Figure 5—figure supplement 2. The endogenous utx-1 mRNA is upregulated in the absence of the PRC2-component MES-6.

Figure 5—figure supplement 2.

Shown are representative in-situ hybridisations against endogenous utx-1 mRNA in dissected gonads. In M+Z- mes-6(bn66) mutants, utx-1 is upregulated throughout the gonads compared to the control wild-type gonads. 'AS' and 'S' indicate antisense and sense probes.
DOI: http://dx.doi.org/10.7554/eLife.15477.021
Figure 5—figure supplement 3. Testing LAG-1 binding to additional genes by ChIP.

Figure 5—figure supplement 3.

Worm lysates (corresponding to 4 mg protein) of animals, with or without lag-1::TY1::EGFP::3xFLAG (wgIs591) transgene, were used for ChIP. Samples were incubated with 50 µl of FLAG (‘specific’ FLAG antibody) or HA antibodies (‘unspecific’ HA antibody) coupled to µMACS microbeads (Milteny). As negative control, lysate N2 or glp-1(ar202) worm lysates, which do not express the recombinant target protein, were used. Both negative control lysates did not show any differences during the ChIP experiment when tested with either specific antibody (anti-FLAG coupled to µMACS beads) or unspecific antibody (anti-HA coupled to µMACS beads). Lysates of worms expressing the recombinant target protein in N2 or glp-1(ar202) background were incubated with specific (anti-FLAG) and unspecific (anti-HA) antibodies coupled to µMACS beads. The qPCR amplicons were tested in a minimum of three independent ChIP-qPCR experiments. Quantification results are shown as fold enrichment of anti-FLAG µMACS™ beads using wgIs591 lysate over anti-FLAG µMACS beads using lysate without wgIs591 (no lag-1::TY1::EGFP::3xFLAG). Primer for qPCRs (sequence details above) were designed using Primer3Plus (Untergasser et al., 2007). The FLAG-beads using lysates with lag-1::TY1::EGFP::3xFLAG show specific enrichment for tested target genes thereby validating the specificity of the ChIP. The asterisks indicates p-values < 0.05 (Students t-test). Error bars represent SEM.
DOI: http://dx.doi.org/10.7554/eLife.15477.022