12 research outputs found

    <i>elt-1</i> mutants have an L4-stage bursting vulva and defective alae formation and <i>elt-1(ku491)</i> is a partial loss-of-function allele.

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    <p><sup>a</sup>Phenotypes on empty-vector control RNAi were similar to the standard <i>E</i>. <i>coli</i> strain OP50.</p><p><sup>b</sup>An allele of <i>elt-1(ku491)</i> linked to mutations in <i>unc-24</i> and <i>dpy-20</i> was used for these strains.</p><p><sup>c</sup>For <i>elt-1(ku491)</i> animals vs <i>elt-1(ku491)-over-elt-1(null)</i> animals at the young adult stage, the p-value for the comparison of seam-cell numbers is 0.0340</p><p><sup>d</sup>For <i>elt-1(ku491);daf-12(rh61rh411)</i> animals vs <i>elt-1(ku491)-over-elt-1(null);daf-12(rh61rh411)</i> animals at the young adult stage, the p-value for the comparison of seam-cell numbers is < 0.0001.</p><p><i>elt-1</i> mutants have an L4-stage bursting vulva and defective alae formation and <i>elt-1(ku491)</i> is a partial loss-of-function allele.</p

    LET-7 family miRNAs are decreased in <i>elt-1(ku491);daf-12(rh61rh411)</i> double-mutant animals and ELT-1 binds to the <i>let-7</i> promoter <i>in vivo</i>.

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    <p>A. ELT-1 and DAF-12 redundantly regulate LET-7, miR-48, miR-84, and miR-241. The expression level of target miRNAs was determined by RT-qPCR using synchronous L4 animals. Graphs show mean ± standard error. Detailed descriptive statistics and p-values are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005099#pgen.1005099.s006" target="_blank">S2 Table</a>. B, ChIP-qPCR data showing enrichment of ELT-1 binding to a region in the <i>let-7</i> promoter. Samples from a strain expressing an ELT-1::GFP fusion transgene were subject to immunoprecipitation using either control IgG or anti-GFP antibody. The primers were specific to a region 1.7kb upstream of the <i>let-7</i> transcription start site (Ch. X, 14747074 to 14747179). Graphs show mean ± standard error of triplicate experiments. ELT-1 also binds to promoters of other <i>let-7</i> family genes (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005099#pgen.1005099.t003" target="_blank">Table 3</a>). p-values: *, < 0.05; **, < 0.01; ***, < 0.001; ****, < 0.0001.</p

    Epistasis analysis of <i>elt-1(ku491); daf-12(rh61rh411)</i> mutant phenotypes with RNAi of other heterochronic genes.

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    <p><sup>a</sup>L4 Bursting Vulva, Alae Formation, and Young Adult Seam cell phenotypes were analyzed for strains of indicated genotypes and RNAi treatment. <i>elt-1(rf)</i> is the partial loss-of-function allele <i>ku491</i>. <i>daf-12(lf)</i> is the loss-of-function allele <i>rh61rh411</i>.</p><p><sup>b</sup>For bursting vulva rate, the p-value for the comparison of <i>elt-1(ku491); daf-12(lf)</i> animals on empty-vector (-) RNAi vs target gene RNAi is < 0.0001 for each of <i>lin-28</i>, <i>hbl-1</i>, <i>lin-14</i>, <i>lin-41 and mab-10</i>, 0.003 for <i>lin-46</i>, 0.0002 for <i>lin-42</i>, 0.983 for <i>kin-20</i>, 0.0003 for <i>ceh-16</i>, and 0.315 for <i>dre-1</i>.</p><p><sup>c</sup>For seam cell numbers, the p-value for the comparison of <i>elt-1(ku491);daf-12(lf)</i> animals on empty-vector (-) RNAi vs on target gene RNAi is <0.0001 for <i>lin-28</i>, <i>hbl-1</i>, <i>lin-14</i>, <i>lin-41</i>, <i>lin-46</i>, <i>lin-42</i>, <i>kin-20</i>, <i>mab-10</i>, <i>ceh-16</i>, and <i>dre-1</i>, and > 0.9999 for <i>lin-29</i>.</p><p>Abbreviations: e.v., empty vector; Abs, absent; Gap, gapped; Pres, present; #, seam cell number measured using the <i>scm</i>::<i>GFP</i> reporter; Std Dev, standard deviation.</p><p>Epistasis analysis of <i>elt-1(ku491); daf-12(rh61rh411)</i> mutant phenotypes with RNAi of other heterochronic genes.</p

    ELT-1/GATA acts in parallel to DAF-12/NHR to regulate developmental timing in <i>C</i>. <i>elegans</i>.

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    <p>A-H, DIC and fluorescence images of representative young-adult stage animals with the indicated genotypes, showing that seam cell numbers, visualized with a <i>scm</i>::<i>GFP</i> marker, are drastically increased in <i>elt-1(ku491); daf-12(rh61rh411)</i> double mutants but not in each single mutant. I-K, Scatter plots showing the distribution of the number of seam cells per lateral side for each genotype at L4 and young adult stages; horizontal bar is mean. L, cellular lineage diagrams for mutants, showing variable cell fate defects in the <i>elt-1(ku491)</i> mutants during the L3 and L4 stages. **** indicates p-value < 0.0001. Data for L1 to L3 stages and summary statistics of data for all developmental stages are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005099#pgen.1005099.s001" target="_blank">S1 Fig</a>.</p

    <i>elt-1/(ku491)</i> mutant animals have defective adult alae formation.

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    <p>A-D, DIC and GFP fluorescence images showing adherins junctions of L4 animals with indicated genotypes. Arrow in D marks the gap of AJM::GFP fluorescence. E, Percentage of L4 animals with AJM::GFP fluorescence gaps in animals with the indicated genotypes.</p

    <i>elt-1/GATA</i> promotes the expression of multiple miRNAs that have key roles in the developmental timing regulatory pathway.

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    <p>Proposed role for <i>elt-1/GATA</i> in the heterochronic gene regulatory network. Arrows and T-bars indicated positive and negative regulatory relationships, respectively. Model adapted from Resnick, McCulloch, and Rougvie (2010) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005099#pgen.1005099.ref007" target="_blank">7</a>].</p

    <i>elt-1(ku491);daf-12(rh61rh411)</i> double-mutant animals fail to down-regulate the heterochronic gene <i>lin-41</i> during development.

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    <p>A, Lin-41 mRNA is down-regulated normally during L4 in wild-type and single-mutant animals as measured with RT-qPCR, but <i>elt-1(ku491);daf-12(rh61rh411)</i> double-mutant animals continue to express it at a level indistinguishable from their L1 level. Detailed descriptive statistics and p-values in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005099#pgen.1005099.s004" target="_blank">S4 Fig</a>. B, <i>elt-1(ku491);daf-12(rh61rh411)</i> double-mutant animals fail to down-regulate a <i>lin-41</i> 3’UTR reporter. The number scored per genotype and stage are noted above each column. Representative images of X-gal stain shown at L4 (C-F) and Young Adult (G-J) stages.</p

    Analysis of modENCODE ChIP-Sequencing with ELT-1::GFP for selected genes.

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    <p><sup>a</sup>Transcription factor binding sites within approximately 5kb of target genes are listed. “-”is used to denote genes without putative transcription factor binding sites nearby.</p><p><sup>b</sup>Out of 327 identified TF binding sites,</p><p><sup>c</sup>Out of 2546 identified TF binding sites. Abbreviations: TF Binding Site, Transcription Factor Binding Site; Ch., Chromosome; IDR, Irreproducible Discovery Rate; Anti., antisense.</p><p>Analysis of modENCODE ChIP-Sequencing with ELT-1::GFP for selected genes.</p

    ACEA induces <i>FGF21</i> gene expression.

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    <p>(A–C) HepG2 cells, rat primary hepatocytes (RPH), and AML12 cells were treated with ACEA (10 μM) for the indicated time periods. (D) Wild-type or CB1<sup>-/-</sup> mouse primary hepatocytes (MPH) were treated with ACEA (10 μM) for 3 h. (E) Mice were treated with ACEA (10 mg/kg) for the indicated number of days. Livers were harvested for mRNA analysis. (A–E) <i>FGF21</i> and <i>ERRγ</i> mRNA levels were measured by quantitative qPCR analysis and normalized to <i>actin</i> mRNA levels. All data are the means ± standard errors of at least three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 by one-way ANOVA.</p

    GSK5182 inhibits ACEA-mediated induction of <i>FGF21</i> gene expression.

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    <p>(A) AML12 cells were transfected with mFGF21-Luc and treated with ACEA (10 μM) for 3 h with or without GSK5182 (10 μM). (B–D) HepG2 cells, AML12 cells, and mouse primary hepatocytes (MPH) were treated with ACEA (10 μM) for 3 h with or without GSK5182 (10 μM). (E and G) GSK5182 (40 mg/kg) was administrated to male C57BL/6J mice (n = 3–4 per group) daily by intraperitoneal injection for 4 days. ACEA (10 mg/kg) was also given by intraperitoneal injection daily during the final 3 days. (A–D) <i>FGF21</i> and <i>ERRγ</i> mRNA levels were measured by qPCR analysis and normalized to <i>actin</i> mRNA levels. (F) AML12 cells were treated with ACEA (10 μM) for 3 h with or without GSK5182 (10 μM). Culture media was recovered for FGF21 secretion analysis. (G) Male C57BL/6J mice (n = 3) were treated with ACEA (10 mg/kg) with and without GSK5182 (40 mg/kg) daily for 3 days. Serum was analyzed for FGF21 secretion. (H) Male C57BL/6J mice (n = 5 per group) were fed an alcohol-containing diet for 4 weeks and GSK5182 (40mg/kg once daily) was given by oral gavage for the final 2 weeks of alcohol feeding. (I) Schematic diagram of ERRγ-mediated <i>FGF21</i> gene expression. GSK5182 inhibits activation of <i>FGF21</i> gene expression and FGF21 secretion mediated by increased ERRγ caused by activation of the hepatic CB1 receptor. All data are the means ± standard errors for at least three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 by one-way ANOVA.</p
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