<i>utx-1</i> postembryonic phenotypes.

<p>(A) Representative DIC images of L1 larvae tails (upper panel) and L1 larvae (lower panel) of N2, <i>utx-1(RNAi)</i> and <i>utx-1(tm3118)</i> animals. Animals are oriented anterior to the left. Scale bar is 50 µm. (B) Representative DIC images of gonads in adults N2, <i>utx-1(RNAi)</i> and <i>utx-1(tm3118)</i> animals. Scale bar is 25 µm. In the upper panels, blacks lines indicate the migration of the gonad arm. In the lower panels, black lines indicate the contours of the oocytes. Animals are oriented anterior to the left. (C) Percentages of posterior (% tail defects) and gonad (% gonadal defects) defects in the indicated strains are shown. For <i>RNAi</i>, F1 larvae and adults from at least three independent experiments were analyzed. (D) <i>utx-1</i> and <i>jmjd-3.1</i> mRNA levels in embryos after control (black bars) or <i>utx-1</i> (white bars) <i>RNAi</i> treatment as measured by qPCR, using <i>rpl-26</i> mRNA as internal control. *P<0.01 (Student's t-test).</p

UTX-1 is an H3K27me2/3 demethylase.

<p>(A) Protein lysates from embryos of the indicated strains were probed with antibodies against H3K27me3 and H3K27me2. H3 was used as loading control. Quantification of the western blot is shown in the graphic on the right. Error bars indicate the standard deviation calculated using at least 2 replicates. The signals were quantified using ImageJ software and normalized to H3. The values are relative to N2 levels. Similar results were obtained with at least two different transgenic lines and in the two <i>utx-1</i> genetic backgrounds (<i>tm3118 and tm3136</i>). (B) Representative images of N2 expressing a translational construct for wild-type <i>(N2+UTX-1::GFP)</i> and catalytically inactive UTX-1 <i>(N2+UTX-1DD::GFP)</i> GFP fusion and GFP-negative siblings, fixed and stained with H3K27me3 antibody and DAPI. The white square encloses an intestinal cell, used for the H3K27me3 quantification. Enlargement of the white square is shown at the bottom of the panel. Quantification of H3K27me3 levels is shown in the graphic on the right. At least 25 cells for each genotype were quantified as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002647#s4" target="_blank">Materials and Methods</a>. Mean fluorescence + s.e.m. values of two independent experiments are displayed. *P<0.01. (Student's t-test). Animals are oriented anterior to the left. (C) Top: Alignment of a part of the Jumonji C domain of human UTX with UTX-1 and with the catalytically inactive UTX-1DD (DD = <u>D</u>emethylase <u>D</u>ead). Asterisks indicate two of the three conserved amino acids in the iron-binding domain (HXD/EX_nH) of the JmjC-domain, modified in the UTX-1DD. Bottom: Epifluorescence of <i>utx-1</i> mutant animals, carrying a translational GFP fusion of wild-type UTX-1 (<i>utx-1+UTX-1::GFP</i>) or catalytically inactive UTX-1 (<i>utx-1+UTX-1DD::GFP</i>). Anterior parts of the animals are shown, with anterior to the left. On the right, lysates from L1 carrying the two transgenes were probed with GFP antibody. Actin was used as loading control. The signal was quantified using ImageJ program and normalized to actin.</p

<i>jmjd-5</i> is not required for DNA damage checkpoints activation.

<p>(A) Number of mitotic cells in N2 and <i>jmjd-5(tm3735)</i> after irradiation of young adults with 40 and 120 Gy, counted 24 h after IR, relative to the non treated (NT) animals. (B) Representative images of germlines dissected from N2 and <i>jmjd-5</i> animals, 24 hours after irradiation with 0 (control) or 80 Gy (+IR), stained with DAPI. Arrowheads indicate arrested and enlarged nuclei. Scale bar: 5 μm. (C) Quantification of apoptotic cells in the indicated strains after staining with SYTO12, 24 hours after IR treatment (80Gy). NT, not treated. Between 40 and 78 animals were scored for genotype and condition. ****<i>p</i>< 0.0001, n.s. = not significant, with two-tailed, unpaired t-test. (D) Relative mRNA expression levels of the pre-apoptotic genes <i>egl-1</i> and <i>ced-13</i> in the indicated strains, 24 hours after IR treatment, measured by qPCR. Values are normalized to untreated samples and <i>act-1</i> is used as internal control. Graph is an average of two biological independent experiments ± sem. <i>cep-1(gk138)</i> is used as control.</p

Aberrant RAD-51 staining in <i>jmjd-5</i> mutant animals.

<p>(A) Schematic representation of a <i>C</i>. <i>elegans</i> gonad arm, showing the position of the 7 zones scored for RAD-51 foci. TZ is the transition zone. Red dots represent RAD-51 foci distribution, according to literature. (B) Top. Histograms showing the quantification of RAD-51 foci in the extracted germlines of N2 and <i>jmjd-5(tm3735)</i> animals. Numbers in parenthesis indicate the number of nuclei analyzed. Bottom. Representative images of RAD-51 (red) and DAPI (blue) staining in germlines dissected from N2 and <i>jmjd-5(tm3735)</i> animals grown at 20°C. Nuclei from zones 4, 5, 6 and 7 are shown. (C) Top. Quantification of RAD-51 foci per nucleus in zones 6 and 7 of N2 and <i>jmjd-5(tm3735)</i> germlines, treated or not with IR (30Gy, 6 hours after the IR). At least 40 nuclei, from a minimum of 4 germlines for each genotype, were quantified for each zone and genotype. ****<i>p</i>< 0.0001, **<i>p</i>< 0.001, with two tailed unpaired t-test. Bottom. Representative images of RAD-51 (red) and DAPI (blue) staining in germlines dissected from N2 and <i>jmjd-5</i> animals, 6h after IR (30Gy). Nuclei from zones 6 and 7 are shown. In (B) and (C), each image shows a projection of multiple z-stacks (0.2 μm spacing) of the entire nuclei. 100X magnification, scale bar 2 μm.</p

UTX-1 expression and <i>utx-1</i> embryonic phenotypes.

<p>(A) Top: Human UTX and the <i>C. elegans</i> homologue UTX-1. TPRs, tetratricopeptide repeats; JmjC, Jumonji C domain. Bottom: Genomic organization of <i>utx-1</i>. Black H-shaped lines indicate the position of the <i>tm3136</i> and <i>tm3118</i> deletions. Black lines indicate the position of the different <i>RNAi</i> fragments (a, b, and c) used in this study. (B) UTX-1 expression during embryogenesis analyzed by immunostaining with an anti-UTX-1 antibody (top panel, right) and by epifluorescence (bottom panel, right). DAPI staining and Nomarski (DIC) images are also shown on the left. (C) UTX-1 expression by epifluorescence (right panels) during larval development. Nomarski (DIC) images are shown on the left panels. Asterisks indicate the distal tip cell, arrow head the forming vulva. Animals are oriented anterior to the left. (D) Brood size of wild type, <i>utx-1</i> mutant worms and rescued <i>utx-1</i> lines. Progeny is given as the average number of viable progeny per worm ± SD. The number of laid, not hatched, eggs counted in <i>utx-1 (m+/z−)</i> mutants is reported in the lower table. <i>utx-1+UTX-1::GFP</i> and <i>utx-1+UTX-1DD::GFP</i>, are <i>utx-1</i> transgenic lines expressing wild-type or catalytically inactive mutant of UTX-1, respectively, as extrachromosomal arrays. (E) Representative Nomarski (DIC) images of a <i>utx-1(tm 3136)</i> mutant embryo and escaper L1 larva. Similar phenotypes are observed in <i>utx-1(tm 3118)</i> (not shown). Bars in B and E are 20 µm, in C 50 µm. Animals are oriented anterior to the left.</p

Functions of the SET-16/UTX-1 complex.

<p>(A) (Upper panel) DIC images of <i>set-16(n4526)</i> and <i>pis-1(ok3720)</i> mutant larvae. Arrowhead indicates misshapen tails. Scale bar is 50 µm. (Lower panel) DIC images of <i>set-16(RNAi)</i> and <i>pis-1(ok3720)</i> adults. Gonadal migration defects are shown. The black line indicates the aberrant gonadal migration. Scale bar is 20 µm. Animals are oriented anterior to the left. The percentage of tail and gonadal defects associated to loss or reduction of <i>set-16</i> and <i>pis-1</i> are reported on the right. (B) Percentage of tail and gonad defects after <i>RNAi</i> of the indicated genes. F1 L1 larvae and adult animals from at least two independent experiments were scored. (C) Percentage of tail and gonad defects after <i>RNAi</i> of the indicated genes. F1 or F2 L1 larvae and adult animals from at least two independent experiments were scored. (D) <i>utx-1</i> and <i>set-16</i> mRNA levels in embryos of worms treated with control, <i>utx-1</i> or <i>set-16(RNAi)</i>. *P<0.01 (Student's t-test). (E) Protein lysates of embryos from worms treated with control, <i>utx-1</i> or <i>set-16(RNAi</i>) were probed with an antibody against UTX-1. Actin was used as loading control. The signal was quantified using ImageJ program and normalized to actin. Indicated values are relative to <i>control (RNAi)</i> and derive from two independent experiments.</p

UTX-1 is part of a MLL-like complex.

<p>(A) Immunoprecipitation of GFP tagged UTX-1 from a mixed population (eggs and adults) of <i>utx-1(tm3118)</i> rescued with UTX-1::GFP. Affinity purified proteins were resolved by SDS-PAGE and stained with colloidal Coomassie. Homologues of the mammalian UTX-MLL complex co-eluted with the bait and identified by LC-MS/MS are listed in grey. Position of the bait protein is shown in black. Molecular weight markers are indicated to the left of the gel. (B) Table summarizing the identified homologues of the components of the mammalian UTX-MLL complex. Gene names, molecular weight in kDa, number of unique peptides and sequence coverage in percentage are reported. (C) Co-immunoprecipitations of WDR-5.1::HA and UTX-1. Total protein extracts from eggs of the indicated strains were immunoprecipitated using anti-HA affinity gel beads. The precipitates were analyzed by SDS-PAGE followed by western blotting using antibodies against HA, GFP or endogenous UTX-1. Input = 30 µg of protein extract. NBF = non bound fraction. (D) UTX-1-associated protein complex assessed by size exclusion chromatography. Superose 6 gel filtration of total protein extracts derived from UTX-1 mutant rescued with wild-type <i>(utx-1+UTX-1::GFP)</i> and catalytically inactive UTX-1 <i>(utx-1+UTX-1DD::GFP)</i>. Fractions were analyzed by western blotting using GFP and actin antibodies.</p

<i>jmjd-5</i> mutant animals are hypersensitive to IR.

<p>(A) Genomic structure of the <i>jmjd-5</i> gene. Dark boxes represent coding sequences and lines represent introns. The black H-shaped bar specifies the deletion in the <i>tm3735</i> allele and the asterisks indicate the position of the two amino acids (H484 and D486) located in the JmjC domain, mutagenized in <i>jmjd-5(DD)</i>. (B) Quantification of <i>jmjd-5</i> mRNA levels by qRT-PCR in the indicated strains. <i>rpl-26</i> is used as internal control. (C) Percent of embryonic lethality in the indicated strains observed after irradiation of young adult worms (24 h post L4) with 0, 40, or 80 Gy of IR. (D) Brood size in N2 and <i>jmjd-5(tm3735)</i> after IR treatment of young adults with 0, 80, or 120 Gy. Deposited embryos are counted from the time of irradiation. (E) Percentage of fertile animals in N2 and <i>jmjd-5(tm3735)</i> (evaluated by the presence of embryos in the uterus) among the living F1 progeny of irradiated mothers. (F) Number of living F1 animals in the indicated strains after irradiation of L1 larvae with 0, 80, or 100 Gy of IR. L1 were irradiated and the total number of living F1 offspring that passed the L1 stage is reported. (G) Percent of embryonic lethality in the indicated strains observed after treatment of young adult worms with 0 or 200 J/m² of UV. (H) Percent of irradiated L1 of the indicated strains that developed to the L4 stage in 48 hours. In C-H, <i>pig-1(gm344)</i>, <i>lin-61(n3809)</i>, <i>atm-1(gk186)</i>, <i>cku-70(tm1524)</i> and <i>xpa-1(ok698)</i> are used as positive controls. In B-H, data are represented as means ± sem from at least 3 biological independent experiments. ****<i>p</i>< 0.0001, ***<i>p</i><0.005, **<i>p</i><0.01, n.s. = not significant, with two-tailed, unpaired t-test.</p

Embryonic lethality and male production in <i>jmjd-5</i> mutant animals.

<p>Embryonic lethality (Emb) and male production of the indicated genotypes were measured at 20°C and after growing the animals at 25°C for 5 generations.</p

The catalytic activity of JMJD-5 is required for proper HR.

<p>(A) Left. Western blots of the indicated histone modifications using lysates from N2 and <i>jmjd-5(tm3735)</i> animals. H3 is used as loading control. Right. Quantification of H3K36 methylation levels, normalized to H3. Data are presented as a mean ± sem of three independent biological experiments. **p<0.01 with two tailed unpaired t-test. (B) Representative images of indicated germline regions of N2, <i>jmjd-5(tm3735)</i> and <i>jmjd-5(DD)</i> animals stained with DAPI (red) and H3K36me2 antibody (green). Overlay is on the right. Asterisks indicate distal tip cells, arrowheads indicate regions devoid of H3K36me2. (C) Quantification of average H3K36me2 intensity per nucleus in mitotic and pachytene regions of N2 and <i>jmjd-5</i> mutants. y-axis shows fluorescence intensity, expressed in arbitrary units. At least 10 gonads were quantified for each strain. Data are presented as mean ± sem. ****p< 0.0001, **p<0.01 with two tailed unpaired t-test. (D) Percent embryonic lethality in the indicated strains after treatment of young adults worms with 80 Gy. <i>pig-1(gm344)</i> is used as positive control. Data are represented as means ± sem of two independent biological experiments. **p<0.01 with two tailed unpaired t-test. (E) Top. Histograms showing the quantification of RAD-51 foci in germlines extracted from <i>jmjd-5(DD)</i> animals grown at 20°C (left) or at 25°C (right) for five generations. Numbers in parenthesis indicate the number of nuclei analyzed. Bottom. Representative images of RAD-51 (red) and DAPI (blue) staining in germlines of <i>jmjd-5(DD)</i> at 20°C (left) and 25°C (right). Nuclei from zones 4, 5, 6 and 7 are shown. In (B) and (E) each panel shows a projection of multiple z-stacks (0.2 μm spacing) of the entire nucleus. 100X magnification, scale bar 2 μm.</p