The catalytic activity of JMJD-5 is required for proper HR.
(A) Left. Western blots of the indicated histone modifications using lysates from N2 and jmjd-5(tm3735) 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, jmjd-5(tm3735) and jmjd-5(DD) 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 jmjd-5 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. pig-1(gm344) 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 jmjd-5(DD) 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 jmjd-5(DD) 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.