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Fig. S1. Comparison of the distributions of H3K36me2 and MES-4. Tissues from wild-type and mes-4 mutant adult hermaphrodites show antibody staining on the left, and on the right a merge of antibody staining (green) and DNA stained with PL4-2 (red). (A) In wild type, anti-H3K36me2 staining is in all nuclei of the germ line, and also in somatic gut nuclei (arrowheads). Arrows in A and B indicate the start of the transition zone. (B) In a wild-type germ line, the MES-4 pattern is dynamic (Fong et al., 2002). MES-4 levels are highest in the distal mitotic region, drop to nearly undetectable levels at the transition zone, rise modestly during late pachytene, and become markedly enhanced after fertilization (see early embryos in the upper half of panel). MES-4 is not detectable in gut nuclei (arrowheads). (C) In a mes-4(bn23) M+Z− hermaphrodite stained with the antibody against MES-4 fusion protein, MES-4 is undetectable in the distal mitotic region of the germ line. bn23 is a protein-null allele. Scale bar: 20 μm for A,B; 10 μm, for C.
Fig. S2. MES-6 is required for proper patterns of H3K36 dimethylation by MES-4, but MES-4 appears to be unnecessary for proper patterns of H3K27 trimethylation by MES-2/MES-3/MES-6. Samples were stained with antibodies to the indicated histone modifications (center panels, and green in the merge). DNA stained with PA3 is red. (A) (Same image as is shown in Fig. 1D.) In wild-type pachytene nuclei, H3K36me2 is distributed on the autosomes in an irregular pattern and is absent from the pair of X chromosomes (arrows) in each germ nucleus. (B) In mes-6(bn38) M+Z− pachytene nuclei, H3K36me2 is more uniformly distributed on the autosomes and lightly distributed on the presumed X-chromosome pair (arrows). Interestingly, in mes-2, mes-3 and mes-6 M+Z− mutant germ lines, MES-4 is not detectable on the X pair prior to or at the stage shown in B; instead, MES-4 appears on the X pair in late pachytene/diplotene (Fong et al., 2002). The altered pattern of H3K36me2 shown here suggests that the distribution of MES-4 is affected earlier than is seen by MES-4 staining. (C) pgl-3 one-cell embryo. H3K36me2 is unevenly distributed on the autosomes. (D) mes-6(bn38) one-cell embryo. H3K36me2 is more intense and more uniformly distributed on the autosomes. P granules are stained with anti-PGL-3 (left, red in merge, marked by arrowheads). pgl-3 mutants were used in place of wild type, so that we could stain embryos of two genotypes on the same slide and distinguish the genotypes by PGL-3 staining, which is present in mes-6 mutants and absent from pgl-3 mutants (Kawasaki et al., 2004). (E) In wild-type pachytene nuclei, H3K27me3 is more concentrated on the pair of X chromosomes (arrows) than on autosomal pairs. (F) mes-4(bn85) M+Z− pachytene nuclei display a similar concentration of H3K27me3 on the X chromosomes (arrows). Scale bars: 5 μm in C,D; 10 μm in all other panels.
Fig. S3. Methylation by the non-MES-4 H3K36 HMT(s) depends on CDK-9 and TLK-1. Analysis of H3K36me2 levels in ∼100-cell embryos lacking MES-4, CDK-1, TLK-1, or combinations of MES-4 and a kinase. DNA is stained red with PA3; H3K36me2 is green. (A) Wild type. (B) mes-4(bn73). (C) cdk-9(RNAi). (D) tlk-1(RNAi). (E) mes-4(bn73); cdk-9(RNAi). (F) mes-4(bn73); tlk-1(RNAi). mes-4 embryos show reduced levels of H3K36me2 compared with wild type. mes-4 embryos depleted of either CDK-9 or TLK-1 lack a detectable H3K36me2 signal. Scale bar: 10 μm.
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