Fig 1: The SET-4 histone methyltransferase is necessary to generate H4K20me2 and H4K20me3.(A) Wild-type (WT) and set-4(n4600) mutant embryos were stained for DNA (DAPI), and H4K20me1, H4K20me2, or H4K20me3. H420me1 levels are higher and H4K20me2 and H4K20me3 levels strongly reduced in set-4 mutant embryos. Scale bar, 10 um. (B) Enlargements of nuclei in wild-type and set-4(n4600) mutant embryos stained for DNA (DAPI), H4K20me1, and DPY-27. In set-4 embryos, H4K20me1 does not show enrichment in DPY-27 positive regions. Confocal settings for H4K20me1 in set-4 were reduced to allow comparison with wild-type images. Scale bar, 2 um. (C) Western blots of extracts made from wild-type and set-4 adults, probed for H4K20me1, H420me2, or H4K20me3. Histone H3 is a loading control. Compared to wild type, set-4 mutants have increased H4K20me1 levels, and reduced H4K20me2 and H4K20me3 levels. (D, E) Western blots of H4K20me1 (D) and H4K20me3 (E) in wild-type and dpy-21 L3 mutant extracts. Lower panels show H3 loading controls. (D) H4K20me1 abundance is lower in dpy-21 than in wild type. (E) H4K20me3 abundance is higher in dpy-21 mutant L3 extract than in wild type. In (D) RNAi of set-4 was additionally performed. H4K20me1 abundance is elevated in both wild-type and dpy-21 after depletion of set-4. The following primary antibodies were used: H4K20me1 (Diagenode SN-147), H420me2 (Kimura 2E2), H4K20me3 (Abcam ab78517).