Fig. 1
Structure and functions of the histone methyltransferases PaKmt1 and PaKmt6. a Domain structure of histone methyltransferases PaKmt1 and PaKmt6. Sizes in amino acid (aa) are given (right). PRE-SET (red, IPR007728), SET (orange, IPR001214) and POST-SET (green, IPR003616) conserved domains are required for H3K9 methyltransferase activity of KMT1 homolog proteins. CXC (yellow, IPR026489) is a cysteine-rich conserved domain located in the H3K27 methyltransferase catalytic domain of KMT6 homologs. b Phylogenetic analysis of KMT1 and KMT6 histone methyltransferase homologs. This tree regroups H3K9 (blue) and H3K27 (red) methyltransferase proteins from fungi, plants and metazoans. They cluster in two different clades of histone methyltransferases. Their evolutionary history is canonical since the topology of the two branches of the tree is consistent with the species tree. Bootstraps are given. Filamentous fungi: Podospora anserina (Pa), Neurospora crassa (Nc), Fusarium graminearum (Fg), Trichoderma reesei (Tr), Epichloë festucae (Ef) Botrytis cinerea (Bc), Magnaporthe oryzae (Mo), Zymoseptoria tritici (Zt), Leptosphaeria maculans (Lm), Aspergillus nidulans (An), Aspergillus fumigatus (Af), Penicillium oxalicum (Po), Ascobolus immersus (Ai), Puccinia graminis (Pg), Pneumocystis jirovecii (Pj), Conidiobolus coronatus (Cc), Mucor circinelloides (Mc); yeasts: Schizosaccharomyces pombe (Sp) and Cryptococcus neoformans (Cn); the worm Caenorhabditis elegans (Ce), the fruit-fly Drosophila melanogaster (Dm), the mouse Mus musculus (Mm) and the model plant Arabidopsis thaliana (At). Accession numbers for proteins used in alignments are listed in Additional file 24: Table S6. c Detection of histone post-translational modifications Western-immunoblot analysis of H3K9me3, H3K27me3 and H3K4me3 modifications in wild-type (WT); single mutants ΔPaKmt1, ΔPaKmt6; double mutant ΔPaKmt1ΔPaKmt6; complemented strains ΔPaKmt1-PaKmt1+ (PaKmt1, PaKmt1-GFP-HA), ΔPaKmt6-PaKmt6+ (PaKmt6, PaKmt6-HA). Nuclear protein samples were extracted from protoplasts. A total of 35 µg of nuclear protein was loaded in each lane. Antibodies were directed against native H3 histone (H3), H3K9me3, H3K27me3 and H3K4me3 modifications. We detected some marginal cross-reactivity between the anti-H3K9me3 antibody and the H3K27me3 modification (evaluated at 3%, see Additional file 7: Fig. S7). Expected size of histone 3 is 17 kDa (arrows indicated 15 kDa)