Comparative Analysis of DNA Methyltransferase Gene Family in Fungi: A Focus on Basidiomycota

Abstract

DNA methylation plays a crucial role in the regulation of gene expression in eukaryotes. Mushrooms belonging to the phylum Basidiomycota are highly valued for both nutritional and pharmaceutical uses. A growing number of studies have demonstrated the significance of DNA methylation in the development of plants and animals. However, our understanding of DNA methylation in mushrooms is limited. In this study, we identified and conducted comprehensive analyses on DNA methyltransferases (DNMtases) in representative species from Basidiomycota and Ascomycota, and obtained new insights into their classification and characterization in fungi. Our results revealed that DNMtases in basidiomycetes can be divided into two classes, the Dnmt1 class and the newly defined Rad8 class. We also demonstrated that the fusion event between the characteristic domains of the DNMtases family and Snf2 family in the Rad8 class is fungi-specific, possibly indicating a functional novelty of Rad8 DNMtases in fungi. Additionally, expression profiles of DNMtases in the edible mushroom Pleurotus ostreatus revealed diverse expression patterns in various organs and developmental stages. For example, DNMtase genes displayed higher expression levels in dikaryons than in monokaryons. Consistent with the expression profiles, we found that dikaryons are more susceptible to the DNA methyltransferase inhibitor 5-azacytidine. Taken together, our findings pinpoint an important role of DNA methylation during the growth of mushrooms and provide a foundation for understanding of DNMtases in basidiomycetes.

Keywords: DNA methyltransferase; DNA methyltransferase inhibitor; Pleurotus ostreatus; basidiomycetes; mushroom development.

Figure 1

Numbers of genes encoding DNA methylase domain-containing proteins in the 39 species investigated. The Taxonomy Common Tree constructed by Taxonomy Browser in NCBI (National Center for Biotechnology Information) is shown on the left. The number detail of each category of each species is listed at the right column. The species lacking gene encoding DNA methyltransferase were also verified by online tblastn toolset of NCBI.

Figure 2

Phylogenetic and domain architecture analysis of DNA methylase domain containing proteins in fungi. The ML tree was constructed with the conserved catalytic domains of the predicted 123 proteins from 39 species (listed in Supplementary Table 1) using the MUSCLE v3.8.31 and MEGA6.0 with 1000 bootstrapping replicates. The three families (Dnmt1, Dnmt2, and Rad8) and 3 subfamilies (Dnmt1/Masc2, Dim-2, Masc1/RID) are marked in different colors. The domain architecture analysis of the identified proteins was performed by the Pfam database.

Figure 3

Phyletic distribution of taxonomic organisms and domain fusion event of characteristic domains between DNMtase family and Snf2 family identified in a genome-wide survey of representative species. The distribution and domain fusion event were mapped to the phyletic tree. Plus (+) and minus (−) indicate the domain presence/absence in the corresponding phylogenetic clade under the cut-off of e < 10⁻⁴ in the hmmpfam toolset.

Figure 4

Expression profiles of DNMtase genes in Pleurotus ostreatus. (A) The schematic diagram for different developmental stages of Pleurotus ostreatus. Mm, Dm, Mp, Cp, Sfp, Ntpp, and Tpp represents monokaryotic mycelium, dikaryotic mycelium, mulberries phase, coral-like phase, sporophor formative phase, not turning pale phase, and turning pale phase, respectively. (B) Expression profiles of DNMtase genes during the development of Pleurotus ostreatus. The expression levels relative to ACTIN were measured by quantitative RT-PCR and displayed in log2 scale. The acronyms from left to the right are representative of Monokaryotic mycelium (Mm), Dicaryotic mycelium (Dm), Mulberries phase (Mp), Coral-like phase (Cp), Fruiting body of formative phase (Fbfp), Stip of formative phase (Sfp), Cap of not turning plae phase (Cntpp), Veil of not turning pale phase (Vntpp), Stip of not turning pale phase (Sntpp), Veil of turning pale phase (Vtpp) that were collected for quantitative RT-PCR analysis. Three biological replicates and three technological replicates were done for each data point. Error bars indicate standard deviations (SDs). Letters indicate significant differences at P < 0.05 according to Duncan's multiple range tests.

Figure 5

Dikaryons are more sensitive to the inhibitory effect of 5-azcytidine than monokaryons. (A) Dikaryons and monokaryons grown on 5-azacytidine -containing medium with 100, 250, or 500 μM 5-azacytidine. Images of dikaryons and monokaryons were taken 10, 14 days after inoculation, respectively. (B) The cell length of 5-azacytidine treated dikaryons is longer than that of the mock. The arrow indicates where the clamp connection forms. (C) Expression profiles of putative DNMtase genes in dikaryons and monokaryons after 5-azacytidine treatment for 10, 14 days. The values shown are means ±SD.