Diversity of cytosine methylation across the fungal tree of life

Abstract

The generation of thousands of fungal genomes is leading to a better understanding of genes and genomic organization within the kingdom. However, the epigenome, which includes DNA and chromatin modifications, remains poorly investigated in fungi. Large comparative studies in animals and plants have deepened our understanding of epigenomic variation, particularly of the modified base 5-methylcytosine (5mC), but taxonomic sampling of disparate groups is needed to develop unifying explanations for 5mC variation. Here, we utilize the largest phylogenetic resolution of 5mC methyltransferases (5mC MTases) and genome evolution to better understand levels and patterns of 5mC across fungi. We show that extant 5mC MTase genotypes are descendent from ancestral maintenance and de novo genotypes, whereas the 5mC MTases DIM-2 and RID are more recently derived, and that 5mC levels are correlated with 5mC MTase genotype and transposon content. Our survey also revealed that fungi lack canonical gene-body methylation, which distinguishes fungal epigenomes from certain insect and plant species. However, some fungal species possess independently derived clusters of contiguous 5mC encompassing many genes. In some cases, DNA repair pathways and the N⁶-methyladenine DNA modification negatively coevolved with 5mC pathways, which additionally contributed to interspecific epigenomic variation across fungi.

Figure 1.

Evolution of 5mC MTases across fungi. (a) Phylogenetic relationships of 5mC DNA and tRNA MTases of fungi. Values at selected nodes indicate posterior probability. Nodes with a star specify duplications, and the single node with a diamond specifies the clade containing ‘DnmtX’ (26). Area of the triangle corresponds to the number of taxa. Branch lengths are in units of amino acid substitutions per amino acid site. Phyla abbreviations: Ascomycota (Asco-), Basidiomycota (Basidio-), Blastocladiomycota (Blastocladio-), Chytridiomycota (Chytridio-), Mucoromycota (Mucoro-), and Zoopagomycota (Zoopago-). (b) Proportion of species within subphyla of fungi absent/present for 5mC DNA and tRNA MTases. Empty circles indicate the evolution of a DIM-2 and RID. Branch lengths of the species tree are in substitutions per site. Subphyla abbreviations: Agaricomycotina (Agarico-), Blastocladiomycotina (Blastocladio-), Chytridiomycetes, Entomophthoromycotina (Entomophthoro-), Glomeromycotina (Glomero-), Kickxellomycotina (Kickxelo-), Mortierellomycotina (Mortierello-), Mucoromycotina (Mucoro-), Neocallimastigomycota (Neocallimastigo-), Pezizomycotina (Pezizo-), Pucciniomycotina (Puccinio-), Saccharomycotina (Saccharo-), Taphrinomycotina (Taphrino-), Ustilaginomycotina (Ustilagino-), and Zoopagomycotina (Zoopago-). The number of species within each subphylum investigated is given at the tips. (c) Number of species for the observed combinations of 5mC MTases (5mC MTase genotypes). Phyla abbreviations are identical to a, with the addition of Cryptomycota (Crypto-), and Neocallimastigomycota (Neocallimastigo-).

Figure 2.

Genome-wide 5mC profiles. (a) Weighted methylation for CG, CH, and CN sites across the genome and various regions of the genome. Empty and filled boxes indicate the absence or presence of 5mC MTases, respectively. Species are ordered based on relationship. Branch lengths of the species tree are in substitutions per site. Phyla abbreviations are identical to Fig. 1a. (b). Weighted 5mC levels at all sequence contexts found across the genome for pairs of fungi with identical 5mC MTase genotypes. Colored boxes beside species names indicate phylum. The dashed line indicates the sodium bisulfite non-conversion rate (estimated background level of methylation).

Figure 3.

5mC profiles of genes. (a) Weighted methylation at CG, CH, and CN sites upstream, within, and downstream of all genes for pairs of fungi with identical 5mC MTase genotypes. The dashed line indicates the sodium bisulfite non-conversion rate (estimated background level of methylation). (b) Distribution of 5mC levels for genes for the same set of ten species in (a). (c) Relationship between gene expression measured as Fragments Per Kilobase of transcript per Million [FPKM] mapped reads and 5mC levels. Empty deciles correspond to missing genic data for those 5mC levels. Boxplot elements: center line, median; upper and lower "hinges", first and third quartiles (the 25th and 75th percentiles), respectively; whiskers, 1.5× interquartile range.

Figure 4.

5mC profiles of DNA transposons and LTRs. (a) Weighted methylation at CG, CH, and CN sites upstream, within, and downstream of all genes for pairs of fungi with identical 5mC MTase genotypes. The dashed line indicates the sodium bisulfite non-conversion rate (estimated background level of methylation). (b) Distribution of 5mC levels for DNA transposons for the same set of ten species in (a). (c) Distribution of 5mC levels for LTRs for the same set of ten species in (a).

Figure 5.

Methylated Cytosine Clusters (MCCs). (a) Examples of MCCs in Pse. destructans, Agaricus bisporus, and Cop. cinerea. 5mC level is given for both strands of DNA (+ and −). (b) 5mC level and length (kbp) distributions of MCCs located within chromosome arms, centromeres, and telomeres of Cop. cinerea. Boxplot elements: center line, median; upper and lower “hinges”, first and third quartiles (the 25th and 75th percentiles), respectively; whiskers, 1.5× interquartile range; large points, outliers. (c) Genetic content distribution of MCCs located within chromosome arms, centromeres, and telomeres of Cop. cinerea. Boxplot elements: center line, median; upper and lower "hinges", first and third quartiles (the 25th and 75th percentiles), respectively; whiskers, 1.5× interquartile range; large points, outliers. (d) Proportion of orthologous genes absent from or present within MCCs across 17 fungi. Abbreviation: OrthoGroup (OG).

Figure 6.

Evolutionary relationships of repetitive DNA and transposons, DNA repair pathways and 6mA pathways with 5mC. (a) Correlations between genome-wide CG methylation, and repeat content, DNA transposon, and LTR content adjusted for non-independence of species. The strength of each correlation is given by a ‘generalized correlation coefficient’ (GCC). Lambda (λ) indicates Pagel’s λ and the amount of phylogenetic signal, ranging from 0 (completely independent random walks) to 1 (Brownian motion). (b) Violin plots of genome-wide CG methylation for fungal species absent and present for ALKBH2 and ALKBH3. Correlations between 5mC and DNA repair enzyme are also given. (c) Solid and empty bars correspond to the proportion of species investigated with or without pathways for the establishment and maintenance of 5mC and 6mA of DNA and RNA, respectively. The number of species within each phylum investigated is given at the tips. Branch lengths of the species tree are in substitutions per site. Phyla abbreviations are identical to Fig. 1a.