DNA Methylation in Basal Metazoans: Insights from Ctenophores

Abstract

Epigenetic modifications control gene expression without altering the primary DNA sequence. However, little is known about DNA methylation in invertebrates and its evolution. Here, we characterize two types of genomic DNA methylation in ctenophores, 5-methyl cytosine (5-mC) and the unconventional form of methylation 6-methyl adenine (6-mA). Using both bisulfite sequencing and an ELISA-based colorimetric assay, we experimentally confirmed the presence of 5-mC DNA methylation in ctenophores. In contrast to other invertebrates studied, Mnemiopsis leidyi has lower levels of genome-wide 5-mC methylation, but higher levels of 5-mC methylation in promoters when compared with gene bodies. Phylogenetic analysis showed that ctenophores have distinct forms of DNA methyltransferase 1 (DNMT1); the zf-CXXC domain type, which localized DNMT1 to CpG sites, and is a metazoan specific innovation. We also show that ctenophores encode the full repertoire of putative enzymes for 6-mA DNA methylation, and these genes are expressed in the aboral organ of Mnemiopsis. Using an ELISA-based colorimetric assay, we experimentally confirmed the presence of 6-mA methylation in the genomes of three different species of ctenophores, M. leidyi, Beroe abyssicola, and Pleurobrachia bachei. The functional role of this novel epigenomic mark is currently unknown. In summary, despite their compact genomes, there is a wide variety of epigenomic mechanisms employed by basal metazoans that provide novel insights into the evolutionary origins of biological novelties.

Fig. 1

Structural organization of DNMT enzymes. Homo DNMT1 (NM_175629.2) gene consists of 41 coding exons and is 61,734 bp long. The Apis mellifera DNMT1 (XM_006562802.1) gene has 15 coding exons and is 6183 bp long. The Pleurobrachia DNMT1-like gene (JX985458.1) has 29 exons and is at least 16,598 bp long (located on two scaffolds). The protein structure of the DNMT1 of humans is comprised of a DNA methyltransferase-associated protein-binding domain (DMAP), replication foci domain (RFD), C-X-X-C domain, 2 Bromo-adjacent homology (BAH) domains, and DNA-methylase (PF00145) domain. The predicted DNMT1 proteins of Apis and Pleurobrachia contain the same domains but each lack a DMAP domain. Crystal structure of mouse DNMT1 (650–1602) in complex with DNA is shown [PDB ID: 3PT6] (Song et al. 2011). Structural organization of the DNMT3 enzyme. There are three DNMT3 genes in humans and we use the DNMT3A as an example. The Homo DNMT3A (NM_175629.2) gene consists of 22 coding exons and is 109,629 bp long. The Apis mellifera DNMT3 (XM_006562802.1) gene has 12 coding exons and is 43,394 bp in length. The DNMT3 enzyme is defined by the presence of the PWWP motif known to be involved in protein–protein interactions with, ADDz domain, a PHD-like zinc-finger domain, and a DNA methylase domain. The crystal structure for DNMT3A complexed with DNMT3L is shown [PDB ID: 3PT62QRV] (Jia et al. 2007). No DNMT3s were found in the sequenced genomes or transcriptomes of ctenophores (Moroz et al. 2014). (This figure is available in black and white in print and in color at Integrative and Comparative Biology online)

Fig. 2

Genealogical relationships of DNMTs. (A) Phylogenetic analysis of DNMT1 was performed by constructing a maximum likelihood using the methyltransferase domains from DNMT1 sequences. All branches with bootstrap support less than 50 have been collapsed. Percent Bootstrap values are indicated by dots. This tree was rooted to the know outgroup sequences from chlorophycean algae Volvox carteri and Chlamydomonas reinhardtii. Within this clade the benthic ctenophores Vallicula multiformis and Coeloplana astericola cluster together. (B) Phylogenetic analysis of TRDMT1 by constructing a maximum likelihood using the methyltranferase domains from DNMT1 sequences. All branches with bootstrap support less than 50 have been collapsed. Percent bootstrap values are indicated by dots. This tree was rooted to known non-metazoan outgroups the choanoflagellate Monosiga brevicollis and the amoebozoan Entamoeba histolytica. Both of these trees demonstrate the basal branching of ctenophore DNMT family sequences. (This figure is available in black and white in print and in color at Integrative and Comparative Biology online)

Fig. 3

Genome-wide rates of contextual methylation are lineage-specific. (A) Percentages of genomic methylation across metazoa. Different contexts of methylation, including CG, CHG, and CHH were obtained from bisulfite sequencing. Mnemiopsis leidyi (CG = 0.09%, CHG = 0.03%, CHH = 0.11%); Nematostella vectensis (CG = 1.44%, CHG = 0.00%, CHH = 0.00%) (Zemach et al. 2010); the data from Apis mellifera (CG = 1.65%, CHG = 0.60%, CHH = 2.00%) are an average from Lyko et al. (2010) and Cingolani et al. (2013). The data from Crassostrea gigas (CG = 1.95%, CHG = 0.00%, CHH = 0.01%) (Wang et al. 2014); the data from Homo sapiens (CG = 4.15%, CHG = 0.35%, CHH = 1.32%) are an average from Lister et al. (2009, 2013) and Li et al. (2010). The pie charts above each species represent the distribution of methylation of CG versus CHG versus CHH in the genome. (B) ELISA-based colorimetric assay to validate presence of 5-mC in Mnemiopsis, Beroe, and Pleurobrachia. The level of 5-mC DNA methylation in Pleurobrachia (∼1.0%) is about 10-fold that of Mnemiopsis and Beroe. (C) The distribution of nucleotides in the genome of Mnemiopsis shows a reduced representation of cytosine and guanine nucleotides and a notable amount of N values. (D). Methylation in Mnemiopsis leidyi in the context of promotor versus gene-body shows that ctenophores have higher promoter methylation (7.44%) than exon or intron methylation (4.59% and 4.95%, respectively). (This figure is available in black and white in print and in color at Integrative and Comparative Biology online)

Fig. 4

RNA expression of DNA methyltransferases (DNMT1) and associated helicases in the ctenophore Pleurobrachia across development and in adult tissues. Expression is presented as normalized transcripts per million (TPMs). (A) The RUVB1 helicase shows several-fold higher expression even in adults’ tissue than does the HELLS helicase which recruits DNMT for de novo methylation. The presence and relative abundance of this enzyme, especially during early-stage development, suggests a role for DNMT1 in de novo methylation in ctenophores. (B) Heatmap of expression of all DNA-associated protein transcripts (TPMs) identified in Pleurobrachia shows that the most abundant expression of DNMT-associated proteins occurs during the 4-cell to 32-cell stages. (This figure is available in black and white in print and in color at Integrative and Comparative Biology online)

Fig. 5

6-mA DNA methylation in ctenophores. (A) Enzymatic mechanisms for 6-mA methylation/demethylation in DNA. Proposed enzymes for 6-mA methylation/demethylation acting on DNA and their resultant products are shown. (B) Quantitative expression of the predicted 6-mA methylation/demethylation in the Mnemiopsis leidyi aboral organ. The putative demethylase (ALKB 4-like) displays twice the amount of expression as the putative methyltransferase 4-like enzyme in the aboral organ of Mnemiopsis (see Supplementary Materials for details). (C) ELISA-based colorimetric assays to validate the presence of 6-mA methylation in the genomes of Mnemiopsis, Beroe, and Pleurobrachia. Both Pleurobrachia and Mnemiopsis genomes contain close to double the amount of 6-mA methylation in their genomes compared with Beroe (see Supplementary Material for details). (This figure is available in black and white in print and in color at Integrative and Comparative Biology online)

Fig. 6

Evolution of the DNMT families of genes. The canonical DNMT1 originated early in eukaryotic evolution but the zf-CXXC domain is a metazoan-specific innovation. Choanoflagellates and the placozoan Trichoplax adhaerens have both lost DNMT1. TRDMT1 is the only gene conserved in every metazoan lineage, but it actually functions as a tRNA methylase. DNMT3s have putative homologs in prokaryotes, and flowering plants. DNMT3 is absent in choanoflagellates and ctenophores but is present in Porifera, cnidarians, and bilaterians. The 6-mA DNA methyltransferase, DAMT-1, is present in unicellular eukaryotes, and has now been confirmed in ctenophores and bilaterians. Although DAMT-1 is known to be present in prokaryotes and algae suggesting a premetazoan origin, the scope of gene loss and gene gain of DAMT-1 has not been systematically evaluated across metazoans. See text for details. (This figure is available in black and white in print and in color at Integrative and Comparative Biology online)