Mitochondrial epigenetics brings new perspectives on doubly uniparental inheritance in bivalves

Mitochondrial epigenetics, particularly mtDNA methylation, is a flourishing field of research. MtDNA methylation appears to play multiple roles, including regulating mitochondrial transcription, cell metabolism and mitochondrial inheritance. In animals, bivalves with doubly uniparental inheritance (DUI) of mitochondria are the exception to the rule of maternal mitochondrial inheritance since DUI also involve a paternal mtDNA transmitted from the father to sons. The mechanisms underlying DUI are still unknown, but mtDNA methylation could play a role in its regulation. Here, we investigated mtDNA methylation levels and machinery in gonads of the mussel Mytilus edulis using methods based on antibodies, enzymatic cleavage and methylome sequencing. Our results confirm the presence in mitochondria of methylated cytosines and adenines and methyltransferases and unveil a more variable cytosine methylation state among males than females. Also, spermatid mtDNA is always methylated, while only few spermatozoa present methylated mtDNA suggesting a relation between cytosine methylation and development stage of male gametes. We propose that mtDNA methylation could play a role in the different fates of the parental mtDNAs in male and female embryos in M. edulis. Our study provides novel insights into the epigenetic landscape of bivalve mtDNA and highlights the multiple roles of mtDNA methylation in animals.

Methods
DNA extractions : DNA extractions used for enzymatic digestions were performed on 9 male and 9 female of Mytilus edulis gonad samples with phenol-chloroform following a protocol adapted to molluscan tissues (Sokolov, 2000). DNA extractions used for methylome sequencing were performed on gonad tissues of 5 males and 3 females of M. edulis following the DNeasy blood & tissue kit (QIAGEN) protocol. DNA samples were then digested to linearize mtDNA using SwaI restriction enzyme (New England BioLabs), following the manufacturer’s protocol. Linearized mtDNA (and digested nuclear DNA) was then purified with a QIAquick PCR purification kit (QIAGEN) according to the manufacturer’s protocol.

Enzymatic digestions : DNA samples were digested with the cytosine methyl-sensitive restriction enzyme (MSRE) HpaII and its insensitive isoschizomer restriction enzyme MspI, as well as with an adenine MSRE, DpnI, and its methyl-insensitive isoschizomer DpnII. Digestions were performed separately for each restriction enzymes in a 50 µl reaction mix containing 1 µg of total DNA, 10 to 20 U of restriction enzymes, i.e. FastDigest MspI, FastDigest HpaII (Thermofisher Scientific), DpnI or DpnII (New England Biolabs) and 1X of their appropriate buffer. Digestions were carried out at 37°C for 10, 20, 30 or 60 min (MspI and HpaII) or 15 min (DpnI and DpnII). Adenine-specific enzymes were inactivated by heat treatment at 80°C (DnpI) or 65°C (DpnII) for 20 min.

Methylome sequencing : DNA samples were submitted to mitochondrial methylome sequencing. Libraries were performed at the Génome Québec Innovation Centre (Montreal, QC, Canada) using 200 ng of linearized and purified DNA samples and a lambda phage DNA (negative control) with the NEBNext® Enzymatic Methyl-Seq kit (New England BioLabs), following the manufacturer’s protocol. Libraries were subjected to 150 bp paired-end sequencing with Illumina NovaSeq 6000, using the NovaSeq Xp protocol. Finally, all reads from the libraries were generated with the Illumina bcl2fastq2 (v2.20) program.