Mitochondrial epigenetics brings new perspectives on doubly uniparental inheritance in bivalves

Leroux, Emelie 1 ; Hosseini Khorami, Hajar1; Angers, Annie1; Angers, Bernard1; Breton, Sophie 1

Research facility: Université de Montréal

Published Jul 26, 2024 on Dryad. https://doi.org/10.5061/dryad.cvdncjtc5

Abstract

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.

https://doi.org/10.5061/dryad.cvdncjtc5

Description of the data and file structure

The dataset comprises text files of enzymatic digestion levels (labelled "ratios.merged.MF.txt", "ratios.enzymes.txt" and "ratios_enzymes_diff.txt.") of Mytilus edulis DNA samples (available on Zenodo), which can be used in the R code (see below). Digestion levels were calculated using ImageJ software program following the DNA band quantification protocol from Davarinejad (2015).

The dataset also comprises fastqsanger files of paired-end sequencing data (R1 and R2) from M. edulis DNA samples (available on Dryad), as well as text files of the processed reads (available on Zenodo), labelled "SampleNumber_RefGenome_seq.txt" (Sample number = M1 to M5 male samples and F3, F5, F6 for female samples), which are also used in the R code. Ref genome = mitotype M, mitotype F, nDNA or Lambda DNA; see below for genome accession numbers in Genbank).

Reads processing includes quality control of reads using FastQC 0.11.8 (Andrews, n.d.) and trimming using Fastp 0.23.2 (Chen et al., 2018) as well as Trim Galore! 0.6.7 (Krueger, 2021). Alignment to M. edulis reference genomes was done using Bismark Mapper with the relaxing stringency function L,0,-0.4 (Krueger and Andrews, 2011). Methylation calls were obtained using Bismark Meth. Extractor 0.22.1 (Krueger and Andrews, 2011).

R Code

The code file ("Script_Lerouxetal.R") can be used on R software (Ihaka and Gentleman, 1996) and includes data calling and formatting, figures build-up and statistical analysis necessary for the article. The R code and the datasets are available in Zenodo.

Statistical analysis :

Enzymatic digestion analysis : Statistical analysis for CCGG methylation determination was made with Student’s T-tests to detect any significant difference in mean digestion level by MspI and HpaII. To assess whether the amount of methylated/unmethylated CCGG or GATC was uniform among males and females, the difference in variability of digestion levels among males and females was statistically evaluated by Fisher’s F-tests.
Methylome sequencing : Statistical analysis for strand-bias coverage was made with a series of Wilcoxon tests.

Reference genomes

Mytilus edulis genomes accession numbers (Genbank):

M mtDNA (Mitotype M): AY823624
F mtDNA (Mitotype F): AY484747
Nuclear DNA (nDNA) contig: CAJPWZ010000084

Lambda spike-in genome accession number (Genbank): J02459

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.

Mitochondrial epigenetics brings new perspectives on doubly uniparental inheritance in bivalves

Data files

Abstract

README: The epigenetic hypothesis of doubly uniparental inheritance of mitochondria: unveiling the mtDNA methylation toolkit in the blue mussel Mytilus edulis

Description of the data and file structure

R Code

Reference genomes

Methods

Works referencing this dataset