DOI: 10.5281/zenodo.18782326
title: N6-methyldeoxyadenosine is a transgenerational epigenetic mark protecting DNA from deletion

description: DNA N6-adenine methylation (6mA) is emerging as an important DNA modification present in all eukaryotic kingdoms, where its roles mostly involve transcriptional regulation and chromatin modification. Here, we demonstrate a distinct role for 6mA in Paramecium tetraurelia. We find that it is a trans-generational epigenetic mark which distinguishes host DNA from invasive elements, protecting the former. During development, Paramecium deletes around 25% of its germline genome, removing TEs, repetitive sequences and transposon-derived elements, to form a functional somatic genome. We show that 6mA is present in the Paramecium germline nucleus, where it is preferentially located on host DNA and excluded from TEs. Ectopic addition of 6mA to germline DNA causes failure to excise TE-derived sequences, leading to lethality. 6mA presence on a synthetic TE-derived element is sufficient to block its excision during development. These effects are inherited via sexual reproduction, demonstrating their trans-generational capacity. 
description: Mass spectrometry analysis of DNA modifications

Macronuclear DNA (see Macronuclear DNA extraction and Sequencing, below) was purified from PGM-KD and empty vector silencing cultures at 2 days post autogamy. Up to 10 µg of sample DNA was hydrolyzed to nucleosides in the following enzymatic digestion process: The samples were added to a mixture containing 0.2 u Benzonase (MilliporeSigma, 70664), 0.5 mU of Phosphodiesterase I from Crotalus adamanteus venom (MilliporeSigma, P3243) and 0.5 U of Alkaline Phosphatase from calf intestine (MilliporeSigma, 524572-5KU) for 2 h at 37°C in a final volume of 100 µl of digestion buffer (5 mM TRIS pH 8.0, 20 mM NaCl and 1 mM MgCl2). As an internal loading control, an equal volume of 13C, 15N‐labelled uridine (MilliporeSigma, 645672) in 0.2% formic acid was added to the samples. These were then filtered through 30 kDa Molecular Weight Cut-Off filters (Amicon® Ultra Centrifugal Filter, 30 kDa MWCO, MilliporeSigma, UFC5030) to remove the proteins. 2 μL of each sample was injected in triplicate for LC-MS/MS analysis.

 

DNA Nucleoside Mass Spectrometry

Nucleoside Standards and Chemical Products

Nucleoside standards were purchased from MillioporeSigma and Biosynth (formerly Carbosynth, Compton, United Kingdom).  All chemicals purchased were grade LC-MS: Acetonitrile (1.00029, CAS: 75-05-8), water (1.15333, CAS: 7732-18-5), and formic acid (5.33002, CAS: 64-18-6) were purchased from MilliporeSigma. 

 

UPLC-MS Method

Samples were resolved over 15 minutes through an Acquity 100 × 2.1 mm C‐18 HSS T3 column using a Thermo Scientific U3000 UPLC system on a gradient of 2–98% (0.1% formic acid/acetonitrile) and analysed on a QExactive‐HF Orbitrap High Resolution Mass Spectrometer (Thermo Fisher Scientific, IQLAAEGAAPFALGMBFZ) in positive full‐scan mode. Initial conditions were regenerated by rinsing with 100% solvent A (0.1% formic acid, 2% Acetonitrile in water) for an additional 2 min. The flow rate was 0.2 mL/min, and the column temperature was 25°C.

The results were deconvoluted using the accompanying Xcalibur Software (Thermo Fisher Scientific). Nucleosides were assigned based on retention time and exact mass, and quantified relative to calibration curves generated from pure standards.  Modified nucleosides were expressed as a percentage of their precursor nucleoside (ie N6mdA as a percentage of dA).

 






Nucleoside



Molecular Formula (M)



Accurate [M+H]+ Mass (Da)



Retention Time (min)





deoxycytidine



C9H13N3O4



228.0984



1.96





deoxyadenosine



C10H13N5O3



252.1097



4.8





deoxyguanosine



C10H13N5O4



268.1046



6.11





thymidine



C10H14N2O5



243.0981



7.5





deoxy5methylcytidine (5-mdC)



C10H15N3O4



242.1141



3.42





deoxy5hydroxymethylcytidine (5-hmdC)



C10H15N3O5



258.109



1.88





deoxy5formylcytidine (5-fdC)



C10H13N3O5



256.0933



6.09





N6methyldeoxyadenosine (N6-mdA)



C11H15N5O3



266.1253



8.04





1-methyldeoxyguanosine (1-mdG)



C11H15N5O4



282.1202



8.38





heavy_uridine (Internal Standard)*



(13C)9H12(15N)2O6



256.1198



3.02