A distinct class of eukaryotic MT-A70 methyltransferases maintain symmetric DNA N6-adenine methylation at the ApT dinucleotides as an epigenetic mark associated with transcription

Abstract

Rediscovered as a potential eukaryotic epigenetic mark, DNA N6-adenine methylation (6mA) varies across species in abundance and its relationships with transcription. Here we characterize AMT1—representing a distinct MT-A70 family methyltransferase—in the ciliate Tetrahymena thermophila. AMT1 loss-of-function leads to severe defects in growth and development. Single Molecule, Real-Time (SMRT) sequencing reveals that AMT1 is required for the bulk of 6mA and all symmetric methylation at the ApT dinucleotides. The detection of hemi-methylated ApT sites suggests a semi-conservative mechanism for maintaining symmetric methylation. AMT1 affects expression of many genes; in particular, RAB46, encoding a Rab family GTPase involved in contractile vacuole function, is likely a direct target. The distribution of 6mA resembles H3K4 methylation and H2A.Z, two conserved epigenetic marks associated with RNA polymerase II transcription. Furthermore, strong 6mA and nucleosome positioning in wild-type cells is attenuated in ΔAMT1 cells. Our results support that AMT1-catalyzed 6mA is an integral part of the transcription-associated epigenetic landscape. AMT1 homologues are generally found in protists and basal fungi featuring ApT hyper-methylation associated with transcription, which are missing in animals, plants, and true fungi. This dichotomy of 6mA functions and the underlying molecular mechanisms may have implications in eukaryotic diversification.

The completed macronuclear genome of a model ciliate Tetrahymena thermophila and its application in genome scrambling and copy number analyses

“…SMRT sub-reads generated in this study and previously (Wang et al, 2017b;Wang et al, 2019b) were assembled into a draft assembly using Canu (Koren et al, 2017) software (correctedErrorRate=0.040, corMaxEvidenceErate=0.15, genomeSize=100 m), including error correction, read trimming and sequence assembly. Subsequently, erroneous contigs (CovStat≤0, contigs more likely to be repetitive; SuggestRepeat=yes, contigs detected as a repeat based on graph topology or read overlaps to other sequences; log 2 (Normalized reads frequency)≤10, contigs with low mapped reads; without telomeres) were filtered.…”

“…As a unicellular eukaryote model organism, Tetrahymena thermophila is the most well-studied of all protozoa and has contributed to fundamental biological discoveries in multiple aspects (Cervantes et al, 2013;Feng et al, 2017;Gao et al, 2013;Mochizuki et al, 2002;Gorovsky, 2004a, 2005;Orias et al, 2017;Wang et al, 2017b;Wang et al, 2019b;Xiong et al, 2016;Xu et al, 2019;Zhao et al, 2019). Like other ciliates, Tetrahymena possess two types of nuclei in a single cell, distinct in their appearance and function (Cheng et al, 2019;Collins and Gorovsky, 2005;Karrer, 2012;Wang et al, 2017a;Yan et al, 2019).…”

Programmed genome rearrangements in ciliates

“…6 mA modifications also localize to linker DNA regions downstream of the transcription start site (TSS of Polymerase II transcribed genes) and directly influence nucleosome positioning [ 133 , 136 – 138 ]. Tetrahymena ’s MT-A70 homologue ATM1 (6mA DNA methyltransferase) is required for the normal growth and development of the cell following sex [ 139 ]. Interestingly, the enzyme MTA1c (DNA methyltransferase) responsible for 6mA modifications in Oxytricha disfavours nucleosome occupancy, contrary to Tetrahymena [ 135 ].…”

Systematics and Multi-Gene Phylogeny of the Subfamily Nothoholostichinae (Ciliophora, Hypotrichia), With Integrative Description of a New Marine Species Nothoholosticha luporinii n. sp.

“…Ciliates (phylum Ciliophora Doflein, 1901), a highly diverse and ubiquitously distributed group of unicellular microbial eukaryotes, play substantial roles in various ecosystems. A lot of attention has been therefore paid to their diversity, function and evolution (e.g., Bharti et al, 2019;Hu et al, 2019;Jung and Berger, 2019;Kaur et al, 2019;Luo et al, 2019;Wang et al, 2019;Yan et al, 2019;Gong et al, 2020;Shao et al, 2020;Sheng et al, 2020;Wang Y. R. et al, 2020;Zhang et al, 2020). Hypotrichs (subclass Hypotrichia Stein, 1859) are not only among the most differentiated ciliate groups, but also among the most confused ones in terms of their systematics and phylogeny (for reviews, see Berger, 1999Berger, , 2006Berger, , 2008Berger, , 2011Luo et al, 2017;Song and Shao, 2017;Luo et al, 2018;Lyu et al, 2018;Kim and Min, 2019;Chen et al, 2020;Dong et al, 2020;Paiva, 2020;Park et al, 2020;Wang J. et al, 2020;Xu et al, 2020).…”