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.

Over 12 years of continuous monitoring of Changbaishan volcano in the border region of China and North Korea by means of volcanic seismicity, ground deformation, and volcanic gas geochemistry yields new evidence for magmatic unrest of the volcano between 2002 and 2006. In this so‐called “active period,” the frequency of volcanic earthquakes increased by about 2 orders of magnitude compared to that of the background “inactive periods.” The active period was also accompanied by ground inflation, high values of CO2, He, H2, and high ratios of N2/O2 and 3He/4He in volcanic gases released from three hot springs near the caldera rim. The monitoring evidence implies pressurization of the magma chamber, possibly caused by incremental magma recharge. The ground deformation data from both GPS and precise leveling are modeled to suggest the corresponding deformation source is at 2–60 km depth beneath the volcano's summit, where earthquake swarms were detected in 2002 and 2003. Our findings suggest that the magma chamber beneath Changbaishan volcano has awakened and resumed activity after remaining dormant since AD 1903. There is an urgent need to keep close watch on this active and very hazardous volcano in northeast China.

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[1] Changbaishan volcano in northeast China, previously dated to have erupted around the mid-10th century A.D., is renowned for producing one of the largest eruptions in history (magnitude 6.8) and thus speculated to have substantial climatic impact. Here we report a new high-precision 14 C wiggle-match age of A.D. 946 AE 3 obtained from a 264 year old tree trunk (with bark) killed during the eruption, using the OxCal's Bayesian modeling approach with 27 sequentially sampled annual rings of decadal intervals. The new chronology conforms well to the calendar date of A.D. 946 for the eruption inferred from historical documentary evidence. We find no stratospherically loaded sulfate spike that might be associated with the A.D. 946 eruption in the global volcanism record from the GISP2 ice core, suggesting the stratospheric sulfate aerosols produced during the eruption were not transported to the arctic region, due probably to its relatively low stratospheric sulfur emission and the seasonal effects of the atmospheric circulation at the time of the eruption that likely occurred in the winter of A.D. 946-947. Since the stratospheric volcanic sulfates are the main cause of large-scale climate perturbations, this finding indicates that the Millennium eruption of Changbaishan volcano might have limited regional climatic effects, rather than global or hemispheric impact as implied by its magnitude.

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While DNA N6-adenine methylation (6mA) is best known in prokaryotes, its presence in eukaryotes has generated great interest recently. Biochemical and genetic evidence supports that AMT1, a MT-A70 family methyltransferase (MTase), is crucial for 6mA deposition in unicellular eukaryotes. Nonetheless, 6mA transmission mechanism remains to be elucidated. Taking advantage of Single Molecule Real-Time Circular Consensus Sequencing (SMRT CCS), here we provide definitive evidence for semi-conservative transmission of 6mA, showcased in the unicellular eukaryote Tetrahymena thermophila. In wildtype (WT) cells, 6mA occurs at the self-complementary ApT dinucleotide, mostly in full methylation (full-6mApT); hemi-methylation (hemi-6mApT) is transiently present on the parental strand of newly replicated DNA. In ΔAMT1 cells, 6mA predominantly occurs as hemi-6mApT. Hemi-to-full conversion in WT cells is fast, robust, and likely processive, while de novo 6mA deposition in ΔAMT1 cells is slow and sporadic. In Tetrahymena, regularly spaced 6mA clusters coincide with linker DNA of the canonical nucleosome arrays in the gene body. Importantly, in vitro methylation of human chromatin by reconstituted AMT1 complex recapitulates preferential targeting of hemi-6mApT sites in linker DNA, supporting AMT1's intrinsic and autonomous role in maintenance methylation. We conclude that 6mA is transmitted by a semi-conservative mechanism: full-6mApT is split by DNA replication into hemi-6mApT, which is restored to full-6mApT by AMT1-dependent maintenance methylation. Our study dissects AMT1-dependent maintenance methylation and AMT1-independent de novo methylation, reveals a molecular pathway for 6mA transmission with striking similarity to 5-methyl cytosine (5mC) transmission at the CpG dinucleotide, and establishes 6mA as a bona fide eukaryotic epigenetic mark.

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