Niu Junhua, Yu Haoze, Liu Yongqiang, Nie Lanheng, Nan Bei, Zhang Wenxin, Song Ni, Rong Shaoqin, Zhou Dan, Gao Shan
Key Laboratory of Evolution and Marine Biodiversity and Institute of Evolution and Marine Biodiversity, Chinese Ministry of Education, Ocean University of China, Qingdao 266003, China.
Laboratory for Marine Biology and Biotechnology, Qingdao Marine Science and Technology Center, Qingdao 266237, China.
Proc Natl Acad Sci U S A. 2026 Mar 10;123(10):e2526398123. doi: 10.1073/pnas.2526398123. Epub 2026 Mar 2.
DNA N-methyladenine (6mA) is a newly recognized transcription-associated epigenetic mark in eukaryotes. While its methylation pathway has been well established, the identity of eukaryotic 6mA demethylase(s) responsible for its removal and dynamic regulation has remained elusive. Here, we identify and characterize DMT3 (TtALKBH5), an AlkB family dioxygenase in , as a 6mA demethylase in ciliates and potentially several other unicellular eukaryotes with abundant 6mA and a defined AMT1 methyltransferase (MTase) complex, supported by genetic and molecular evidence. DMT3 acts on both fully and hemimethylated ApT dinucleotides, an activity partially facilitated by a naturally occurring cysteine-to-serine substitution. Genome profiling shows that DMT3 is enriched at transcription start sites (TSSs) of 6mA-enriched genes, complementary to the occupancy pattern of the AMT1 complex, where it selectively removes spurious 6mA deposited by AMT1. Genetic disruption of DMT3-mediated demethylation, either by knockout or catalytic inactivation, leads to aberrant 6mA accumulation at TSS regions, transcriptional dysregulation, altered chromatin accessibility, and impaired initiation of sexual reproduction. Notably, simultaneous removal of DMT3 and AMT1 eliminates these defects, indicating that spurious TSS 6mA underlies transcriptional and developmental impairment.
DNA N-甲基腺嘌呤（6mA）是真核生物中一种新发现的与转录相关的表观遗传标记。虽然其甲基化途径已得到充分确立，但负责去除和动态调节6mA的真核生物6mA去甲基化酶的身份仍然难以捉摸。在这里，我们鉴定并表征了嗜热栖热菌中的一种AlkB家族双加氧酶DMT3（TtALKBH5），它是纤毛虫以及其他几种可能具有丰富6mA和明确的AMT1甲基转移酶（MTase）复合物的单细胞真核生物中的6mA去甲基化酶，这一结论得到了遗传学和分子证据的支持。DMT3作用于完全甲基化和半甲基化的ApT二核苷酸，一种天然存在的半胱氨酸到丝氨酸的取代部分促进了这种活性。基因组分析表明，DMT3在富含6mA的基因的转录起始位点（TSS）处富集，这与AMT1复合物的占据模式互补，在那里它选择性地去除由AMT1沉积的假6mA。通过敲除或催化失活对DMT3介导的去甲基化进行基因破坏，会导致TSS区域异常积累6mA、转录失调、染色质可及性改变以及有性生殖起始受损。值得注意的是，同时去除DMT3和AMT1可消除这些缺陷，表明假TSS 6mA是转录和发育受损的基础。
