RESUMO
Although DNA N 6-adenine methylation (6mA) is best known in prokaryotes, its presence in eukaryotes has recently generated great interest. Biochemical and genetic evidence supports that AMT1, an MT-A70 family methyltransferase (MTase), is crucial for 6mA deposition in unicellular eukaryotes. Nonetheless, the 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 semiconservative transmission of 6mA in Tetrahymena thermophila In wild-type (WT) cells, 6mA occurs at the self-complementary ApT dinucleotide, mostly in full methylation (full-6mApT); after DNA replication, hemi-methylation (hemi-6mApT) is transiently present on the parental strand, opposite to the daughter strand readily labeled by 5-bromo-2'-deoxyuridine (BrdU). In ΔAMT1 cells, 6mA predominantly occurs as hemi-6mApT. Hemi-to-full conversion in WT cells is fast, robust, and processive, whereas de novo methylation in ΔAMT1 cells is slow and sporadic. In Tetrahymena, regularly spaced 6mA clusters coincide with the linker DNA of nucleosomes arrayed in the gene body. Importantly, in vitro methylation of human chromatin by the 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 semiconservative 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 6mA transmission pathway with a striking similarity to 5-methylcytosine (5mC) transmission at the CpG dinucleotide, and establishes 6mA as a bona fide eukaryotic epigenetic mark.
Assuntos
Adenina , Metilação de DNA , Tetrahymena thermophila , Tetrahymena thermophila/genética , Tetrahymena thermophila/metabolismo , Adenina/metabolismo , Adenina/análogos & derivados , Replicação do DNA , DNA de Protozoário/genética , DNA de Protozoário/metabolismoRESUMO
DNA methylation is one of the major epigenetic mechanisms crucial for gene regulation and genome stability. De novo DNA methyltransferase DNMT3C is required for silencing evolutionarily young transposons during mice spermatogenesis. Mutation of DNMT3C led to a sterility phenotype that cannot be rescued by its homologues DNMT3A and DNMT3B. However, the structural basis of DNMT3C-mediated DNA methylation remains unknown. Here, we report the structure and mechanism of DNMT3C-mediated DNA methylation. The DNMT3C methyltransferase domain recognizes CpG-containing DNA in a manner similar to that of DNMT3A and DNMT3B, in line with their high sequence similarity. However, two evolutionary covariation sites, C543 and E590, diversify the substrate interaction among DNMT3C, DNMT3A and DNMT3B, resulting in distinct DNA methylation activity and specificity between DNMT3C, DNMT3A and DNMT3B in vitro. In addition, our combined structural and biochemical analysis reveals that the disease-causing rahu mutation of DNMT3C compromises its oligomerization and DNA-binding activities, providing an explanation for the loss of DNA methylation activity caused by this mutation. This study provides a mechanistic insight into DNMT3C-mediated DNA methylation that complements DNMT3A- and DNMT3B-mediated DNA methylation in mice, unravelling a regulatory mechanism by which evolutionary conservation and diversification fine-tunes the activity of de novo DNA methyltransferases.
RESUMO
Functional crosstalk between DNA methylation, histone H3 lysine-9 trimethylation (H3K9me3) and heterochromatin protein 1 (HP1) is essential for proper heterochromatin assembly and genome stability. However, how repressive chromatin cues guide DNA methyltransferases for region-specific DNA methylation remains largely unknown. Here, we report structure-function characterizations of DNA methyltransferase Defective-In-Methylation-2 (DIM2) in Neurospora. The DNA methylation activity of DIM2 requires the presence of both H3K9me3 and HP1. Our structural study reveals a bipartite DIM2-HP1 interaction, leading to a disorder-to-order transition of the DIM2 target-recognition domain that is essential for substrate binding. Furthermore, the structure of DIM2-HP1-H3K9me3-DNA complex reveals a substrate-binding mechanism distinct from that for its mammalian orthologue DNMT1. In addition, the dual recognition of H3K9me3 peptide by the DIM2 RFTS and BAH1 domains allosterically impacts the DIM2-substrate binding, thereby controlling DIM2-mediated DNA methylation. Together, this study uncovers how multiple heterochromatin factors coordinately orchestrate an activity-switching mechanism for region-specific DNA methylation.
Assuntos
Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona , Metilação de DNA , Proteínas Fúngicas , Heterocromatina , Histonas , Heterocromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Histonas/metabolismo , Histonas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Ligação Proteica , Neurospora crassa/genética , Neurospora crassa/metabolismoRESUMO
Flaviviruses, including Zika virus (ZIKV) and Dengue virus (DENV), rely on their non-structural protein 5 (NS5) for both replication of viral genome and suppression of host IFN signaling. DENV and ZIKV NS5s were shown to facilitate proteosome-mediated protein degradation of human STAT2 (hSTAT2). However, how flavivirus NS5s have evolved for species-specific IFN-suppression remains unclear. Here we report structure-function characterization of the DENV serotype 2 (DENV2) NS5-hSTAT2 complex. The MTase and RdRP domains of DENV2 NS5 form an extended conformation to interact with the coiled-coil and N-terminal domains of hSTAT2, thereby promoting hSTAT2 degradation in cells. Disruption of the extended conformation of DENV2/ZIKV NS5, but not the alternative compact state, impaired their hSTAT2 binding. Our comparative structural analysis of flavivirus NS5s further reveals a conserved protein-interaction platform with subtle amino-acid variations likely underpinning diverse IFN-suppression mechanisms. Together, this study uncovers a conformational selection mechanism underlying species-specific hSTAT2 inhibition by flavivirus NS5.
Assuntos
Flavivirus , Fator de Transcrição STAT2 , Proteínas não Estruturais Virais , Infecção por Zika virus , Zika virus , Humanos , Proteólise , Especificidade da Espécie , Fator de Transcrição STAT2/metabolismo , Proteínas não Estruturais Virais/metabolismoRESUMO
Isoform 1 of DNA methyltransferase DNMT3A (DNMT3A1) specifically recognizes nucleosome monoubiquitylated at histone H2A lysine-119 (H2AK119ub1) for establishment of DNA methylation. Mis-regulation of this process may cause aberrant DNA methylation and pathogenesis. However, the molecular basis underlying DNMT3A1-nucleosome interaction remains elusive. Here we report the cryo-EM structure of DNMT3A1's ubiquitin-dependent recruitment (UDR) fragment complexed with H2AK119ub1-modified nucleosome. DNMT3A1 UDR occupies an extensive nucleosome surface, involving the H2A-H2B acidic patch, a surface groove formed by H2A and H3, nucleosomal DNA, and H2AK119ub1. The DNMT3A1 UDR's interaction with H2AK119ub1 affects the functionality of DNMT3A1 in cells in a context-dependent manner. Our structural and biochemical analysis also reveals competition between DNMT3A1 and JARID2, a cofactor of polycomb repression complex 2 (PRC2), for nucleosome binding, suggesting the interplay between different epigenetic pathways. Together, this study reports a molecular basis for H2AK119ub1-dependent DNMT3A1-nucleosome association, with important implications in DNMT3A1-mediated DNA methylation in development.
Assuntos
DNA (Citosina-5-)-Metiltransferases , Metilação de DNA , DNA Metiltransferase 3A , Histonas , Nucleossomos , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/genética , Histonas/metabolismo , Humanos , Ligação Proteica , Microscopia Crioeletrônica , Animais , Camundongos , Ubiquitinação , Complexo Repressor Polycomb 2/metabolismo , Complexo Repressor Polycomb 2/química , Complexo Repressor Polycomb 2/genética , Células HEK293 , Modelos MolecularesRESUMO
DNA methyltransferases DNMT3A- and DNMT3B-mediated DNA methylation critically regulate epigenomic and transcriptomic patterning during development. The hotspot DNMT3A mutations at the site of Arg822 (R882) promote polymerization, leading to aberrant DNA methylation that may contribute to the pathogenesis of acute myeloid leukemia (AML). However, the molecular basis underlying the mutation-induced functional misregulation of DNMT3A remains unclear. Here, we report the crystal structures of the DNMT3A methyltransferase domain, revealing a molecular basis for its oligomerization behavior distinct to DNMT3B, and the enhanced intermolecular contacts caused by the R882H or R882C mutation. Our biochemical, cellular, and genomic DNA methylation analyses demonstrate that introducing the DNMT3B-converting mutations inhibits the R882H-/R882C-triggered DNMT3A polymerization and enhances substrate access, thereby eliminating the dominant-negative effect of the DNMT3A R882 mutations in cells. Together, this study provides mechanistic insights into DNMT3A R882 mutations-triggered aberrant oligomerization and DNA hypomethylation in AML, with important implications in cancer therapy.
Assuntos
DNA (Citosina-5-)-Metiltransferases , Leucemia Mieloide Aguda , Humanos , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Mutação , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Metilação de DNA/genética , DNA/metabolismoRESUMO
DNA methylation is an important epigenetic mechanism essential for transposon silencing and genome integrity. Across evolution, the substrates of DNA methylation have diversified between kingdoms to account for genome complexity. In plants, Chromomethylase3 (CMT3) and CMT2 are the major methyltransferases mediating CHG and CHH methylation, respectively. However, how these two enzymes diverge on substrate specificities during evolution remains unknown. Here, we reveal that CMT2 originates from a duplication of the evolutionarily more ancient CMT3 in flowering plants. Lacking a key arginine residue recognizing CHG in CMT2 impairs its CHG methylation activity in most flowering plants. An engineered V1200R mutation empowers CMT2 to restore both CHG and CHH methylation in Arabidopsis cmt2cmt3 mutant, testifying a loss-of-function effect for CMT2 after â¼200 million years of evolution. Interestingly, CMT2 has evolved a long and unstructured N-terminus critical for balancing protein stability, especially under heat stress. Furthermore, CMT2 N-terminus is plastic and can be tolerant to various natural mutations. Together, this study reveals the mechanism of chromomethylase divergence for context-specific DNA methylation in plants and sheds important lights on DNA methylation evolution and function.