Suppression of Type I Interferon Signaling by Flavivirus NS5.

Type I interferon (IFN-I) is the first line of mammalian host defense against viral infection. To counteract this, the flaviviruses, like other viruses, have encoded a variety of antagonists, and use a multi-layered molecular defense strategy to establish their infections. Among the most potent antagonists is non-structural protein 5 (NS5), which has been shown for all disease-causing flaviviruses to target different steps and players of the type I IFN signaling pathway. Here, we summarize the type I IFN antagonist mechanisms used by flaviviruses with a focus on the role of NS5 in regulating one key regulator of type I IFN, signal transducer and activator of transcription 2 (STAT2)

Structure and regulation of ZCCHC4 in m6A-methylation of 28S rRNA.

N6-methyladenosine (m6A) modification provides an important epitranscriptomic mechanism that critically regulates RNA metabolism and function. However, how m6A writers attain substrate specificities remains unclear. We report the 3.1 Å-resolution crystal structure of human CCHC zinc finger-containing protein ZCCHC4, a 28S rRNA-specific m6A methyltransferase, bound to S-adenosyl-L-homocysteine. The methyltransferase (MTase) domain of ZCCHC4 is packed against N-terminal GRF-type and C2H2 zinc finger domains and a C-terminal CCHC domain, creating an integrated RNA-binding surface. Strikingly, the MTase domain adopts an autoinhibitory conformation, with a self-occluded catalytic site and a fully-closed cofactor pocket. Mutational and enzymatic analyses further substantiate the molecular basis for ZCCHC4-RNA recognition and a role of the stem-loop structure within substrate in governing the substrate specificity. Overall, this study unveils unique structural and enzymatic characteristics of ZCCHC4, distinctive from what was seen with the METTL family of m6A writers, providing the mechanistic basis for ZCCHC4 modulation of m6A RNA methylation

Multi-layered heterochromatin interaction as a switch for DIM2-mediated DNA methylation.

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

Structure and function of Zika virus NS5 protein: perspectives for drug design.

Zika virus (ZIKV) belongs to the positive-sense single-stranded RNA-containing Flaviviridae family. Its recent outbreak and association with human diseases (e.g. neurological disorders) have raised global health concerns, and an urgency to develop a therapeutic strategy against ZIKV infection. However, there is no currently approved antiviral against ZIKV. Here we present a comprehensive overview on recent progress in structure-function investigation of ZIKV NS5 protein, the largest non-structural protein of ZIKV, which is responsible for replication of the viral genome, RNA capping and suppression of host interferon responses. Structural comparison of the N-terminal methyltransferase domain and C-terminal RNA-dependent RNA polymerase domain of ZIKV NS5 with their counterparts from related viruses provides mechanistic insights into ZIKV NS5-mediated RNA replication, and identifies residues critical for its enzymatic activities. Finally, a collection of recently identified small molecule inhibitors against ZIKV NS5 or its closely related flavivirus homologues are also discussed

Arsenite binds to the RING finger domains of RNF20-RNF40 histone E3 ubiquitin ligase and inhibits DNA double-strand break repair.

Arsenic is a widespread environmental contaminant. However, the exact molecular mechanisms underlying the carcinogenic effects of arsenic remain incompletely understood. Core histones can be ubiquitinated by RING finger E3 ubiquitin ligases, among which the RNF20-RNF40 heterodimer catalyzes the ubiquitination of histone H2B at lysine 120. This ubiquitination event is important for the formation of open and biochemically accessible chromatin fiber that is conducive for DNA repair. Herein, we found that arsenite could bind directly to the RING finger domains of RNF20 and RNF40 in vitro and in cells, and treatment with arsenite resulted in substantially impaired H2B ubiquitination in multiple cell lines. Exposure to arsenite also diminished the recruitment of BRCA1 and RAD51 to laser-induced DNA double-strand break (DSB) sites, compromised DNA DSB repair in human cells, and rendered cells sensitive toward a radiomimetic agent, neocarzinostatin. Together, the results from the present study revealed, for the first time, that arsenite may exert its carcinogenic effect by targeting cysteine residues in the RING finger domains of histone E3 ubiquitin ligase, thereby altering histone epigenetic mark and compromising DNA DSB repair. Our results also suggest arsenite as a general inhibitor for RING finger E3 ubiquitin ligases

Structural basis for DNMT3A-mediated de novo DNA methylation.

DNA methylation by de novo DNA methyltransferases 3A (DNMT3A) and 3B (DNMT3B) at cytosines is essential for genome regulation and development. Dysregulation of this process is implicated in various diseases, notably cancer. However, the mechanisms underlying DNMT3 substrate recognition and enzymatic specificity remain elusive. Here we report a 2.65-ångström crystal structure of the DNMT3A-DNMT3L-DNA complex in which two DNMT3A monomers simultaneously attack two cytosine-phosphate-guanine (CpG) dinucleotides, with the target sites separated by 14 base pairs within the same DNA duplex. The DNMT3A-DNA interaction involves a target recognition domain, a catalytic loop, and DNMT3A homodimeric interface. Arg836 of the target recognition domain makes crucial contacts with CpG, ensuring DNMT3A enzymatic preference towards CpG sites in cells. Haematological cancer-associated somatic mutations of the substrate-binding residues decrease DNMT3A activity, induce CpG hypomethylation, and promote transformation of haematopoietic cells. Together, our study reveals the mechanistic basis for DNMT3A-mediated DNA methylation and establishes its aetiological link to human disease

A DNA aptamer for binding and inhibition of DNA methyltransferase 1.

DNA methyltransferases (DNMTs) are enzymes responsible for establishing and maintaining DNA methylation in cells. DNMT inhibition is actively pursued in cancer treatment, dominantly through the formation of irreversible covalent complexes between small molecular compounds and DNMTs that suffers from low efficacy and high cytotoxicity, as well as no selectivity towards different DNMTs. Herein, we discover aptamers against the maintenance DNA methyltransferase, DNMT1, by coupling Asymmetrical Flow Field-Flow Fractionation (AF4) with Systematic Evolution of Ligands by EXponential enrichment (SELEX). One of the identified aptamers, Apt. #9, contains a stem-loop structure, and can displace the hemi-methylated DNA duplex, the native substrate of DNMT1, off the protein on sub-micromolar scale, leading for effective enzymatic inhibition. Apt. #9 shows no inhibition nor binding activity towards two de novo DNMTs, DNMT3A and DNMT3B. Intriguingly, it can enter cancer cells with over-expression of DNMT1, colocalize with DNMT1 inside the nuclei, and inhibit the activity of DNMT1 in cells. This study opens the possibility of exploring the aptameric DNMT inhibitors being a new cancer therapeutic approach, by modulating DNMT activity selectively through reversible interaction. The aptamers could also be valuable tools for study of the functions of DNMTs and the related epigenetic mechanisms

Structural basis for the H2AK119ub1-specific DNMT3A-nucleosome interaction.

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 DNMT3A1s 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 UDRs 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