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.

Age-based diagnostic reference levels and achievable doses for paediatric CT: a survey in Shanghai, China.

Computed tomography (CT) is extensively utilised in medical diagnostics due to its notable radiographic superiority. However, the cancer risk associated with CT examinations, particularly in children, is of significant concern. The assessment of cancer risk relies on the radiation dose to examinees. Diagnostic reference levels (DRLs) and achievable doses (ADs) were used to assess the level of radiation dose in CT examinations widely. Although the national DRLs of paediatric CT have been explored in China, few local DRLs at the city level have been assessed. To set up the local DRLs and ADs of paediatric CT, we investigated the radiation dose level for paediatric CT in Shanghai. In this survey, a total of 3061 paediatric CT examinations underwent in Shanghai in 2022 were selected by stratified sampling, and the dose levels in terms of volume CT dose index (CTDIvol) and the dose-length product (DLP) were analysed by 4 age groups. The DRLs and ADs were set at the 75th and 50th percentile of the distribution and compared with the previous studies at home and abroad. The survey results revealed that, for head scan, the DRLs of CTDIvolwere from 25 to 46 mGy, and the levels of DLP were from 340 to 663 mGy·cm. For chest, the DRLs of CTDIvolwere from 2.2 to 8.3 mGy, and the levels of DLP were from 42 to 223 mGy·cm. For abdomen, the DRLs of CTDIvolwere from 6.3 to 16 mGy, and the levels of DLP were from 181 to 557 mGy·cm. The ADs were about 60% lower than their corresponding DRLs. The levels of radiation doses in children-based hospitals were higher than those in other medical institutions (P< 0.001). In conclusion, there was still potential for reducing radiation dose of paediatric CT, emphasising the urgent need for optimising paediatric CT dose in Shanghai.

Direct readout of heterochromatic H3K9me3 regulates DNMT1-mediated maintenance DNA methylation.

In mammals, repressive histone modifications such as trimethylation of histone H3 Lys9 (H3K9me3), frequently coexist with DNA methylation, producing a more stable and silenced chromatin state. However, it remains elusive how these epigenetic modifications crosstalk. Here, through structural and biochemical characterizations, we identified the replication foci targeting sequence (RFTS) domain of maintenance DNA methyltransferase DNMT1, a module known to bind the ubiquitylated H3 (H3Ub), as a specific reader for H3K9me3/H3Ub, with the recognition mode distinct from the typical trimethyl-lysine reader. Disruption of the interaction between RFTS and the H3K9me3Ub affects the localization of DNMT1 in stem cells and profoundly impairs the global DNA methylation and genomic stability. Together, this study reveals a previously unappreciated pathway through which H3K9me3 directly reinforces DNMT1-mediated maintenance DNA methylation.

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.

Domino Effect: Gold Electrocatalyzing Lithium Reduction to Accelerate Nitrogen Fixation.

Green production of NH3 , especially the Li-mediated electrochemical N2 reduction reaction (NRR) in non-aqueous solutions, is attracting research interest. Controversies regarding the NRR mechanism greatly impede its optimization and wide applications. To understand the electrocatalytic process, we treated Au coated carbon fibrous paper (Au/CP) as the model catalyst. In situ XRD confirmed the transformation of lithium intermediates during NRR. Au greatly improved electron transfer kinetics to catalyze metallic Li formation, and accordingly highly accelerated spontaneous NRR. The Faradaic efficiency of NRR on Au/CP reached 34.0 %, and NH3 yield was as high as 50 µg h-1 cm-2 . Our research shows that the key step of Li-mediated non-aqueous NRR is electrocatalytic Li reduction and offers a novel electrocatalyst design method for Li reduction.

Chemical Proteomic Profiling of Lysophosphatidic Acid-Binding Proteins.

Lysophosphatidic acid (LPA) is an endogenous cell signaling molecule, and dysregulation of LPA signaling pathways is accompanied by several types of cancer. Herein, we developed a chemical proteomic method for the proteome-wide identification of LPA-binding proteins. The method involves the synthesis of a desthiobiotin-conjugated LPA acyl phosphate probe for the covalent labeling, enrichment, and subsequent LC-MS/MS identification of LPA-binding proteins at the proteome-wide level. By conducting labeling reactions at two different probe concentrations (10 and 100 µM) in conjunction with an SILAC (stable isotope labeling by amino acids in cell culture)-based workflow, we characterized the LPA-binding capabilities of these proteins at the entire proteome scale, which led to the identification of 86 candidate LPA-binding proteins in HEK293T cells. Moreover, we validated that two of these proteins, annexin A5 and phosphoglycerate kinase 1, can bind directly with LPA. Together, we developed a novel LPA probe for the identification and characterizations of LPA-binding proteins from the entire human proteome. The method should be adaptable for the identification of other lipid-binding proteins.

Developing carbon-nitride nanosheets for mode-locking ytterbium fiber lasers.

Graphitic carbon nitrides (CNs) have appeared as a new type of photocatalyst for water splitting, but their optical properties (e.g., nonlinear absorption), to the best of our knowledge, have been seldom explored. Here, we report the saturable absorption effects of novel 2D carbon-nitride-type nanosheets and use them as saturable absorbers to passively mode-lock Yb-doped fiber lasers. The CN-based saturable absorber is manufactured by solution coating of 2D CN nanosheets on a gold mirror and has a modulation depth and saturation intensity of 12.5% and 7.5 MW/cm2, respectively. Two different output couplers are employed to construct ring laser cavities. With the 10% coupler, the mode-locked fiber laser produces pulses with duration of ∼310 ps, average power of 1.24 mW, and repetition rate of 7.65 MHz. The laser spectrum is centered at 1066 nm with a bandwidth of 2.4 nm. Increasing the coupling ratio to 50% improves the output power to 2.58 mW but at the same time broadens the pulse width to 420 ps. As a new kind of 2D material with strong saturable absorption, CN nanosheets will open a new way for novel photonic and optoelectronic devices.

NTNH protein: more than a bodyguard for botulinum neurotoxins.

As one of the most fatal substances, botulinum neurotoxins (BoNTs) have never acted solo to accomplish their formidable missions. Most notably, nontoxic nonhemagglutinin (NTNH), a protein co-secreted with BoNT by bacteria, plays critical roles to stabilize and protect BoNT by tightly associating with it to form the minimal progenitor toxin complex (M-PTC). A new cryo-EM structure of the M-PTC of a BoNT-like toxin from Weissella oryzae (BoNT/Wo) reveals similar assembly modes between M-PTC/Wo and that of other BoNTs, yet also reveals some unique structural features of NTNH/Wo. These findings shed new light on the potential versatile roles of NTNH during BoNT intoxication.

Crystal structures of OrfX1, OrfX2 and the OrfX1-OrfX3 complex from the orfX gene cluster of botulinum neurotoxin E1.

Botulinum neurotoxins (BoNTs) are among the most lethal toxins known to humans, comprising seven established serotypes termed BoNT/A-G encoded in two types of gene clusters (ha and orfX) in BoNT-producing clostridia. The ha cluster encodes four non-toxic neurotoxin-associated proteins (NAPs) that assemble with BoNTs to protect and enhance their oral toxicity. However, the structure and function of the orfX-type NAPs remain largely unknown. Here, we report the crystal structures for OrfX1, OrfX2, and an OrfX1-OrfX3 complex, which are encoded in the orfX cluster of a BoNT/E1-producing Clostridium botulinum strain associated with human foodborne botulism. These structures lay the foundation for future studies on the potential roles of OrfX proteins in oral intoxication and pathogenesis of BoNTs.

Blind detection of circular image rotation angle based on ensemble transfer regression and fused HOG.

Introduction: Aiming at the problems of low accuracy in estimating the rotation angle after the rotation of circular image data within a wide range (0°-360°) and difficulty in blind detection without a reference image, a method based on ensemble transfer regression network, fused HOG, and Rotate Loss is adopted to solve such problems. Methods: The proposed Rotate Loss was combined to solve the angle prediction error, especially the huge error when near 0°. Fused HOG was mainly used to extract directional features. Then, the feature learning was conducted by the ensemble transfer regression model combined with the feature extractor and the ensemble regressors to estimate an exact rotation angle. Based on miniImageNet and Minist, we made the circular random rotation dataset Circular-ImageNet and random rotation dataset Rot-Minist, respectively. Results: Experiments showed that for the proposed evaluation index MSE_Rotate, the best single regressor could be as low as 28.79 on the training set of Circular-ImageNet and 2686.09 on the validation set. For MSE_Rotate, MSE, MAE, and RMSE on the test set were 1,702.4325, 0.0263, 0.0881, and 0.1621, respectively. And under the ensemble transfer regression network, it could continue to decrease by 15%. The mean error rate on Rot-Minist could be just 0.59%, significantly working easier in a wide range than other networks in recent years. Based on the ensemble transfer regression model, we also completed the application of image righting blindly.

Impact of Protein Nitration on Influenza Virus Infectivity and Immunogenicity.

Influenza viruses are deadly respiratory pathogens of special importance due to their long history of global pandemics. During influenza virus infections, the host responds by producing interferons, which activate interferon-stimulated genes (ISGs) inside target cells. One of these ISGs is inducible nitric oxide synthase (iNOS). iNOS produces nitric oxide (NO) from arginine and molecular oxygen inside the cell. NO can react with superoxide radicals to form reactive nitrogen species, principally peroxynitrite. While much work has been done studying the many roles of nitric oxide in influenza virus infections, the direct effect of peroxynitrite on influenza virus proteins has not been determined. Manipulations of NO, either by knocking out iNOS or chemically inhibiting NO, produced no change in virus titers in mouse models of influenza infection. However, peroxynitrite has a known antimicrobial effect on various bacteria and parasites, and the reason for its lack of antimicrobial effect on influenza virus titers in vivo remains unclear. Therefore, we wished to test the direct effect of nitration of influenza virus proteins. We examined the impact of nitration on virus infectivity, replication, and immunogenicity. We observed that the nitration of influenza A virus proteins decreased virus infectivity and replication ex vivo. We also determined that the nitration of influenza virus hemagglutinin protein can reduce antibody responses to native virus protein. However, our study also suggests that nitration of influenza virus proteins in vivo is likely not extensive enough to inhibit virus functions substantially. These findings will help clarify the role of peroxynitrite during influenza virus infections. IMPORTANCE Nitric oxide and peroxynitrite produced during microbial infections have diverse and seemingly paradoxical functions. While nitration of lung tissue during influenza virus infection has been observed in both mice and humans, the direct effect of protein nitration on influenza viruses has remained elusive. We addressed the impact of nitration of influenza virus proteins on virus infectivity, replication, and immunogenicity. We observed that ex vivo nitration of influenza virus proteins reduced virus infectivity and immunogenicity. However, we did not detect nitration of influenza virus hemagglutinin protein in vivo. These results contribute to our understanding of the roles of nitric oxide and peroxynitrite in influenza virus infections.

Structure of DNMT3B homo-oligomer reveals vulnerability to impairment by ICF mutations.

DNA methyltransferase DNMT3B plays an essential role in establishment of DNA methylation during embryogenesis. Mutations of DNMT3B are associated with human diseases, notably the immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome. How ICF mutations affect DNMT3B activity is not fully understood. Here we report the homo-oligomeric structure of DNMT3B methyltransferase domain, providing insight into DNMT3B-mediated DNA methylation in embryonic stem cells where the functional regulator DNMT3L is dispensable. The interplay between one of the oligomer interfaces (FF interface) and the catalytic loop renders DNMT3B homo-oligomer a conformation and activity distinct from the DNMT3B-DNMT3L heterotetramer, and a greater vulnerability to certain ICF mutations. Biochemical and cellular analyses further reveal that the ICF mutations of FF interface impair the DNA binding and heterochromatin targeting of DNMT3B, leading to reduced DNA methylation in cells. Together, this study provides a mechanistic understanding of DNMT3B-mediated DNA methylation and its dysregulation in disease.

DNMT1 reads heterochromatic H4K20me3 to reinforce LINE-1 DNA methylation.

DNA methylation and trimethylated histone H4 Lysine 20 (H4K20me3) constitute two important heterochromatin-enriched marks that frequently cooperate in silencing repetitive elements of the mammalian genome. However, it remains elusive how these two chromatin modifications crosstalk. Here, we report that DNA methyltransferase 1 (DNMT1) specifically 'recognizes' H4K20me3 via its first bromo-adjacent-homology domain (DNMT1BAH1). Engagement of DNMT1BAH1-H4K20me3 ensures heterochromatin targeting of DNMT1 and DNA methylation at LINE-1 retrotransposons, and cooperates with the previously reported readout of histone H3 tail modifications (i.e., H3K9me3 and H3 ubiquitylation) by the RFTS domain to allosterically regulate DNMT1's activity. Interplay between RFTS and BAH1 domains of DNMT1 profoundly impacts DNA methylation at both global and focal levels and genomic resistance to radiation-induced damage. Together, our study establishes a direct link between H4K20me3 and DNA methylation, providing a mechanism in which multivalent recognition of repressive histone modifications by DNMT1 ensures appropriate DNA methylation patterning and genomic stability.

Dissect the DNMT3A- and DNMT3B-mediated DNA Co-methylation through a Covalent Complex Approach.

DNA methylation plays a critical role in regulating gene expression, genomic stability, and cell fate commitment. Mammalian DNA methylation, which mostly occurs in the context of CpG dinucleotide, is installed by two denovo DNA methyltransferases, DNMT3A and DNMT3B. Oligomerization of DNMT3A and DNMT3B permits both enzymes to comethylate two CpG sites located on the same DNA substrates. However, how DNMT3A- and DNMT3B-mediated co-methylation contributes to the DNA methylation patterns remain unclear. Here we generated covalent enzyme-substrate complexes of DNMT3A and DNMT3B, and performed bisulfite sequencing-based single-turnover methylation analysis on both complexes. Our results showed that both DNMT3A- and DNMT3B-mediated co-methylation preferentially gives rise to a methylation spacing of 14 base pairs, consistent with the previous structural observation for DNMT3A in complex with regulatory protein DNMT3L and CpG DNA. This study provides a novel method for mechanistic investigation of DNMT3A- and DNMT3B-mediated DNA co-methylation.

The Agpat4/LPA axis in colorectal cancer cells regulates antitumor responses via p38/p65 signaling in macrophages.

Lipid metabolic reprogramming plays an essential role in regulating the progression of colorectal cancer (CRC). However, the effect of lysophosphatidic acid (LPA) metabolism on CRC development is incompletely characterized. Here, we compared the mRNA levels of human CRC tissues to those of paracarcinoma tissues and focused on the notably enriched LPA metabolic pathways. We identified and verified that 1-acylglycerol-3-phosphate O-acyltransferase 4 (Agpat4) was aberrantly expressed in CRC tissues and predicted poor survival in CRC patients. Manipulating Agpat4 expression in CRC cells did not affect the growth or migration of CRC cells in vitro, whereas Agpat4 silencing suppressed CRC cell growth in subcutaneous and peritoneal xenograft models. Mechanistically, Agpat4 silencing-induced LPA release from CRC cells and polarized macrophages to an M1-like phenotype through LPA receptors 1 and 3. This M1 activation, characterized by elevated p38/p65 signaling and increased proinflammatory cytokines, promoted the infiltration and activation of CD4+ and CD8+ T cells in the tumor microenvironment. Modulation of the Agpat4/LPA/p38/p65 axis regulated macrophage polarization, T-cell activity and CRC progression. Notably, combined therapy with LPA and regular chemotherapy drugs synergistically suppressed CRC development. Taken together, our results showed that the Agpat4/LPA axis in CRC cells regulated p38/p65 signaling-dependent macrophage polarization, T-cell activation, and CRC progression. The Agpat4/LPA/p38/p65 axis might represent a potential target for therapy in the clinic.

Structural basis for STAT2 suppression by flavivirus NS5.

Suppressing cellular signal transducers of transcription 2 (STAT2) is a common strategy that viruses use to establish infections, yet the detailed mechanism remains elusive, owing to a lack of structural information about the viral-cellular complex involved. Here, we report the cryo-EM and crystal structures of human STAT2 (hSTAT2) in complex with the non-structural protein 5 (NS5) of Zika virus (ZIKV) and dengue virus (DENV), revealing two-pronged interactions between NS5 and hSTAT2. First, the NS5 methyltransferase and RNA-dependent RNA polymerase (RdRP) domains form a conserved interdomain cleft harboring the coiled-coil domain of hSTAT2, thus preventing association of hSTAT2 with interferon regulatory factor 9. Second, the NS5 RdRP domain also binds the amino-terminal domain of hSTAT2. Disruption of these ZIKV NS5-hSTAT2 interactions compromised NS5-mediated hSTAT2 degradation and interferon suppression, and viral infection under interferon-competent conditions. Taken together, these results clarify the mechanism underlying the functional antagonism of STAT2 by both ZIKV and DENV.

Comprehensive structure-function characterization of DNMT3B and DNMT3A reveals distinctive de novo DNA methylation mechanisms.

Mammalian DNA methylation patterns are established by two de novo DNA methyltransferases, DNMT3A and DNMT3B, which exhibit both redundant and distinctive methylation activities. However, the related molecular basis remains undetermined. Through comprehensive structural, enzymology and cellular characterization of DNMT3A and DNMT3B, we here report a multi-layered substrate-recognition mechanism underpinning their divergent genomic methylation activities. A hydrogen bond in the catalytic loop of DNMT3B causes a lower CpG specificity than DNMT3A, while the interplay of target recognition domain and homodimeric interface fine-tunes the distinct target selection between the two enzymes, with Lysine 777 of DNMT3B acting as a unique sensor of the +1 flanking base. The divergent substrate preference between DNMT3A and DNMT3B provides an explanation for site-specific epigenomic alterations seen in ICF syndrome with DNMT3B mutations. Together, this study reveals distinctive substrate-readout mechanisms of the two DNMT3 enzymes, implicative of their differential roles during development and pathogenesis.

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.

Structural Basis of DNMT1 and DNMT3A-Mediated DNA Methylation.

DNA methylation, one of the major epigenetic mechanisms, plays critical roles in regulating gene expression, genomic stability and cell lineage commitment. The establishment and maintenance of DNA methylation in mammals is achieved by two groups of DNA methyltransferases (DNMTs): DNMT3A and DNMT3B, which are responsible for installing DNA methylation patterns during gametogenesis and early embryogenesis, and DNMT1, which is essential for propagating DNA methylation patterns during replication. Both groups of DNMTs are multi-domain proteins, containing a large N-terminal regulatory region in addition to the C-terminal methyltransferase domain. Recent structure-function investigations of the individual domains or large fragments of DNMT1 and DNMT3A have revealed the molecular basis for their substrate recognition and specificity, intramolecular domain-domain interactions, as well as their crosstalk with other epigenetic mechanisms. These studies highlight a multifaceted regulation for both DNMT1 and DNMT3A/3B, which is essential for the precise establishment and maintenance of lineage-specific DNA methylation patterns in cells. This review summarizes current understanding of the structure and mechanism of DNMT1 and DNMT3A-mediated DNA methylation, with emphasis on the functional cooperation between the methyltransferase and regulatory domains.

An all-inorganic lead halide perovskite-based photocathode for stable water reduction.

Lead halide perovskites (LHPs) have been investigated for photoelectrochemical hydrogen generation from water splitting. However, the harsh requirements in preparing the environment, i.e. isolating water and oxygen, hinder the wide applications of lead halide perovskites. Herein, an all-inorganic perovskite, i.e. a CsPbBr3-based photocathode, has been prepared to generate hydrogen. It is notable that as a valuable trial for a potential large-scale production, the whole preparation process was completed in an open-air environment. The LHP photocathode achieved the highest photocurrent of about 1.2 mA cm-2 at 0 VRHE. And the photocurrent remains around 94% after continuous illumination for 1 h with the Faradaic efficiency of 90%, illustrating a good photoelectrochemical stability. The all-inorganic LHP photocathodes are facile to prepare with a relatively good performance, and can be improved via band engineering and structure optimization, of which large-scale applications can be expected.