Reshaping the Landscape of the Genome: Toolkits for Precise DNA Methylation Manipulation and Beyond.

DNA methylation plays a pivotal role in various biological processes and is highly related to multiple diseases. The exact functions of DNA methylation are still puzzling due to its uneven distribution, dynamic conversion, and complex interactions with other substances. Current methods such as chemical- and enzyme-based sequencing techniques have enabled us to pinpoint DNA methylation at single-base resolution, which necessitated the manipulation of DNA methylation at comparable resolution to precisely illustrate the correlations and causal relationships between the functions of DNA methylation and its spatiotemporal patterns. Here a perspective on the past, recent process, and future of precise DNA methylation tools is provided. Specifically, genome-wide and site-specific manipulation of DNA methylation methods is discussed, with an emphasis on their principles, limitations, applications, and future developmental directions.

Stimuli-Responsive Protein Hydrogels: Their Design, Properties, and Biomedical Applications.

Protein-based hydrogels are considered ideal biomaterials due to their high biocompatibility, diverse structure, and their improved bioactivity and biodegradability. However, it remains challenging to mimic the native extracellular matrices that can dynamically respond to environmental stimuli. The combination of stimuli-responsive functionalities with engineered protein hydrogels has facilitated the development of new smart hydrogels with tunable biomechanics and biological properties that are triggered by cyto-compatible stimuli. This review summarizes the recent advancements of responsive hydrogels prepared from engineered proteins and integrated with physical, chemical or biological responsive moieties. We underscore the design principles and fabrication approaches of responsive protein hydrogels, and their biomedical applications in disease treatment, drug delivery, and tissue engineering are briefly discussed. Finally, the current challenges and future perspectives in this field are highlighted.

Circulating Transforming Growth Factor-ß1 Levels in Preeclamptic Women: a Meta-analysis.

Transforming growth factor (TGF)-ß1, an angiogenic factor in the maternal circulation, has been suggested to be related to preeclampsia. However, the findings from previous studies were inconsistent. Thus, we conducted a meta-analysis to assess the difference in circulating TGF-ß1 levels between women with preeclampsia and normal pregnancies. Twenty-four studies including 1748 women with PE and 1404 women with normal pregnancy were included in our study. The results showed that circulating TGF-ß1 levels were not different before the time of active disease (standardized mean differences, - 0.46 [95% CI, - 0.16 to 0.15]; P = 0.000). At the time of active disease, women with preeclampsia (n = 1207) had higher circulating TGF-ß1 levels than normotensive controls (n = 912; standardized mean differences, 0.94 [95% CI, 0.52 to 1.35]; P = 0.000). Circulating TGF-ß1 levels were higher in both early-onset/severe and late-onset/mild types of preeclampsia. No publication biases were found. We conclude that preeclamptic women have higher circulating TGF-ß1 than those with normal pregnancy at the time of preeclampsia diagnosis.

METTL4-mediated nuclear N6-deoxyadenosine methylation promotes metastasis through activating multiple metastasis-inducing targets.

BACKGROUND: DNA N6-methyldeoxyadenosine (6mA) is rarely present in mammalian cells and its nuclear role remains elusive. RESULTS: Here we show that hypoxia induces nuclear 6mA modification through a DNA methyltransferase, METTL4, in hypoxia-induced epithelial-mesenchymal transition (EMT) and tumor metastasis. Co-expression of METTL4 and 6mA represents a prognosis marker for upper tract urothelial cancer patients. By RNA sequencing and 6mA chromatin immunoprecipitation-exonuclease digestion followed by sequencing, we identify lncRNA RP11-390F4.3 and one novel HIF-1α co-activator, ZMIZ1, that are co-regulated by hypoxia and METTL4. Other genes involved in hypoxia-mediated phenotypes are also regulated by 6mA modification. Quantitative chromatin isolation by RNA purification assay shows the occupancy of lncRNA RP11-390F4.3 on the promoters of multiple EMT regulators, indicating lncRNA-chromatin interaction. Knockdown of lncRNA RP11-390F4.3 abolishes METTL4-mediated tumor metastasis. We demonstrate that ZMIZ1 is an essential co-activator of HIF-1α. CONCLUSIONS: We show that hypoxia results in enriched 6mA levels in mammalian tumor cells through METTL4. This METTL4-mediated nuclear 6mA deposition induces tumor metastasis through activating multiple metastasis-inducing genes. METTL4 is characterized as a potential therapeutic target in hypoxic tumors.

Relationship between the molecular structure of different dithiocarbamates and their oil removal performance.

Six kinds of dithiocarbamates (DTCs) were synthesized from three linear amines with different amino numbers, two polyether amines with different molecular weights, and one branched amine with benzene rings, respectively. The conditions affecting oil removal rate and floc rising time of DTC were studied using simulated oily wastewater. Furthermore, the effects of the molecular structure of DTC on oil removal efficiency, floc morphology, floc rising time, and floc adhesion were investigated. When the conditions were optimal, the oil removal efficiency of DTC synthesized from polyethylene polyamine was 95.14%, which was higher than other DTCs. Meanwhile, the ferrous ion was the most suitable chelating metal ion for DTC than other transition metal ions. The increase of amino groups in the initiators improves the oil removal efficiency of DTC, while the linear structural DTC exhibits a low oil removal efficiency due to a lack of network structural flocs. The introduction of polyether structure helps reduce the volume of the flocs and make them compact, but it also increases the adhesion of the floc on the metal surface. The introduction of bisphenol A phenol amino resin structure induces the generation of the flocs in oil wastewater and improves the oil removal efficiency.

Bearing Fault Diagnosis Based on an Enhanced Image Representation Method of Vibration Signal and Conditional Super Token Transformer.

Multipoint Optimal Minimum Entropy Deconvolution Adjusted (MOMEDA) is an advanced deconvolution method, which can effectively inhibit the interference of background noise and distinguish the fault period by calculating the multipoint kurtosis values. However, multipoint kurtosis (MKurt) could lead to misjudgment since it is sensitive to spurious noise spikes. Considering that L-kurtosis has good robustness with noise, this paper proposes a multipoint envelope L-kurtosis (MELkurt) method for establishing the temporal features. Then, an enhanced image representation method of vibration signals is proposed by employing the Gramian Angular Difference Field (GADF) method to convert the MELkurt series into images. Furthermore, to effectively learn and extract the features of GADF images, this paper develops a deep learning method named Conditional Super Token Transformer (CSTT) by incorporating the Super Token Transformer block, Super Token Mixer module, and Conditional Positional Encoding mechanism into Vision Transformer appropriately. Transfer learning is introduced to enhance the diagnostic accuracy and generalization capability of the designed CSTT. Consequently, a novel bearing fault diagnosis framework is established based on the presented enhanced image representation and CSTT. The proposed method is compared with Vision Transformer and some CNN-based models to verify the recognition effect by two experimental datasets. The results show that MELkurt significantly improves the fault feature enhancement ability with superior noise robustness to kurtosis, and the proposed CSTT achieves the highest diagnostic accuracy and stability.

FTO mediates LINE1 m6A demethylation and chromatin regulation in mESCs and mouse development.

N6-methyladenosine (m6A) is the most abundant internal modification on mammalian messenger RNA. It is installed by a writer complex and can be reversed by erasers such as the fat mass and obesity-associated protein FTO. Despite extensive research, the primary physiological substrates of FTO in mammalian tissues and development remain elusive. Here, we show that FTO mediates m6A demethylation of long-interspersed element-1 (LINE1) RNA in mouse embryonic stem cells (mESCs), regulating LINE1 RNA abundance and the local chromatin state, which in turn modulates the transcription of LINE1-containing genes. FTO-mediated LINE1 RNA m6A demethylation also plays regulatory roles in shaping chromatin state and gene expression during mouse oocyte and embryonic development. Our results suggest broad effects of LINE1 RNA m6A demethylation by FTO in mammals.

m6A RNA modifications are measured at single-base resolution across the mammalian transcriptome.

Functional studies of the RNA N6-methyladenosine (m6A) modification have been limited by an inability to map individual m6A-modified sites in whole transcriptomes. To enable such studies, here, we introduce m6A-selective allyl chemical labeling and sequencing (m6A-SAC-seq), a method for quantitative, whole-transcriptome mapping of m6A at single-nucleotide resolution. The method requires only ~30 ng of poly(A) or rRNA-depleted RNA. We mapped m6A modification stoichiometries in RNA from cell lines and during in vitro monocytopoiesis from human hematopoietic stem and progenitor cells (HSPCs). We identified numerous cell-state-specific m6A sites whose methylation status was highly dynamic during cell differentiation. We observed changes of m6A stoichiometry as well as expression levels of transcripts encoding or regulated by key transcriptional factors (TFs) critical for HSPC differentiation. m6A-SAC-seq is a quantitative method to dissect the dynamics and functional roles of m6A sites in diverse biological processes using limited input RNA.

ALKBH7-mediated demethylation regulates mitochondrial polycistronic RNA processing.

Members of the mammalian AlkB family are known to mediate nucleic acid demethylation1,2. ALKBH7, a mammalian AlkB homologue, localizes in mitochondria and affects metabolism3, but its function and mechanism of action are unknown. Here we report an approach to site-specifically detect N1-methyladenosine (m1A), N3-methylcytidine (m3C), N1-methylguanosine (m1G) and N2,N2-dimethylguanosine (m22G) modifications simultaneously within all cellular RNAs, and discovered that human ALKBH7 demethylates m22G and m1A within mitochondrial Ile and Leu1 pre-tRNA regions, respectively, in nascent polycistronic mitochondrial RNA4-6. We further show that ALKBH7 regulates the processing and structural dynamics of polycistronic mitochondrial RNAs. Depletion of ALKBH7 leads to increased polycistronic mitochondrial RNA processing, reduced steady-state mitochondria-encoded tRNA levels and protein translation, and notably decreased mitochondrial activity. Thus, we identify ALKBH7 as an RNA demethylase that controls nascent mitochondrial RNA processing and mitochondrial activity.

N6-Deoxyadenosine Methylation in Mammalian Mitochondrial DNA.

N6-Methyldeoxyadenosine (6mA) has recently been shown to exist and play regulatory roles in eukaryotic genomic DNA (gDNA). However, the biological functions of 6mA in mammals have yet to be adequately explored, largely due to its low abundance in most mammalian genomes. Here, we report that mammalian mitochondrial DNA (mtDNA) is enriched for 6mA. The level of 6mA in HepG2 mtDNA is at least 1,300-fold higher than that in gDNA under normal growth conditions, corresponding to approximately four 6mA modifications on each mtDNA molecule. METTL4, a putative mammalian methyltransferase, can mediate mtDNA 6mA methylation, which contributes to attenuated mtDNA transcription and a reduced mtDNA copy number. Mechanistically, the presence of 6mA could repress DNA binding and bending by mitochondrial transcription factor (TFAM). Under hypoxia, the 6mA level in mtDNA could be further elevated, suggesting regulatory roles for 6mA in mitochondrial stress response. Our study reveals DNA 6mA as a regulatory mark in mammalian mtDNA.

Biomimetic nanoscale metal-organic framework harnesses hypoxia for effective cancer radiotherapy and immunotherapy.

Tumor hypoxia presents a major impediment to effective cancer therapy with ionizing radiation and immune checkpoint inhibitors. Here we report the design of a biomimetic nanoscale metal-organic-framework (nMOF), Hf-DBP-Fe, with catalase-like activity to decompose elevated levels of H2O2 in hypoxic tumors to generate oxygen and hydroxyl radical. The generated oxygen attenuates hypoxia to enable radiodynamic therapy upon X-ray irradiation and fixes DNA damage while hydroxyl radical inflicts direct damage to tumor cells to afford chemodynamic therapy. Hf-DBP-Fe thus mediates effective local therapy of hypoxic cancer with low-dose X-ray irradiation, leading to highly immunogenic tumor microenvironments for synergistic combination with anti-PD-L1 immune checkpoint blockade. This combination treatment not only eradicates primary tumors but also rejects distant tumors through systemic anti-tumor immunity. We have thus advanced an nMOF-based strategy to harness hypoxic tumor microenvironments for highly effective cancer therapy using a synergistic combination of low dose radiation and immune checkpoint blockade.

Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA.

BACKGROUND: Directed DNA methylation on N6-adenine (6mA), N4-cytosine (4mC), and C5-cytosine (5mC) can potentially increase DNA coding capacity and regulate a variety of biological functions. These modifications are relatively abundant in bacteria, occurring in about a percent of all bases of most bacteria. Until recently, 5mC and its oxidized derivatives were thought to be the only directed DNA methylation events in metazoa. New and more sensitive detection techniques (ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-ms/ms) and single molecule real-time sequencing (SMRTseq)) have suggested that 6mA and 4mC modifications could be present in a variety of metazoa. RESULTS: Here, we find that both of these techniques are prone to inaccuracies, which overestimate DNA methylation concentrations in metazoan genomic DNA. Artifacts can arise from methylated bacterial DNA contamination of enzyme preparations used to digest DNA and contaminating bacterial DNA in eukaryotic DNA preparations. Moreover, DNA sonication introduces a novel modified base from 5mC that has a retention time near 4mC that can be confused with 4mC. Our analyses also suggest that SMRTseq systematically overestimates 4mC in prokaryotic and eukaryotic DNA and 6mA in DNA samples in which it is rare. Using UHPLC-ms/ms designed to minimize and subtract artifacts, we find low to undetectable levels of 4mC and 6mA in genomes of representative worms, insects, amphibians, birds, rodents and primates under normal growth conditions. We also find that mammalian cells incorporate exogenous methylated nucleosides into their genome, suggesting that a portion of 6mA modifications could derive from incorporation of nucleosides from bacteria in food or microbiota. However, gDNA samples from gnotobiotic mouse tissues found rare (0.9-3.7 ppm) 6mA modifications above background. CONCLUSIONS: Altogether these data demonstrate that 6mA and 4mC are rarer in metazoa than previously reported, and highlight the importance of careful sample preparation and measurement, and need for more accurate sequencing techniques.

N6-methyldeoxyadenosine directs nucleosome positioning in Tetrahymena DNA.

BACKGROUND: N6-methyldeoxyadenosine (6mA or m6dA) was shown more than 40 years ago in simple eukaryotes. Recent studies revealed the presence of 6mA in more prevalent eukaryotes, even in vertebrates. However, functional characterizations have been limited. RESULTS: We use Tetrahymena thermophila as a model organism to examine the effects of 6mA on nucleosome positioning. Independent methods reveal the enrichment of 6mA near and after transcription start sites with a periodic pattern and anti-correlation relationship with the positions of nucleosomes. The distribution pattern can be recapitulated by in vitro nucleosome assembly on native Tetrahymena genomic DNA but not on DNA without 6mA. Model DNA containing artificially installed 6mA resists nucleosome assembling compared to unmodified DNA in vitro. Computational simulation indicates that 6mA increases dsDNA rigidity, which disfavors nucleosome wrapping. Knockout of a potential 6mA methyltransferase leads to a transcriptome-wide change of gene expression. CONCLUSIONS: These findings uncover a mechanism by which DNA 6mA assists to shape the nucleosome positioning and potentially affects gene expression.

Mapping and characterizing N6-methyladenine in eukaryotic genomes using single-molecule real-time sequencing.

N6-Methyladenine (m6dA) has been discovered as a novel form of DNA methylation prevalent in eukaryotes; however, methods for high-resolution mapping of m6dA events are still lacking. Single-molecule real-time (SMRT) sequencing has enabled the detection of m6dA events at single-nucleotide resolution in prokaryotic genomes, but its application to detecting m6dA in eukaryotic genomes has not been rigorously examined. Herein, we identified unique characteristics of eukaryotic m6dA methylomes that fundamentally differ from those of prokaryotes. Based on these differences, we describe the first approach for mapping m6dA events using SMRT sequencing specifically designed for the study of eukaryotic genomes and provide appropriate strategies for designing experiments and carrying out sequencing in future studies. We apply the novel approach to study two eukaryotic genomes. For green algae, we construct the first complete genome-wide map of m6dA at single-nucleotide and single-molecule resolution. For human lymphoblastoid cells (hLCLs), it was necessary to integrate SMRT sequencing data with independent sequencing data. The joint analyses suggest putative m6dA events are enriched in the promoters of young full-length LINE-1 elements (L1s), but call for validation by additional methods. These analyses demonstrate a general method for rigorous mapping and characterization of m6dA events in eukaryotic genomes.

Genetically encoded fluorescent sensors for measuring transition and heavy metals in biological systems.

Great progress has been made in expanding the repertoire of genetically encoded fluorescent sensors for monitoring intracellular transition metals (TMs). This powerful toolkit permits dynamic and non-invasive detection of TMs with high spatial-temporal resolution, which enables us to better understand the roles of TM homeostasis in both physiological and pathological settings. Here we summarize the recent development of genetically encoded fluorescent sensors for intracellular detection of TMs such as zinc and copper, as well as heavy metals including lead, cadmium, mercury, and arsenic.

Genetically encoded releasable photo-cross-linking strategies for studying protein-protein interactions in living cells.

Although protein-protein interactions (PPIs) have crucial roles in virtually all cellular processes, the identification of more transient interactions in their biological context remains challenging. Conventional photo-cross-linking strategies can be used to identify transient interactions, but these approaches often suffer from high background due to the cross-linked bait proteins. To solve the problem, we have developed membrane-permeable releasable photo-cross-linkers that allow for prey-bait separation after protein complex isolation and can be installed in proteins of interest (POIs) as unnatural amino acids. Here we describe the procedures for using two releasable photo-cross-linkers, DiZSeK and DiZHSeC, in both living Escherichia coli and mammalian cells. A cleavage after protein photo-cross-linking (CAPP ) strategy based on the photo-cross-linker DiZSeK is described, in which the prey protein pool is released from a POI after affinity purification. Prey proteins are analyzed using mass spectrometry or 2D gel electrophoresis for global comparison of interactomes from different experimental conditions. An in situ cleavage and mass spectrometry (MS)-label transfer after protein photo-cross-linking (IMAPP) strategy based on the photo-cross-linker DiZHSeC is also described. This strategy can be used for the identification of cross-linking sites to allow detailed characterization of PPI interfaces. The procedures for photo-cross-linker incorporation, photo-cross-linking of interaction partners and affinity purification of cross-linked complexes are similar for the two photo-cross-linkers. The final section of the protocol describes prey-bait separation (for CAPP) and MS-label transfer and identification (for IMAPP). After plasmid construction, the CAPP and IMAPP strategies can be completed within 6 and 7 d, respectively.

Structural characterization of the DNA-binding mechanism underlying the copper(II)-sensing MarR transcriptional regulator.

Multiple antibiotic resistance regulator (MarR) family proteins are widely conserved transcription factors that control bacterial resistance to antibiotics, environmental stresses, as well as the regulation of virulence determinants. Escherichia coli MarR, the prototype member of this family, has recently been shown to undergo copper(II)-catalyzed inter-dimer disulfide bond formation via a unique cysteine residue (Cys80) residing in its DNA-binding domain. However, despite extensive structural characterization of the MarR family proteins, the structural mechanism for DNA binding of this copper(II)-sensing MarR factor remains elusive. Here, we report the crystal structures of DNA-bound forms of MarR, which revealed a unique, concerted generation of two new helix-loop-helix motifs that facilitated MarR's DNA binding. Structural analysis and electrophoretic mobility shift assays (EMSA) show that the flexibility of Gly116 in the center of helix α5 and the extensive hydrogen-bonding interactions at the N-terminus of helix α1 together assist the reorientation of the wHTH domains and stabilize MarR's DNA-bound conformation.