CTCF-Mediated Human 3D Genome Architecture Reveals Chromatin Topology for Transcription.

Spatial genome organization and its effect on transcription remains a fundamental question. We applied an advanced chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) strategy to comprehensively map higher-order chromosome folding and specific chromatin interactions mediated by CCCTC-binding factor (CTCF) and RNA polymerase II (RNAPII) with haplotype specificity and nucleotide resolution in different human cell lineages. We find that CTCF/cohesin-mediated interaction anchors serve as structural foci for spatial organization of constitutive genes concordant with CTCF-motif orientation, whereas RNAPII interacts within these structures by selectively drawing cell-type-specific genes toward CTCF foci for coordinated transcription. Furthermore, we show that haplotype variants and allelic interactions have differential effects on chromosome configuration, influencing gene expression, and may provide mechanistic insights into functions associated with disease susceptibility. 3D genome simulation suggests a model of chromatin folding around chromosomal axes, where CTCF is involved in defining the interface between condensed and open compartments for structural regulation. Our 3D genome strategy thus provides unique insights in the topological mechanism of human variations and diseases.

Interactome maps of mouse gene regulatory domains reveal basic principles of transcriptional regulation.

A key finding of the ENCODE project is that the enhancer landscape of mammalian cells undergoes marked alterations during ontogeny. However, the nature and extent of these changes are unclear. As part of the NIH Mouse Regulome Project, we here combined DNaseI hypersensitivity, ChIP-seq, and ChIA-PET technologies to map the promoter-enhancer interactomes of pluripotent ES cells and differentiated B lymphocytes. We confirm that enhancer usage varies widely across tissues. Unexpectedly, we find that this feature extends to broadly transcribed genes, including Myc and Pim1 cell-cycle regulators, which associate with an entirely different set of enhancers in ES and B cells. By means of high-resolution CpG methylomes, genome editing, and digital footprinting, we show that these enhancers recruit lineage-determining factors. Furthermore, we demonstrate that the turning on and off of enhancers during development correlates with promoter activity. We propose that organisms rely on a dynamic enhancer landscape to control basic cellular functions in a tissue-specific manner.

Extensive promoter-centered chromatin interactions provide a topological basis for transcription regulation.

Higher-order chromosomal organization for transcription regulation is poorly understood in eukaryotes. Using genome-wide Chromatin Interaction Analysis with Paired-End-Tag sequencing (ChIA-PET), we mapped long-range chromatin interactions associated with RNA polymerase II in human cells and uncovered widespread promoter-centered intragenic, extragenic, and intergenic interactions. These interactions further aggregated into higher-order clusters, wherein proximal and distal genes were engaged through promoter-promoter interactions. Most genes with promoter-promoter interactions were active and transcribed cooperatively, and some interacting promoters could influence each other implying combinatorial complexity of transcriptional controls. Comparative analyses of different cell lines showed that cell-specific chromatin interactions could provide structural frameworks for cell-specific transcription, and suggested significant enrichment of enhancer-promoter interactions for cell-specific functions. Furthermore, genetically-identified disease-associated noncoding elements were found to be spatially engaged with corresponding genes through long-range interactions. Overall, our study provides insights into transcription regulation by three-dimensional chromatin interactions for both housekeeping and cell-specific genes in human cells.

MethMarkerDB: a comprehensive cancer DNA methylation biomarker database.

DNA methylation plays a crucial role in tumorigenesis and tumor progression, sparking substantial interest in the clinical applications of cancer DNA methylation biomarkers. Cancer-related whole-genome bisulfite sequencing (WGBS) data offers a promising approach to precisely identify these biomarkers with differentially methylated regions (DMRs). However, currently there is no dedicated resource for cancer DNA methylation biomarkers with WGBS data. Here, we developed a comprehensive cancer DNA methylation biomarker database (MethMarkerDB, https://methmarkerdb.hzau.edu.cn/), which integrated 658 WGBS datasets, incorporating 724 curated DNA methylation biomarker genes from 1425 PubMed published articles. Based on WGBS data, we documented 5.4 million DMRs from 13 common types of cancer as candidate DNA methylation biomarkers. We provided search and annotation functions for these DMRs with different resources, such as enhancers and SNPs, and developed diagnostic and prognostic models for further biomarker evaluation. With the database, we not only identified known DNA methylation biomarkers, but also identified 781 hypermethylated and 5245 hypomethylated pan-cancer DMRs, corresponding to 693 and 2172 genes, respectively. These novel potential pan-cancer DNA methylation biomarkers hold significant clinical translational value. We hope that MethMarkerDB will help identify novel cancer DNA methylation biomarkers and propel the clinical application of these biomarkers.

Regulation by distinct MYB transcription factors defines the roles of OsCYP86A9 in anther development and root suberin deposition.

Cytochrome P450 proteins (CYPs) play critical roles in plant development and adaptation to fluctuating environments. Previous reports have shown that CYP86A proteins are involved in the biosynthesis of suberin and cutin in Arabidopsis. However, the functions of these proteins in rice remain obscure. In this study, a rice mutant with incomplete male sterility was identified. Cytological analyses revealed that this mutant was defective in anther development. Cloning of the mutant gene indicated that the responsible mutation was on OsCYP86A9. OsMYB80 is a core transcription factor in the regulation of rice anther development. The expression of OsCYP86A9 was abolished in the anther of osmyb80 mutant. In vivo and in vitro experiments showed that OsMYB80 binds to the MYB-binding motifs in OsCYP86A9 promoter region and regulates its expression. Furthermore, the oscyp86a9 mutant exhibited an impaired suberin deposition in the root, and was more susceptible to drought stress. Interestingly, genetic and biochemical analyses revealed that OsCYP86A9 expression was regulated in the root by certain MYB transcription factors other than OsMYB80. Moreover, mutations in the MYB genes that regulate OsCYP86A9 expression in the root did not impair the male fertility of the plant. Taken together, these findings revealed the critical roles of OsCYP86A9 in plant development and proposed that OsCYP86A9 functions in anther development and root suberin formation via two distinct tissue-specific regulatory pathways.

Dynamic Phytomeric Growth Contributes to Local Adaptation in Barley.

Vascular plants have segmented body axes with iterative nodes and internodes. Appropriate node initiation and internode elongation are fundamental to plant fitness and crop yield; however, how these events are spatiotemporally coordinated remains elusive. We show that in barley (Hordeum vulgare L.), selections during domestication have extended the apical meristematic phase to promote node initiation, but constrained subsequent internode elongation. In both vegetative and reproductive phases, internode elongation displays a dynamic proximal-distal gradient, and among subpopulations of domesticated barleys worldwide, node initiation and proximal internode elongation are associated with latitudinal and longitudinal gradients, respectively. Genetic and functional analyses suggest that, in addition to their converging roles in node initiation, flowering-time genes have been repurposed to specify the timing and duration of internode elongation. Our study provides an integrated view of barley node initiation and internode elongation and suggests that plant architecture should be recognized as a collection of dynamic phytomeric units in the context of crop adaptive evolution.

ScSmOP: a universal computational pipeline for single-cell single-molecule multiomics data analysis.

Single-cell multiomics techniques have been widely applied to detect the key signature of cells. These methods have achieved a single-molecule resolution and can even reveal spatial localization. These emerging methods provide insights elucidating the features of genomic, epigenomic and transcriptomic heterogeneity in individual cells. However, they have given rise to new computational challenges in data processing. Here, we describe Single-cell Single-molecule multiple Omics Pipeline (ScSmOP), a universal pipeline for barcode-indexed single-cell single-molecule multiomics data analysis. Essentially, the C language is utilized in ScSmOP to set up spaced-seed hash table-based algorithms for barcode identification according to ligation-based barcoding data and synthesis-based barcoding data, followed by data mapping and deconvolution. We demonstrate high reproducibility of data processing between ScSmOP and published pipelines in comprehensive analyses of single-cell omics data (scRNA-seq, scATAC-seq, scARC-seq), single-molecule chromatin interaction data (ChIA-Drop, SPRITE, RD-SPRITE), single-cell single-molecule chromatin interaction data (scSPRITE) and spatial transcriptomic data from various cell types and species. Additionally, ScSmOP shows more rapid performance and is a versatile, efficient, easy-to-use and robust pipeline for single-cell single-molecule multiomics data analysis.

ChromLoops: a comprehensive database for specific protein-mediated chromatin loops in diverse organisms.

Chromatin loops (or chromatin interactions) are important elements of chromatin structures. Disruption of chromatin loops is associated with many diseases, such as cancer and polydactyly. A few methods, including ChIA-PET, HiChIP and PLAC-Seq, have been proposed to detect high-resolution, specific protein-mediated chromatin loops. With rapid progress in 3D genomic research, ChIA-PET, HiChIP and PLAC-Seq datasets continue to accumulate, and effective collection and processing for these datasets are urgently needed. Here, we developed a comprehensive, multispecies and specific protein-mediated chromatin loop database (ChromLoops, https://3dgenomics.hzau.edu.cn/chromloops), which integrated 1030 ChIA-PET, HiChIP and PLAC-Seq datasets from 13 species, and documented 1 491 416 813 high-quality chromatin loops. We annotated genes and regions overlapping with chromatin loop anchors with rich functional annotations, such as regulatory elements (enhancers, super-enhancers and silencers), variations (common SNPs, somatic SNPs and eQTLs), and transcription factor binding sites. Moreover, we identified genes with high-frequency chromatin interactions in the collected species. In particular, we identified genes with high-frequency interactions in cancer samples. We hope that ChromLoops will provide a new platform for studying chromatin interaction regulation in relation to biological processes and disease.

Integrated 3D genome, epigenome and transcriptome analyses reveal transcriptional coordination of circadian rhythm in rice.

Photoperiods integrate with the circadian clock to coordinate gene expression rhythms and thus ensure plant fitness to the environment. Genome-wide characterization and comparison of rhythmic genes under different light conditions revealed delayed phase under constant darkness (DD) and reduced amplitude under constant light (LL) in rice. Interestingly, ChIP-seq and RNA-seq profiling of rhythmic genes exhibit synchronous circadian oscillation in H3K9ac modifications at their loci and long non-coding RNAs (lncRNAs) expression at proximal loci. To investigate how gene expression rhythm is regulated in rice, we profiled the open chromatin regions and transcription factor (TF) footprints by time-series ATAC-seq. Although open chromatin regions did not show circadian change, a significant number of TFs were identified to rhythmically associate with chromatin and drive gene expression in a time-dependent manner. Further transcriptional regulatory networks mapping uncovered significant correlation between core clock genes and transcription factors involved in light/temperature signaling. In situ Hi-C of ZT8-specific expressed genes displayed highly connected chromatin association at the same time, whereas this ZT8 chromatin connection network dissociates at ZT20, suggesting the circadian control of gene expression by dynamic spatial chromatin conformation. These findings together implicate the existence of a synchronization mechanism between circadian H3K9ac modifications, chromatin association of TF and gene expression, and provides insights into circadian dynamics of spatial chromatin conformation that associate with gene expression rhythms.

Co-targeting SKP2 and KDM5B inhibits prostate cancer progression by abrogating AKT signaling with induction of senescence and apoptosis.

BACKGROUND: Prostate cancer (PCa) is the second-leading cause of cancer mortalities in the United States and is the most commonly diagnosed malignancy in men. While androgen deprivation therapy (ADT) is the first-line treatment option to initial responses, most PCa patients invariably develop castration-resistant PCa (CRPC). Therefore, novel and effective treatment strategies are needed. The goal of this study was to evaluate the anticancer effects of the combination of two small molecule inhibitors, SZL-P1-41 (SKP2 inhibitor) and PBIT (KDM5B inhibitor), on PCa suppression and to delineate the underlying molecular mechanisms. METHODS: Human CRPC cell lines, C4-2B and PC3 cells, were treated with small molecular inhibitors alone or in combination, to assess effects on cell proliferation, migration, senescence, and apoptosis. RESULTS: SKP2 and KDM5B showed an inverse regulation at the translational level in PCa cells. Cells deficient in SKP2 showed an increase in KDM5B protein level, compared to that in cells expressing SKP2. By contrast, cells deficient in KDM5B showed an increase in SKP2 protein level, compared to that in cells with KDM5B intact. The stability of SKP2 protein was prolonged in KDM5B depleted cells as measured by cycloheximide chase assay. Cells deficient in KDM5B were more vulnerable to SKP2 inhibition, showing a twofold greater reduction in proliferation compared to cells with KDM5B intact (p < 0.05). More importantly, combined inhibition of KDM5B and SKP2 significantly decreased proliferation and migration of PCa cells as compared to untreated controls (p < 0.005). Mechanistically, combined inhibition of KDM5B and SKP2 in PCa cells abrogated AKT activation, resulting in an induction of both cellular senescence and apoptosis, which was measured via Western blot analysis and senescence-associated ß-galactosidase (SA-ß-Gal) staining. CONCLUSIONS: Combined inhibition of KDM5B and SKP2 was more effective at inhibiting proliferation and migration of CRPC cells, and this regimen would be an ideal therapeutic approach of controlling CRPC malignancy.

Coordinated demethylation of H3K9 and H3K27 is required for rapid inflammatory responses of endothelial cells.

Histone H3 lysine-9 di-methylation (H3K9me2) and lysine-27 tri-methylation (H3K27me3) are linked to repression of gene expression, but the functions of repressive histone methylation dynamics during inflammatory responses remain enigmatic. Here, we report that lysine demethylases 7A (KDM7A) and 6A (UTX) play crucial roles in tumor necrosis factor (TNF)-α signaling in endothelial cells (ECs), where they are regulated by a novel TNF-α-responsive microRNA, miR-3679-5p. TNF-α rapidly induces co-occupancy of KDM7A and UTX at nuclear factor kappa-B (NF-κB)-associated elements in human ECs. KDM7A and UTX demethylate H3K9me2 and H3K27me3, respectively, and are both required for activation of NF-κB-dependent inflammatory genes. Chromosome conformation capture-based methods furthermore uncover increased interactions between TNF-α-induced super enhancers at NF-κB-relevant loci, coinciding with KDM7A and UTX recruitments. Simultaneous pharmacological inhibition of KDM7A and UTX significantly reduces leukocyte adhesion in mice, establishing the biological and potential translational relevance of this mechanism. Collectively, these findings suggest that rapid erasure of repressive histone marks by KDM7A and UTX is essential for NF-κB-dependent regulation of genes that control inflammatory responses of ECs.

MCIBox: a toolkit for single-molecule multi-way chromatin interaction visualization and micro-domains identification.

The emerging ligation-free three-dimensional (3D) genome mapping technologies can identify multiplex chromatin interactions with single-molecule precision. These technologies not only offer new insight into high-dimensional chromatin organization and gene regulation, but also introduce new challenges in data visualization and analysis. To overcome these challenges, we developed MCIBox, a toolkit for multi-way chromatin interaction (MCI) analysis, including a visualization tool and a platform for identifying micro-domains with clustered single-molecule chromatin complexes. MCIBox is based on various clustering algorithms integrated with dimensionality reduction methods that can display multiplex chromatin interactions at single-molecule level, allowing users to explore chromatin extrusion patterns and super-enhancers regulation modes in transcription, and to identify single-molecule chromatin complexes that are clustered into micro-domains. Furthermore, MCIBox incorporates a two-dimensional kernel density estimation algorithm to identify micro-domains boundaries automatically. These micro-domains were stratified with distinctive signatures of transcription activity and contained different cell-cycle-associated genes. Taken together, MCIBox represents an invaluable tool for the study of multiple chromatin interactions and inaugurates a previously unappreciated view of 3D genome structure.

Recent advances of nanopore technique in single cell analysis.

Single cells and their dynamic behavior are closely related to biological research. Monitoring their dynamic behavior is of great significance for disease prevention. How to achieve rapid and non-destructive monitoring of single cells is a major issue that needs to be solved urgently. As an emerging technology, nanopores have been proven to enable non-destructive and label-free detection of single cells. The structural properties of nanopores enable a high degree of sensitivity and accuracy during analysis. In this article, we summarize and classify the different types of solid-state nanopores that can be used for single-cell detection and illustrate their specific applications depending on the size of the analyte. In addition, their research progress in material transport and microenvironment monitoring is also highlighted. Finally, a brief summary of existing research challenges and future trends in nanopore single-cell analysis is tentatively provided.

MNAzyme catalyzed signal amplification-mediated lateral flow biosensor for portable and sensitive detection of mycotoxin in food samples.

Here, an enzyme-free lateral flow aptasensor was designed by target-induced strand-displacement effect and followed by the activation of multi-component nucleic acid enzyme (MNAzyme)-mediated cleavage to enable rapid and portable ochratoxin A (OTA) detection. The substrate was prepared as an oligonucleotide strand modified with magnetic beads (MB) and human chorionic gonadotropin (hCG). The interaction of OTA with the aptamer induces the release of blocking DNA, which hybridized with three separated subunits of DNA, forming a sequence-specific MNAzyme catalytic core. This core subsequently initiated an enzyme-free MNAzyme cleavage reaction in the presence of the Mg2+ cofactor, cleaving a special substrate and releasing both the incomplete MNAzyme catalytic core and hCG-DNA probe. The incomplete MNAzyme catalytic core was then recognized by substrates once again, triggering a cascade recycling cleavage and resulting in the generation of a larger number of hCG-DNA probes. After magnetic enrichment, the free hCG-DNA probes flow through the pregnancy test strip (PTS) to the T line, generating a colorimetric readout that unequivocally confirms the presence of the target OTA. This work leverages the efficient enzyme-free cleavage amplification of MNAzyme and the PTS-based portable detection device, presenting a biosensing strategy with significant potential for sensitive and portable OTA detection. This method exhibited remarkable sensitivity and selectivity for OTA detection, boasting a detection limit of 5 nM. The present study successfully demonstrated the practical application of this method on real samples, offering a viable alternative for rapid and portable detection of mycotoxins.

Review on magnetic adsorbents for removal of elemental mercury from coal combustion flue gas.

Under the influence of human activities, atmospheric mercury (Hg) concentrations have increased by 450% compared with natural levels. In the context of the Minamata Convention on Mercury, which came into effect in August 2017, it is imperative to strengthen Hg emission controls. Existing Air Pollution Control Devices (APCDs) combined with collaborative control technology can effectively remove Hg2+ and Hgp; however, Hg0 removal is substandard. Compared with the catalytic oxidation method, Hg0 removal through adsorbent injection carries the risk of secondary release and is uneconomical. Magnetic adsorbents exhibit excellent recycling and Hg0 recovery performance and have recently attracted the attention of researchers. This review summarizes the existing magnetic materials for Hg0 adsorption and discusses the removal performances and mechanisms of iron, carbon, mineral-based, and magnetosphere materials. The effects of temperature and different flue gas components, including O2, NO, SO2, H2O, and HCl, on the adsorption performance of Hg0 are also summarized. Finally, different regeneration methods are discussed in detail. Although the research and development of magnetic adsorbents has progressed, significant challenges remain regarding their application. This review provides theoretical guidance for the improvement of existing and development of new magnetic adsorbents.

ASMdb: a comprehensive database for allele-specific DNA methylation in diverse organisms.

DNA methylation is known to be the most stable epigenetic modification and has been extensively studied in relation to cell differentiation, development, X chromosome inactivation and disease. Allele-specific DNA methylation (ASM) is a well-established mechanism for genomic imprinting and regulates imprinted gene expression. Previous studies have confirmed that certain special regions with ASM are susceptible and closely related to human carcinogenesis and plant development. In addition, recent studies have proven ASM to be an effective tumour marker. However, research on the functions of ASM in diseases and development is still extremely scarce. Here, we collected 4400 BS-Seq datasets and 1598 corresponding RNA-Seq datasets from 47 species, including human and mouse, to establish a comprehensive ASM database. We obtained the data on DNA methylation level, ASM and allele-specific expressed genes (ASEGs) and further analysed the ASM/ASEG distribution patterns of these species. In-depth ASM distribution analysis and differential methylation analysis conducted in nine cancer types showed results consistent with the reported changes in ASM in key tumour genes and revealed several potential ASM tumour-related genes. Finally, integrating these results, we constructed the first well-resourced and comprehensive ASM database for 47 species (ASMdb, www.dna-asmdb.com).

Pyrococcus furiosus argonaute based Alicyclobacillus acidoterrestrsis detection in fruit juice.

Alicyclobacillus acidoterrestris is the major threat to fruit juice for its off-odor producing characteristic. In this study, Pyrococcus furiosus Argonaute (PfAgo), a novel endonuclease with precise DNA cleavage activity, was used for A. acidoterrestrisdetection, termed as PAD. The partially amplified 16 S rRNA gene of A. acidoterrestris can be cleaved by PfAgo activated by a short 5'-phosphorylated single strand DNA, producing a new guide DNA (gDNA). Then, PfAgo was activated by the new gDNA to cut a molecular beacon (MB) with fluorophore-quencher reporter, resulting in the recovery of fluorescence. The fluorescent intensity is positively related with the concentration of A. acidoterrestris. The PAD assay showed excellent specificity and sensitivity as low as 101 CFU/mL, which can be a powerful tool for on-site detection of A. acidoterrestris in fruit juice industry in the future, reducing the economic loss.

A rate threshold mechanism regulates MAPK stress signaling and survival.

Cells are exposed to changes in extracellular stimulus concentration that vary as a function of rate. However, how cells integrate information conveyed from stimulation rate along with concentration remains poorly understood. Here, we examined how varying the rate of stress application alters budding yeast mitogen-activated protein kinase (MAPK) signaling and cell behavior at the single-cell level. We show that signaling depends on a rate threshold that operates in conjunction with stimulus concentration to determine the timing of MAPK signaling during rate-varying stimulus treatments. We also discovered that the stimulation rate threshold and stimulation rate-dependent cell survival are sensitive to changes in the expression levels of the Ptp2 phosphatase, but not of another phosphatase that similarly regulates osmostress signaling during switch-like treatments. Our results demonstrate that stimulation rate is a regulated determinant of cell behavior and provide a paradigm to guide the dissection of major stimulation rate dependent mechanisms in other systems.

Timing outweighs magnitude of rainfall in shaping population dynamics of a small mammal species in steppe grassland.

Climate change-induced shifts in species phenology differ widely across trophic levels, which may lead to consumer-resource mismatches with cascading population and ecosystem consequences. Here, we examined the effects of different rainfall patterns (i.e., timing and amount) on the phenological asynchrony of population of a generalist herbivore and their food sources in semiarid steppe grassland in Inner Mongolia. We conducted a 10-y (2010 to 2019) rainfall manipulation experiment in 12 0.48-ha field enclosures and found that moderate rainfall increases during the early rather than late growing season advanced the timing of peak reproduction and drove marked increases in population size through increasing the biomass of preferred plant species. By contrast, greatly increased rainfall produced no further increases in vole population growth due to the potential negative effect of the flooding of burrows. The increases in vole population size were more coupled with increased reproduction of overwintered voles and increased body mass of young-of-year than with better survival. Our results provide experimental evidence for the fitness consequences of phenological mismatches at the population level and highlight the importance of rainfall timing on the population dynamics of small herbivores in the steppe grassland environment.

Investigations on the defect structures for Mn2+ in CdSe nanocrystals and bulk materials and the criterion of occupation for Mn2+ in CdX (X = S, Se, Te) nanocrystals.

The spin Hamiltonian parameters and defect structures are theoretically studied for the substitutional Mn2+ at the core of CdSe nanocrystals and in the bulk materials from the perturbation calculations of spin Hamiltonian parameters for trigonal tetrahedral 3d5 clusters. Both the crystal-field and charge transfer contributions are taken into account in the calculations from the cluster approach. The impurity-ligand bond angles are found to be about 1.84° larger and 0.10° smaller in the CdSe:Mn2+ nanocrystals and bulk materials, respectively, than those (≈109.37°) of the host Cd2+ sites. The quantitative criterion of occupation (at the core or surface) for Mn2+ in CdX (X = S, Se, Te) nanocrystals is presented for the first time based on the inequations of hyperfine structure constants (HSCs). This criterion is well supported by the experimental HSCs data of Mn2+ in CdX nanocrystals. The previous assignments of signals SI as Mn2+ at the core of CdS nanocrystals are renewed as Mn2+ at the surface based on the above criterion. The present studies would be helpful to achieve convenient determination of occupation for Mn2+ impurities in CdX semiconductor nanocrystals by means of spectral (e.g., HSCs) analysis.