The zebrafish (Danio rerio) has been widely used in the study of human disease and development, and about 70% of the protein-coding genes are conserved between the two species
. However, studies in ...zebrafish remain constrained by the sparse annotation of functional control elements in the zebrafish genome. Here we performed RNA sequencing, assay for transposase-accessible chromatin using sequencing (ATAC-seq), chromatin immunoprecipitation with sequencing, whole-genome bisulfite sequencing, and chromosome conformation capture (Hi-C) experiments in up to eleven adult and two embryonic tissues to generate a comprehensive map of transcriptomes, cis-regulatory elements, heterochromatin, methylomes and 3D genome organization in the zebrafish Tübingen reference strain. A comparison of zebrafish, human and mouse regulatory elements enabled the identification of both evolutionarily conserved and species-specific regulatory sequences and networks. We observed enrichment of evolutionary breakpoints at topologically associating domain boundaries, which were correlated with strong histone H3 lysine 4 trimethylation (H3K4me3) and CCCTC-binding factor (CTCF) signals. We performed single-cell ATAC-seq in zebrafish brain, which delineated 25 different clusters of cell types. By combining long-read DNA sequencing and Hi-C, we assembled the sex-determining chromosome 4 de novo. Overall, our work provides an additional epigenomic anchor for the functional annotation of vertebrate genomes and the study of evolutionarily conserved elements of 3D genome organization.
Fusion transcripts are used as biomarkers in companion diagnoses. Although more than 15,000 fusion RNAs have been identified from diverse cancer types, few common features have been reported. Here, ...we compared 16,410 fusion transcripts detected in cancer (from a published cohort of 9,966 tumor samples of 33 cancer types) with genome-wide RNA–DNA interactions mapped in two normal, noncancerous cell types using iMARGI, an enhanced version of the mapping of RNA–genome interactions (MARGI) assay. Among the top 10 most significant RNA–DNA interactions in normal cells, 5 colocalized with the gene pairs that formed fusion RNAs in cancer. Furthermore, throughout the genome, the frequency of a gene pair to exhibit RNA–DNA interactions is positively correlated with the probability of this gene pair to present documented fusion transcripts in cancer. To test whether RNA–DNA interactions in normal cells are predictive of fusion RNAs, we analyzed these in a validation cohort of 96 lung cancer samples using RNA sequencing (RNA-seq). Thirty-seven of 42 fusion transcripts in the validation cohort were found to exhibit RNA–DNA interactions in normal cells. Finally, by combining RNA-seq, single-molecule RNA FISH, and DNA FISH, we detected a cancer sample with EML4-ALK fusion RNA without forming the EML4-ALK fusion gene. Collectively, these data suggest an RNA-poise model, where spatial proximity of RNA and DNA could poise for the creation of fusion transcripts.
Nearly 70% of Uterine fibroid (UF) tumors are driven by recurrent MED12 hotspot mutations. Unfortunately, no cellular models could be generated because the mutant cells have lower fitness in 2D ...culture conditions. To address this, we employ CRISPR to precisely engineer MED12 Gly44 mutations in UF-relevant myometrial smooth muscle cells. The engineered mutant cells recapitulate several UF-like cellular, transcriptional, and metabolic alterations, including altered Tryptophan/kynurenine metabolism. The aberrant gene expression program in the mutant cells is, in part, driven by a substantial 3D genome compartmentalization switch. At the cellular level, the mutant cells gain enhanced proliferation rates in 3D spheres and form larger lesions in vivo with elevated production of collagen and extracellular matrix deposition. These findings indicate that the engineered cellular model faithfully models key features of UF tumors and provides a platform for the broader scientific community to characterize genomics of recurrent MED12 mutations.
Muscle-invasive bladder cancers are characterized by their distinct expression of luminal and basal genes, which could be used to predict key clinical features such as disease progression and overall ...survival. Transcriptionally, FOXA1, GATA3, and PPARG are shown to be essential for luminal subtype-specific gene regulation and subtype switching, while TP63, STAT3, and TFAP2 family members are critical for regulation of basal subtype-specific genes. Despite these advances, the underlying epigenetic mechanisms and 3D chromatin architecture responsible for subtype-specific regulation in bladder cancer remain unknown. RESULT: We determine the genome-wide transcriptome, enhancer landscape, and transcription factor binding profiles of FOXA1 and GATA3 in luminal and basal subtypes of bladder cancer. Furthermore, we report the first-ever mapping of genome-wide chromatin interactions by Hi-C in both bladder cancer cell lines and primary patient tumors. We show that subtype-specific transcription is accompanied by specific open chromatin and epigenomic marks, at least partially driven by distinct transcription factor binding at distal enhancers of luminal and basal bladder cancers. Finally, we identify a novel clinically relevant transcription factor, Neuronal PAS Domain Protein 2 (NPAS2), in luminal bladder cancers that regulates other subtype-specific genes and influences cancer cell proliferation and migration. CONCLUSION: In summary, our work identifies unique epigenomic signatures and 3D genome structures in luminal and basal urinary bladder cancers and suggests a novel link between the circadian transcription factor NPAS2 and a clinical bladder cancer subtype.
A recognition motif is vital in determining the specificity and sensitivity of the fluorescence polarization assay (FPA) for detecting chemical contaminants in food. Four candidates (Gyrase, GyrBA, ...TopIV, and QepA) were prepared for this study. The applicability of QepA was confirmed through DNA cleavage assay, inhibition effects, and mechanism investigations using molecular docking, compared to other counterparts. Finally, a novel FPA based on QepA and a CIP-FITC tracer for the detection of fluoroquinolones (FQs) in eggs was developed. The limits of detection (LODs) for eight fluoroquinolones ranged from 2.2 to 5.1 ng g–1, with enrofloxacin, danofloxacin, and difloxacin meeting the maximum residue limits (MRLs). The spiked recoveries ranged from 65.8 to 103.6% with coefficients of variation (CVs) of 5.4–12.8%. Therefore, a new recognition motif for FQs that did not belong to conventional antibodies was identified, and QepA-based FPA could be a potential tool for rapid, homogeneous, and sensitive monitoring of the residue of FQs in eggs.
This paper studies the dynamic Routing and Spectrum Assignment (RSA) problem in Bandwidth Flexible Optical networks. We propose a k-path Signaling-based RSA scheme and simulation results show that it ...performs much better than other RSA schemes in Bandwidth Flexible Optical networks.
Acute myeloid leukaemia (AML) represents a set of heterogeneous myeloid malignancies, and hallmarks include mutations in epigenetic modifiers, transcription factors and kinases
. The extent to which ...mutations in AML drive alterations in chromatin 3D structure and contribute to myeloid transformation is unclear. Here we use Hi-C and whole-genome sequencing to analyse 25 samples from patients with AML and 7 samples from healthy donors. Recurrent and subtype-specific alterations in A/B compartments, topologically associating domains and chromatin loops were identified. RNA sequencing, ATAC with sequencing and CUT&Tag for CTCF, H3K27ac and H3K27me3 in the same AML samples also revealed extensive and recurrent AML-specific promoter-enhancer and promoter-silencer loops. We validated the role of repressive loops on their target genes by CRISPR deletion and interference. Structural variation-induced enhancer-hijacking and silencer-hijacking events were further identified in AML samples. Hijacked enhancers play a part in AML cell growth, as demonstrated by CRISPR screening, whereas hijacked silencers have a downregulating role, as evidenced by CRISPR-interference-mediated de-repression. Finally, whole-genome bisulfite sequencing of 20 AML and normal samples revealed the delicate relationship between DNA methylation, CTCF binding and 3D genome structure. Treatment of AML cells with a DNA hypomethylating agent and triple knockdown of DNMT1, DNMT3A and DNMT3B enabled the manipulation of DNA methylation to revert 3D genome organization and gene expression. Overall, this study provides a resource for leukaemia studies and highlights the role of repressive loops and hijacked cis elements in human diseases.
Comprehensive profiling of the enhancer landscape and 3D genome structure in liposarcoma identifies extensive enhancer-oncogene coamplification and enhancer hijacking events, deepening the ...understanding of how oncogenes are regulated in cancer.
High manganese steels offer exceptional combinations of high strength and ductility, resulting in weight reduction when utilized in structural applications. Nevertheless, the conventional ...manufacturing routes of these steels is hindered by many production problems. Additive Manufacturing (AM) has emerged as a reliable solution to fabricate thin or complex shape compounds using these steel grades. Indeed, several studies have demonstrated the success to fabricate high manganese twinning-induced plasticity (TWIP) and transformation-induced plasticity (TRIP) steels by laser-powder bed fusion (L-PBF). However, a recent study on a high manganese triplex steel composition has revealed the occurrence of both hot cracking and micro segregation during rapid solidification in L-PBF, highlighting potential processability issues of this family of high alloy steels. In this study, the hot cracking susceptibility of different triplex steels is evaluated, focusing on the impact of the composition on the solidification paths and final microstructures. A Computer Coupling of Phase Diagrams and Thermochemistry (CALPHAD) approach is employed to predict alloy-dependent hot cracking susceptibility so as to establish guidelines for preventing hot cracking and provide insights into alloy design for AM. Microstructural observations are used to determine the accuracy of CALPHAD predictions in terms of elemental segregation, phase formation and hot cracking susceptibility. To this end, scanning transmission electron microscopy is used to evaluate elemental segregation at grain boundaries, while the local distribution of phases and their relative amount is measured by electron backscattered and X-ray diffraction, respectively. Hot cracking susceptibility is experimentally evaluated by measuring the length of cracks (when present) in the cross section of printed specimens.
•Fabrication of crack-free high-Mn triplex steels in laser-powder bed fusion.•Evaluation of fast solidification segregation and microstructures in gas-atomized triplex steel powders.•CALPHAD-based criteria proposed to prevent hot cracking.•Evaluation of hot cracking susceptibility criteria with respect to experimental observations.
Materials with both high thermal conductivity (TC) and low coefficient of thermal expansion (CTE) are critically needed in high-density, multi-functional, and high-power electronic devices. Cu-Gr ...composites are among the materials that potentially satisfy such requirements. However, processing Cu-Gr composites by powder metallurgical (PM) methods with sufficiently uniform Gr distribution has been challenging due to the inherent agglomeration of Gr particles. Various deagglomeration methods were previously investigated as a pre-consolidation step, but none had proven effective. In this work, an innovative PM route was investigated that combined controlled pre-ball milling of Cu-Gr composite powders as a means of deagglomeration of Gr powder with ultrasonic powder consolidation (UPC), a new rapid, low-temperature powder consolidation process. Various routines and parameters of ball-milling (BM), sonication, ultrasonic powder consolidation (UPC) were tested to optimize the UPC route. Cu-Gr composites consolidated under optimal conditions (500℃, 100 MPa, 5 s, air) are fully dense and exhibit a well-consolidated lamellar composite microstructure of 50 - 100 nm thick Cu lamellae with much finer Gr particles between the Cu lamellae. The Cu-Gr composites have a TC as high as 100 W/mK and exhibit almost negligible CTE up to about 400 °C.
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