Pervasive Chromatin-RNA Binding Protein Interactions Enable RNA-Based Regulation of Transcription.

Increasing evidence suggests that transcriptional control and chromatin activities at large involve regulatory RNAs, which likely enlist specific RNA-binding proteins (RBPs). Although multiple RBPs have been implicated in transcription control, it has remained unclear how extensively RBPs directly act on chromatin. We embarked on a large-scale RBP ChIP-seq analysis, revealing widespread RBP presence in active chromatin regions in the human genome. Like transcription factors (TFs), RBPs also show strong preference for hotspots in the genome, particularly gene promoters, where their association is frequently linked to transcriptional output. Unsupervised clustering reveals extensive co-association between TFs and RBPs, as exemplified by YY1, a known RNA-dependent TF, and RBM25, an RBP involved in splicing regulation. Remarkably, RBM25 depletion attenuates all YY1-dependent activities, including chromatin binding, DNA looping, and transcription. We propose that various RBPs may enhance network interaction through harnessing regulatory RNAs to control transcription.

Comprehensive Characterization of Cancer Driver Genes and Mutations.

Identifying molecular cancer drivers is critical for precision oncology. Multiple advanced algorithms to identify drivers now exist, but systematic attempts to combine and optimize them on large datasets are few. We report a PanCancer and PanSoftware analysis spanning 9,423 tumor exomes (comprising all 33 of The Cancer Genome Atlas projects) and using 26 computational tools to catalog driver genes and mutations. We identify 299 driver genes with implications regarding their anatomical sites and cancer/cell types. Sequence- and structure-based analyses identified >3,400 putative missense driver mutations supported by multiple lines of evidence. Experimental validation confirmed 60%-85% of predicted mutations as likely drivers. We found that >300 MSI tumors are associated with high PD-1/PD-L1, and 57% of tumors analyzed harbor putative clinically actionable events. Our study represents the most comprehensive discovery of cancer genes and mutations to date and will serve as a blueprint for future biological and clinical endeavors.

Non-coding RNA Generated following Lariat Debranching Mediates Targeting of AID to DNA.

Transcription through immunoglobulin switch (S) regions is essential for class switch recombination (CSR), but no molecular function of the transcripts has been described. Likewise, recruitment of activation-induced cytidine deaminase (AID) to S regions is critical for CSR; however, the underlying mechanism has not been fully elucidated. Here, we demonstrate that intronic switch RNA acts in trans to target AID to S region DNA. AID binds directly to switch RNA through G-quadruplexes formed by the RNA molecules. Disruption of this interaction by mutation of a key residue in the putative RNA-binding domain of AID impairs recruitment of AID to S region DNA, thereby abolishing CSR. Additionally, inhibition of RNA lariat processing leads to loss of AID localization to S regions and compromises CSR; both defects can be rescued by exogenous expression of switch transcripts in a sequence-specific manner. These studies uncover an RNA-mediated mechanism of targeting AID to DNA.

BRD4 isoforms have distinct roles in tumour progression and metastasis in rhabdomyosarcoma.

BRD4, a bromodomain and extraterminal (BET) protein, is deregulated in multiple cancers and has emerged as a promising drug target. However, the function of the two main BRD4 isoforms (BRD4-L and BRD4-S) has not been analysed in parallel in most cancers. This complicates determining therapeutic efficacy of pan-BET inhibitors. In this study, using functional and transcriptomic analysis, we show that BRD-L and BRD4-S isoforms play distinct roles in fusion negative embryonal rhabdomyosarcoma. BRD4-L has an oncogenic role and inhibits myogenic differentiation, at least in part, by activating myostatin expression. Depletion of BRD4-L in vivo impairs tumour progression but does not impact metastasis. On the other hand, depletion of BRD4-S has no significant impact on tumour growth, but strikingly promotes metastasis in vivo. Interestingly, BRD4-S loss results in the enrichment of BRD4-L and RNA Polymerase II at integrin gene promoters resulting in their activation. In fusion positive alveolar rhabdomyosarcoma, BRD4-L is unrestricted in its oncogenic role, with no evident involvement of BRD4-S. Our work unveils isoform-specific functions of BRD4 in rhabdomyosarcoma.

A large-scale binding and functional map of human RNA-binding proteins.

Many proteins regulate the expression of genes by binding to specific regions encoded in the genome1. Here we introduce a new data set of RNA elements in the human genome that are recognized by RNA-binding proteins (RBPs), generated as part of the Encyclopedia of DNA Elements (ENCODE) project phase III. This class of regulatory elements functions only when transcribed into RNA, as they serve as the binding sites for RBPs that control post-transcriptional processes such as splicing, cleavage and polyadenylation, and the editing, localization, stability and translation of mRNAs. We describe the mapping and characterization of RNA elements recognized by a large collection of human RBPs in K562 and HepG2 cells. Integrative analyses using five assays identify RBP binding sites on RNA and chromatin in vivo, the in vitro binding preferences of RBPs, the function of RBP binding sites and the subcellular localization of RBPs, producing 1,223 replicated data sets for 356 RBPs. We describe the spectrum of RBP binding throughout the transcriptome and the connections between these interactions and various aspects of RNA biology, including RNA stability, splicing regulation and RNA localization. These data expand the catalogue of functional elements encoded in the human genome by the addition of a large set of elements that function at the RNA level by interacting with RBPs.

The Augmented R-Loop Is a Unifying Mechanism for Myelodysplastic Syndromes Induced by High-Risk Splicing Factor Mutations.

Mutations in several general pre-mRNA splicing factors have been linked to myelodysplastic syndromes (MDSs) and solid tumors. These mutations have generally been assumed to cause disease by the resultant splicing defects, but different mutations appear to induce distinct splicing defects, raising the possibility that an alternative common mechanism is involved. Here we report a chain of events triggered by multiple splicing factor mutations, especially high-risk alleles in SRSF2 and U2AF1, including elevated R-loops, replication stress, and activation of the ataxia telangiectasia and Rad3-related protein (ATR)-Chk1 pathway. We further demonstrate that enhanced R-loops, opposite to the expectation from gained RNA binding with mutant SRSF2, result from impaired transcription pause release because the mutant protein loses its ability to extract the RNA polymerase II (Pol II) C-terminal domain (CTD) kinase-the positive transcription elongation factor complex (P-TEFb)-from the 7SK complex. Enhanced R-loops are linked to compromised proliferation of bone-marrow-derived blood progenitors, which can be partially rescued by RNase H overexpression, suggesting a direct contribution of augmented R-loops to the MDS phenotype.

Novel mechanisms of diversity generation in Acinetobacter baumannii resistance islands driven by Tn7-like elements.

Mobile genetic elements play an important role in the acquisition of antibiotic and biocide resistance, especially through the formation of resistance islands in bacterial chromosomes. We analyzed the contribution of Tn7-like transposons to island formation and diversification in the nosocomial pathogen Acinetobacter baumannii and identified four separate families that recognize different integration sites. One integration site is within the comM gene and coincides with the previously described Tn6022 elements suggested to account for the AbaR resistance island. We established Tn6022 in a heterologous E. coli host and confirmed basic features of transposition into the comM attachment site and the use of a novel transposition protein. By analyzing population features within Tn6022 elements we identified two potential novel transposon-encoded diversification mechanisms with this dynamic genetic island. The activities of these diversification features were confirmed in E. coli. One was a novel natural gain-of-activity allele that could function to broaden transposition targeting. The second was a transposon-encoded hybrid dif-like site that parasitizes the host dimer chromosome resolution system to function with its own tyrosine recombinase. This work establishes a highly active Tn7-like transposon that harnesses novel features allowing the spread and diversification of genetic islands in pathogenic bacteria.

PHF2 regulates genome topology and DNA replication in neural stem cells via cohesin.

Cohesin plays a crucial role in the organization of topologically-associated domains (TADs), which influence gene expression and DNA replication timing. Whether epigenetic regulators may affect TADs via cohesin to mediate DNA replication remains elusive. Here, we discover that the histone demethylase PHF2 associates with RAD21, a core subunit of cohesin, to regulate DNA replication in mouse neural stem cells (NSC). PHF2 loss impairs DNA replication due to the activation of dormant replication origins in NSC. Notably, the PHF2/RAD21 co-bound genomic regions are characterized by CTCF enrichment and epigenomic features that resemble efficient, active replication origins, and can act as boundaries to separate adjacent domains. Accordingly, PHF2 loss weakens TADs and chromatin loops at the co-bound loci due to reduced RAD21 occupancy. The observed topological and DNA replication defects in PHF2 KO NSC support a cohesin-dependent mechanism. Furthermore, we demonstrate that the PHF2/RAD21 complex exerts little effect on gene regulation, and that PHF2's histone-demethylase activity is dispensable for normal DNA replication and proliferation of NSC. We propose that PHF2 may serve as a topological accessory to cohesin for cohesin localization to TADs and chromatin loops, where cohesin represses dormant replication origins directly or indirectly, to sustain DNA replication in NSC.

The polyA tail facilitates splicing of last introns with weak 3' splice sites via PABPN1.

The polyA tail of mRNAs is important for many aspects of RNA metabolism. However, whether and how it regulates pre-mRNA splicing is still unknown. Here, we report that the polyA tail acts as a splicing enhancer for the last intron via the nuclear polyA binding protein PABPN1 in HeLa cells. PABPN1-depletion induces the retention of a group of introns with a weaker 3' splice site, and they show a strong 3'-end bias and mainly locate in nuclear speckles. The polyA tail is essential for PABPN1-enhanced last intron splicing and functions in a length-dependent manner. Tethering PABPN1 to nonpolyadenylated transcripts also promotes splicing, suggesting a direct role for PABPN1 in splicing regulation. Using TurboID-MS, we construct the PABPN1 interactome, including many spliceosomal and RNA-binding proteins. Specifically, PABPN1 can recruit RBM26&27 to promote splicing by interacting with the coiled-coil and RRM domain of RBM27. PABPN1-regulated terminal intron splicing is conserved in mice. Together, our study establishes a novel mode of post-transcriptional splicing regulation via the polyA tail and PABPN1.

The Dawn of a New Era: Targeting the "Undruggables" with Antibody-Based Therapeutics.

The high selectivity and affinity of antibodies toward their antigens have made them a highly valuable tool in disease therapy, diagnosis, and basic research. A plethora of chemical and genetic approaches have been devised to make antibodies accessible to more "undruggable" targets and equipped with new functions of illustrating or regulating biological processes more precisely. In this Review, in addition to introducing how naked antibodies and various antibody conjugates (such as antibody-drug conjugates, antibody-oligonucleotide conjugates, antibody-enzyme conjugates, etc.) work in therapeutic applications, special attention has been paid to how chemistry tools have helped to optimize the therapeutic outcome (i.e., with enhanced efficacy and reduced side effects) or facilitate the multifunctionalization of antibodies, with a focus on emerging fields such as targeted protein degradation, real-time live-cell imaging, catalytic labeling or decaging with spatiotemporal control as well as the engagement of antibodies inside cells. With advances in modern chemistry and biotechnology, well-designed antibodies and their derivatives via size miniaturization or multifunctionalization together with efficient delivery systems have emerged, which have gradually improved our understanding of important biological processes and paved the way to pursue novel targets for potential treatments of various diseases.

R-ChIP Using Inactive RNase H Reveals Dynamic Coupling of R-loops with Transcriptional Pausing at Gene Promoters.

R-loop, a three-stranded RNA/DNA structure, has been linked to induced genome instability and regulated gene expression. To enable precision analysis of R-loops in vivo, we develop an RNase-H-based approach; this reveals predominant R-loop formation near gene promoters with strong G/C skew and propensity to form G-quadruplex in non-template DNA, corroborating with all biochemically established properties of R-loops. Transcription perturbation experiments further indicate that R-loop induction correlates to transcriptional pausing. Interestingly, we note that most mapped R-loops are each linked to a nearby free RNA end; by using a ribozyme to co-transcriptionally cleave nascent RNA, we demonstrate that such a free RNA end coupled with a G/C-skewed sequence is necessary and sufficient to induce R-loop. These findings provide a topological solution for RNA invasion into duplex DNA and suggest an order for R-loop initiation and elongation in an opposite direction to that previously proposed.

RNase H1 facilitates recombinase recruitment by degrading DNA-RNA hybrids during meiosis.

DNA-RNA hybrids play various roles in many physiological progresses, but how this chromatin structure is dynamically regulated during spermatogenesis remains largely unknown. Here, we show that germ cell-specific knockout of Rnaseh1, a specialized enzyme that degrades the RNA within DNA-RNA hybrids, impairs spermatogenesis and causes male infertility. Notably, Rnaseh1 knockout results in incomplete DNA repair and meiotic prophase I arrest. These defects arise from the altered RAD51 and DMC1 recruitment in zygotene spermatocytes. Furthermore, single-molecule experiments show that RNase H1 promotes recombinase recruitment to DNA by degrading RNA within DNA-RNA hybrids and allows nucleoprotein filaments formation. Overall, we uncover a function of RNase H1 in meiotic recombination, during which it processes DNA-RNA hybrids and facilitates recombinase recruitment.

The Saururus chinensis genome provides insights into the evolution of pollination strategies and herbaceousness in magnoliids.

Saururus chinensis, an herbaceous magnoliid without perianth, represents a clade of early-diverging angiosperms that have gone through woodiness-herbaceousness transition and pollination obstacles: the characteristic white leaves underneath inflorescence during flowering time are considered a substitute for perianth to attract insect pollinators. Here, using the newly sequenced S. chinensis genome, we revisited the phylogenetic position of magnoliids within mesangiosperms, and recovered a sister relationship for magnoliids and Chloranthales. By considering differentially expressed genes, we identified candidate genes that are involved in the morphogenesis of the white leaves in S. chinensis. Among those genes, we verified - in a transgenic experiment with Arabidopsis - that increasing the expression of the "pseudo-etiolation in light" gene (ScPEL) can inhibit the biosynthesis of chlorophyll. ScPEL is thus likely responsible for the switches between green and white leaves, suggesting that changes in gene expression may underlie the evolution of pollination strategies. Despite being an herbaceous plant, S. chinensis still has vascular cambium and maintains the potential for secondary growth as a woody plant, because the necessary machinery, i.e., the entire gene set involved in lignin biosynthesis, is well preserved. However, similar expression levels of two key genes (CCR and CAD) between the stem and other tissues in the lignin biosynthesis pathway are possibly associated with the herbaceous nature of S. chinensis. In conclusion, the S. chinensis genome provides valuable insights into the adaptive evolution of pollination in Saururaceae and reveals a possible mechanism for the evolution of herbaceousness in magnoliids.

The MYB family and their response to abiotic stress in ginger (Zingiber officinale Roscoe).

BACKGROUND: Zingiber officinale Roscoe, colloquially known as ginger, is a crop of significant medicinal and culinary value that frequently encounters adversity stemming from inhospitable environmental conditions. The MYB transcription factors have garnered recognition for their pivotal role in orchestrating a multitude of plant biological pathways. Nevertheless, the enumeration and characterization of the MYBs within Z. officinale Roscoe remains unknown. This study embarks on a genome-wide scrutiny of the MYB gene lineage in ginger, with the aim of cataloging all ZoMYB genes implicated in the biosynthesis of gingerols and curcuminoids, and elucidating their potential regulatory mechanisms in counteracting abiotic stress, thereby influencing ginger growth and development. RESULTS: In this study, we identified an MYB gene family comprising 231 members in ginger genome. This ensemble comprises 74 singular-repeat MYBs (1R-MYB), 156 double-repeat MYBs (R2R3-MYB), and a solitary triple-repeat MYB (R1R2R3-MYB). Moreover, a comprehensive analysis encompassing the sequence features, conserved protein motifs, phylogenetic relationships, chromosome location, and gene duplication events of the ZoMYBs was conducted. We classified ZoMYBs into 37 groups, congruent with the number of conserved domains and gene structure analysis. Additionally, the expression profiles of ZoMYBs during development and under various stresses, including ABA, cold, drought, heat, and salt, were investigated in ginger utilizing both RNA-seq data and qRT-PCR analysis. CONCLUSION: This work provides a comprehensive understanding of the MYB family in ginger and lays the foundation for the future investigation of the potential functions of ZoMYB genes in ginger growth, development and abiotic stress tolerance of ginger.

Electrochemical Monitoring of Sphingosine-1-phosphate-Induced ATP Release Using a Microsensor Based on an Entropy-Driven Bipedal DNA Walker.

Neuropathic pain is a chronic and severe syndrome for which effective therapy is insufficient and the release of ATP from microglia induced by sphingosine-1-phosphate (S1P) plays a vital role in neuropathic pain. Therefore, there is an urgent demand to develop highly sensitive and selective ATP biosensors for quantitative monitoring of low-concentration ATP in the complex nervous system, which helps in understanding the mechanism involved in neuropathic pain. Herein, we developed an electrochemical microsensor based on an entropy-driven bipedal DNA walker. First, the microsensor specifically recognized ATP via ATP aptamers, initiating the entropy-driven bipedal DNA walker. Subsequently, the bipedal DNA walker autonomously traversed the microelectrode interface, introducing methylene blue to the electrode surface and achieving cascade signal amplification. This microsensor showed excellent selectivity, stability, and a low limit of detection at 1.13 nM. The S1P-induced ATP release from BV2 cells was successfully monitored, and it was observed that dicumarol could inhibit this release, suggesting dicumarol as a potential treatment for neuropathic pain. The microsensor's small size exhibited significant potential for monitoring ATP level changes in neuropathic pain in vivo, which provides a new strategy for in situ and quantitative monitoring of nonelectroactive biomolecules associated with neurological diseases.

Amorphization of MXenes: Boosting Electrocatalytic Hydrogen Evolution.

The emergence of amorphous 2D materials has opened up new avenue for materials science and nanotechnology in the recent years. Their unique disordered structure, excellent large-area uniformity, and low fabrication cost make them important for various industrial applications. However, there have no reports on the amorphous MXene materials. In this work, the amorphous Ti2C-MXene (a-Ti2C-MXene) model is built by ab initio molecular dynamics (AIMD) approach. This model is a unique amorphous model, which is totally different from continuous random network (CRN) model for silicate glass and amorphous model for amorphous 2D BN and graphene. The structure analysis shows that the a-Ti2C-MXene composited by [Ti5C] and [Ti6C] cluster, which are surrounded by the region of mixed cluster [TixC], [Ti-Ti] cluster, and [C-C] cluster. There is a high chemical activity for hydrogen evolution reaction (HER) in a-Ti2C-MXene with |ΔGH| 0.001 eV, implying that they serve as the potential boosting HER performance. The work provides insights that can pave the way for future research on novel MXene materials, leading to their increased applications in various fields.

Scalable Dual In Situ Synthesis of Polyester Nanocomposites for High-Energy Storage.

Incorporating ultralow loading of nanoparticles into polymers has realized increases in dielectric constant and breakdown strength for excellent energy storage. However, there are still a series of tough issues to be dealt with, such as organic solvent uses, which face enormous challenges in scalable preparation. Here, a new strategy of dual in situ synthesis is proposed, namely polymerization of polyethylene terephthalate (PET) synchronizes with growth of calcium borate nanoparticles, making polyester nanocomposites from monomers directly. Importantly, this route is free of organic solvents and surface modification of nanoparticles, which is readily accessible to scalable synthesis of polyester nanocomposites. Meanwhile, uniform dispersion of as ultralow as 0.1 wt% nanoparticles and intense bonding at interfaces have been observed. Furthermore, the PET-based nanocomposite displays obvious increases in both dielectric constant and breakdown strength as compared to the neat PET. Its maximum discharged energy density reaches 15 J cm-3 at 690 MV m-1 and power density attains 218 MW cm-3 under 150 Ω resistance at 300 MV m-1, which is far superior to the current dielectric polymers that can be produced at large scales. This work presents a scalable, safe, low-cost, and environment-friendly route toward polymer nanocomposites with superior capacitive performance.

Effect of high-fat diet on the fatty acid profiles of brain in offspring mice exposed to maternal gestational diabetes mellitus.

OBJECTIVE: Fatty acids play a critical role in the proper functioning of the brain. This study investigated the effects of a high-fat (HF) diet on brain fatty acid profiles of offspring exposed to maternal gestational diabetes mellitus (GDM). METHODS: Insulin receptor antagonist (S961) and HF diet were used to establish the GDM animal model. Brain fatty acid profiles of the offspring mice were measured by gas chromatography at weaning and adulthood. Protein expressions of the fatty acid transport pathway Wnt3/ß-catenin and the target protein major facilitator superfamily domain-containing 2a (MFSD2a) were measured in the offspring brain by Western blot. RESULTS: Maternal GDM increased the body weight of male offspring (P < 0.05). In weaning offspring, factorial analysis showed that maternal GDM increased the monounsaturated fatty acid (MUFA) percentage of the weaning offspring's brain (P < 0.05). Maternal GDM decreased offspring brain arachidonic acid (AA), but HF diet increased brain linoleic acid (LA) (P < 0.05). Maternal GDM and HF diet reduced offspring brain docosahexaenoic acid (DHA), and the male offspring had higher DHA than the female offspring (P < 0.05). In adult offspring, factorial analysis showed that HF diet increased brain MUFA in offspring, and male offspring had higher brain MUFA than female offspring (P < 0.05). The HF diet increased brain LA in the offspring. Male offspring had higher level of AA than female offspring (P < 0.05). HF diet reduced DHA in the brains of female offspring. The brain protein expression of ß-catenin and MFSD2a in both weaning and adult female offspring was lower in the HF + GDM group than in the CON group (P < 0.05). CONCLUSIONS: Maternal GDM increased the susceptibility of male offspring to HF diet-induced obesity. HF diet-induced adverse brain fatty acid profiles in both male and female offspring exposed to GDM.

Mixed active metabolites of the SNP-6 series of novel compounds mitigate metabolic dysfunction-associated steatohepatitis and fibrosis: promising results from pre-clinical and clinical trials.

BACKGROUND: Metabolic dysfunction-associated steatohepatitis (MASH) is a growing global health concern with no effective pharmacological treatments. SNP-630, a newly developed synthetic molecule with multiple mechanisms of action, and a mixture of two of its active metabolites (SNP-630-MS) inhibit CYP2E1 expression to prevent reactive oxygen species generation, thereby reducing the accumulation of hepatic triglycerides and lowering chemokine levels. This study investigated the SNP-630's potential to alleviate the liver injury in MASH and its efficacy in both a mouse model and patients with MASH to identify a drug candidate that targets multiple pathways implicated in MASH. METHODS: SNP-630 and SNP-630-MS were separately administered to the MASH mouse model. The tolerability, safety, and efficacy of SNP-630-MS were also evaluated in 35 patients with MASH. The primary endpoint of the study was assessment of the changes in serum alanine aminotransferase (ALT) levels from baseline to week 12, while the secondary endpoints included the evaluation of liver inflammation, steatosis, and fibrosis parameters and markers. RESULTS: SNP-630 treatment in mice improved inflammation, liver steatosis, and fibrosis compared with that in the MASH control group. Both SNP-630 and SNP-630-MS treatments markedly reduced ALT levels, hepatic triglyceride content, and the expression of inflammatory cytokines monocyte chemoattractant protein 1 and fibrotic collagen (i.e., Col1a1, Col3a1, and Timp1) in mice. In the clinical trial, patients treated with SNP-630-MS exhibited significant improvement in ALT levels at week 12 compared with baseline levels, with no reports of severe adverse events. This improvement in ALT levels surpassed that achieved with most other MASH candidates. SNP-630-MS demonstrated potential antifibrotic effects, as evidenced by a significant decrease in the levels of fibrogenesis-related biomarkers such as CCL4, CCL5, and caspase 3. Subgroup analysis using FibroScan measurements further indicated the efficacy of SNP-630-MS in ameliorating liver fibrosis. CONCLUSIONS: SNP-630 and SNP-630-MS demonstrated favorable results in mice. SNP-630-MS showed excellent tolerability in mice and patients with MASH. Efficacy analyses indicated that SNP-630-MS improved liver steatosis and injury in patients with MASH, suggesting that SNP-630 and 630-MS are promising therapeutic options for MASH. Larger scale clinical trials remain warranted to assess the efficacy and safety of SNP-630 in MASH. TRIAL REGISTRATION: ClinicalTrials.gov NCT03868566. Registered 06 March 2019-Retrospectively registered, https://clinicaltrials.gov/study/NCT03868566.