Biology subjects

Zhang, C.

Publications and source records attributed to Zhang, C..

At least 613 records · Page 34Linked to original sources

bioRxiv2022

Cell type-specific attenuation of brassinosteroid signalling drives stomatal asymmetric cell division

In Arabidopsis thaliana, the negative brassinosteroid (BR) signalling regulator, BR INSENSITIVE2 (BIN2) promotes and restricts stomatal asymmetric cell division (ACD) depending on its subcellular localization, which is regulated by the stomatal lineage-specific scaffolding protein POLAR. BRs inactivate BIN2, but how they govern stomatal development remains unclear. Mapping the single-cell transcriptome of stomatal lineages with either exogenous BRs or the specific BIN2 inhibitor revealed that the two modes of BR activation triggered spatiotemporally distinct transcriptional responses. We established that when in a complex with POLAR and its closest homolog POLAR-LIKE1, BIN2 is insulated from BR-mediated inactivation, nevertheless, it remains sensitive to the inhibitor. Subsequently, BR signalling is attenuated in ACD precursors, whereas it remains active in epidermal cells that would undergo differentiation. Our study demonstrates how scaffold proteins contribute to cellular signal specificity of hormonal responses in plants.

plant biology↗

bioRxiv2022

A multi-omics atlas of the human retina at single-cell resolution

Cell classes in the human retina are highly heterogeneous with their abundance varying by several orders of magnitude. Although previous studies reported the profiles of the retinal cell types as the transcriptome level, there is no study regarding the open-chromatin profiles at a similar resolution. Here, we generated and integrated a multi-omics single-cell atlas of the adult human retina, including over 250K nuclei for single-nuclei RNA-seq and 137K nuclei for single-nuclei ATAC-seq. Through enrichment of rare cell types, this single cell multiome atlas is more comprehensive than previous human retina studies. Cross species comparison of the retina atlas among human, monkey, mice, and chicken revealed relatively conserved and non-conserved types. Interestingly, the overall cell heterogeneity in primate retina decreases compared to that of rodent and chicken retina. Furthermore, integrative analysis of the single cell multi-omics data identified 35k distal cis-element-gene pairs with most of these cis-elements being cell type specific. We also showed that the cis-element-gene relationship in different cell types within the same class could be highly heterogenous. Moreover, we constructed transcription factor (TF)-target regulons for over 200 TFs, partitioned the TFs into distinct co-active modules, and annotated each module based on their cell-type specificity. Taken together, we present the most comprehensive single-cell multi-omics atlas of the human retina as a valuable resource that enables systematic in-depth molecular characterization at individual cell type resolution.

genomics↗

bioRxiv2022

Deep-learning-assisted Sort-Seq enables high-throughput profiling of gene expression characteristics with high precision

As an essential physiological process, gene expression determines the function of each cell. However, owing to the complex nondeterministic and nonlinear nature of gene expression, the steady-state intracellular protein abundance of a clonal population forms a distribution. The characteristics of this distribution, including expression strength and noise, are closely related to cellular behavior. Therefore, quantitative description of these characteristics is an important goal in biology. This task, however, has so far relied on arrayed methods, which are time-consuming and labor-intensive. To address this issue, we propose a deep-learning-assisted Sort-Seq approach (dSort-Seq) in this work, enabling high-throughput profiling of expression properties with high precision. We demonstrated the validity of dSort-Seq for large-scale assaying of the dose-response relationships of biosensors. In addition, we comprehensively investigated the contribution of transcription and translation to noise production in E. coli, from which we discovered that the expression noise is strongly coupled with the mean expression level instead of translation strength, even in the case of weak transcription. We also discovered that the transcriptional interference caused by overlapping RpoD-binding sites contributes to noise production, which suggested the existence of a simple and feasible noise control strategy in E. coli. Overall, dSort-Seq is able to efficiently determine the strength-noise landscape, which has promising applications in studies related to gene expression.

genetics↗

bioRxiv2022

CRISPRi-FGP: web-based genome-scale CRISPRi sgRNA design and validation tool in prokaryotes

CRISPRi screening has become a powerful approach for functional genomic research. However, the off-target effects resulting from the mismatch tolerance between sgRNAs and their intended targets is a primary concern in CRISPRi applications. To address this issue, we introduce Guide Library Designer (GLiDe), a web-based tool specifically created for the genome-scale design of sgRNA libraries tailored for CRISPRi screening in prokaryotic organisms. GLiDe incorporates a robust quality control framework, rooted in prior experimental knowledge, ensuring the accurate identification of off-target hits. It boasts an extensive built-in database, encompassing 1,397 common prokaryotic species as a comprehensive design resource. In addition, GLiDe provides the capability to design sgRNAs for newly discovered organisms. We further demonstrated that GLiDe exhibits enhanced precision in identifying off-target binding sites for the CRISPRi system. Availability and ImplementationFreely available on the web at: https://www.thu-big.net/sgRNA_design/. AUTHOR SUMMARYCRISPRi screening is a powerful strategy for functional genomic study in prokaryotes. However, its applicability has been hindered by false positive results arising from off-target binding of sgRNAs. To tackle this challenge head-on, we present GLiDe, a web-based tool meticulously crafted for the genome-scale CRISPRi sgRNA library design in prokaryotes. GLiDe integrates a stringent quality control mechanism grounded in prior experimental insights. It has a built-in database and accepts uploaded reference files for new organisms. Moreover, we have demonstrated that our tool outperforms existing tools in CRISPRi sgRNA design for prokaryotes through cytometry assay. In summary, GLiDe emerges as a robust solution for sgRNA library design, poised to significantly advance functional genomic studies.

bioinformatics↗

bioRxiv2022

Thermodynamic Parameters Contributions of Single Internal Mismatches In RNA/DNA Hybrid Duplexes

Nucleic acid is a fundamental component of life. The thermodynamic is a basic physical quantity for nucleic acid and has been adopted in varieties of biotechnological applications. However, the parameters predicting thermodynamic parameters of RNA/DNA hybrid duplexes is incomplete and thus lead to the inability to proper predicting the properties. To address this problem, we measured for 99 RNA/DNA hybrid duplexes with 11 nucleotide length in a 1 M NaCl buffer using UV absorbance thermal denaturation. Thermodynamic parameters were obtained by fitting absorbance versus temperature profiles using SciPy non-linear curve fit. A dataset of 228 duplexes were constructed combining with literature experimental thermodynamic parameters for fitting a full set of all 113 possible Watson-Crick and single internal mismatch involving nearest-neighbor parameters using singular value decomposition. The new nearest-neighbor parameters predict thermodynamic parameters of RNA/DNA hybrid duplexes within an average error of 0.26 kcal/mol (3.55%) in free energy at 37{degrees}C ({Delta}G{degrees}37), 5.05 kcal/mol (6.70%) in enthalpy ({Delta}H{degrees}), 16.3 cal/mol{middle dot}K (7.50%) in entropy ({Delta}S{degrees}) and 1.14{degrees}C (2.91%) in melting temperature (Tm). The highest accuracy of all parameters reported. We observed a trend of increased stability for mismatch involving pairs of rG/dT >> rU/dG > rG/dA > rG/dG {approx} rA/dC {approx} rA/dG > rC/dA {approx} rC/dT > rU/dT {approx} rU/dC >> rA/dA >> rC/dC, which can be explained by different hydrogen bond conditions in mismatch pairs. We further compared the stability with RNA/RNA and DNA/DNA duplexes and found a backbone structure depended pattern in these three kinds of nucleic acid duplexes. This nearest-neighbor parameter set could enhance thermodynamical understanding of RNA/DNA combination and have potential usage in predicting on- and off-target RNA-DNA binding activity when applied to biotechnological applications such as CRISPR/Cas systems, RNAi and qPCR.

molecular biology↗

bioRxiv2022

Novel and unexpected genetic and microbial diversity for arsenic cycling in deep sea cold seep sediments

Cold seeps, where cold hydrocarbon-rich fluid escapes from the seafloor, showed strong enrichment of toxic metalloid arsenic (As). The toxicity and mobility of As can be greatly altered by microbial processes that play an important role in global As biogeochemical cycling. However, a global overview of genes and microbes involved in As transformation at seeps remains to be fully unveiled. Using 87 sediment metagenomes and 33 metatranscriptomes derived from 13 globally distributed cold seeps, we show that As detoxification genes (arsM, arsP, arsC1/arsC2, acr3) were prevalent at seeps and more phylogenetically diverse than previously expected. Asgardarchaeota and a variety of unidentified bacterial phyla (e.g. 4484-113, AABM5-125-24 and RBG-13-66-14) may also function as the key players in As transformation. The abundances of As-cycling genes and the compositions of As-associated microbiome shifted across different sediment depths or types of cold seep. The energy-conserving arsenate reduction or arsenite oxidation could impact biogeochemical cycling of carbon and nitrogen, via supporting carbon fixation, hydrocarbon degradation and nitrogen fixation. Overall, this study provides a comprehensive overview of As-cycling genes and microbes at As-enriched cold seeps, laying a solid foundation for further studies of As cycling in deep sea microbiome at the enzymatic and processual levels.

microbiology↗

bioRxiv2022

SLC3A2 N-glycosylation and alternate evolutionary trajectories for amino acid metabolism

SLC3A2 (4F2hc, CD98) is an adaptor to the SLC7A exchangers and has undergone extensive repositioning of N-glycosylation sites with vertebrate evolution, presumably in synchrony with the species-specific demands of metabolism. The SLC3A2*SLC7A5 heterodimer imports essential amino acids (AA) and thereby stimulates mTOR signaling, while SLC3A2*SLC7A11 imports cystine required for glutathione synthesis and mitigation of oxidative stress. Analysis of SLC3A2 N-glycans revealed stable site-specific profiles of Golgi remodeling, apart from the conserved N365 site where branching and poly-N-acetylglucosamine content were sensitive to the insertion of lost ancestral sites and to metabolism. N-glycans at N381 and N365 stabilized SLC3A2 in the galectin lattice and opposed endocytosis, while N365 which is nearest the membrane, also promoted down-regulation by galectin-driven clathrin-independent endocytosis (glycolipid-lectin GL-Lect). This is the first report of both positive and negative regulation by galectin binding to N-glycans that are strategically positioned in the same membrane glycoproteins. Proteomics analysis in SLC3A2 mutant HeLa cells with induced re-expression of SLC3A2 as bait revealed the canonical non-N-glycosylated interactors, SLC7A5 and SLC7A11 exchangers, but also AA transporters that were dependent on SLC3A2 N-glycosylation, and are themselves, N-glycosylated AA/Na+ symporters (SLC7A1, SLC38A1, SLC38A2, SLC1A4, SLC1A5). The results suggest that the N-glycans on SLC3A2 regulate clustering of SLC7A exchangers with AA/Na+ symporters, thereby promoting Gln/Glu export-driven import of essential AA and cystine, with the potential to adversely impact redox balance. The evolution of modern birds (Neoaves) led to improved control of bioenergetics with the loss of genes including SLC3A2, SLC7A-5, -7, -8, -10, BCAT2, KEAP1, as well as duplications of SLC7A9, SLC7A11 and the Golgi branching enzymes MGAT4B and MGAT4C known to enhance affinities for galectins. Analyzing the fate of these and other genes in the down-sized genomes of birds, spanning [~]10,000 species and >100 Myr of evolution, may reveal the mystery of their longevity with prolonged vitality. Key PointsO_LIGolgi N-glycan remodeling at each site on SLC3A2 differs with the microenvironment. C_LIO_LIThe galectin lattice and GL-Lect mediated endocytosis act as opposing forces on trafficking, controlled by N-glycans at the distal N381 and membrane proximal N365 sites, respectively. C_LIO_LIMutation at N381 or N365 decreased SLC3A2 association with SLC7A5, SLC7A11 and N-glycosylated AA/Na+ symporters as well as the capacity to mitigate stress. C_LIO_LIClustering of SLC3A2*SLC7A exchangers, with AA/Na+ symporter and ATPase Na+/K+ exchanger promotes growth but continuously consumes ATP in non-proliferating cells. C_LIO_LIBird evolution has improved bioenergetics with the deletion of SLC3A2 and associated transporters; - replaced by transporters of keto acids and a re-enforced galectin lattice. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=73 SRC="FIGDIR/small/516651v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1cb7958org.highwire.dtl.DTLVardef@18ba32dorg.highwire.dtl.DTLVardef@861269org.highwire.dtl.DTLVardef@22df0e_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

The GxcM-Fbp17/RacC-WASP signaling cascade regulates polarized cortex assembly in migrating cells

The actin-rich cortex plays a fundamental role in many cellular processes. Its architecture and molecular composition vary across cell types and physiological states. The full complement of actin assembly factors driving cortex formation and how their activities are spatiotemporally regulated remain to be fully elucidated. Using Dictyostelium as a model for polarized and rapidly migrating cells, we show that GxcM, a RhoGEF localized specifically in the rear of migrating cells, functions together with F-BAR protein Fbp17, a small GTPase RacC, and the actin nucleation-promoting factor WASP to coordinately promote Arp2/3 complex-mediated cortical actin assembly. Over-activation of this signaling cascade leads to excessive actin polymerization in the rear cortex, whereas its disruption causes defects in cortical integrity and function. Therefore, different from its well-defined role in the formation of the front protrusions, the Arp2/3 complex-based actin carries out a previously unappreciated function in building the rear cortical subcompartment in rapidly migrating cells.

cell biology↗

bioRxiv2022

Rate of neuronal turnover in the healthy adult murine myenteric ganglia varies with ganglia size

Maintenance of normal structure of the enteric nervous system (ENS), which regulates key gastrointestinal functions, requires robust homeostatic mechanisms, since by virtue of its location within the gut wall, the ENS is subject to constant mechanical, chemical, and biological stressors. Using transgenic and thymidine analogue-based experiments, we previously discovered that neuronal turnover - where continual neurogenesis offsets ongoing neuronal loss at steady state - represents one such mechanism. Although other studies confirmed that neuronal death continues into adulthood in the myenteric plexus of the enteric nervous system (ENS), the complicated nature of thymidine analogue presents challenges in substantiating the occurrence of adult neurogenesis. Therefore, its vital to employ alternative, well-recognized techniques to substantiate the existence of adult enteric neurogenesis in the healthy gut. Here, by using established methods of assessing nuclear DNA content and detecting known mitotic marker phosphor-histone H3 (pH3) in Hu+ adult ENS cells, we show that [~]10% of adult murine small intestinal myenteric Hu+ cells, and [~]20% of adult human small intestinal myenteric Hu+ cells show evidence of mitosis and hence are cycling neuroblasts. We observe that proportions of Hu+ cycling neuroblasts in the adult murine ENS neither vary with ganglia size, nor do they differ significantly between two intestinal regions - duodenum and ileum, or between sexes. Confocal microscopy provides further evidence of cytokinesis in Hu+ cells. The presence of a significant population of cycling neuroblasts in adult ENS provide further evidence of steady state neurogenesis in the adult ENS.

neuroscience↗

bioRxiv2022

Dendritic degeneration and altered synaptic innervation of a central auditory neuron during age-related hearing loss

Cellular morphology and synaptic configuration are key determinants of neuronal function and are often modified under pathological conditions. In the first nucleus of the central auditory system, the cochlear nucleus (CN), principal bushy neurons specialize in processing temporal information of sound critical for hearing. These neurons alter their physiological properties during aging that contribute to age-related hearing loss (ARHL). The structural basis of such changes remains unclear, especially age-related modifications in their dendritic morphology and the innervating auditory nerve (AN) synapses. Using young (2-5 months) and aged (28-33 months) CBA/CaJ mice of either sex, we filled individual bushy neurons with fluorescent dye in acute brain slices to characterize their dendritic morphology, followed by immunostaining against vesicular glutamate transporter 1 (VGluT1) and calretinin (CR) to identify innervating AN synapses. We found that dendritic morphology of aged bushy neurons had significantly reduced complexity, suggesting age-dependent dendritic degeneration, especially in neurons with predominantly non-CR-expressing synapses on the soma. These dendrites were innervated by AN bouton synapses, which were predominantly non-CR-expressing in young mice but had increased proportion of CR-expressing synapses in old mice. While somatic AN synapses degenerated substantially with age, as quantified by VGluT1-labeled puncta volume, no significant difference was observed in the total volume of dendritic synapses between young and old mice. Consequently, synaptic density on dendrites was significantly higher in old mice. The findings suggest that dendritic degeneration and altered synaptic innervation in bushy neurons during aging may underlie their changed physiological activity and contribute to the development of ARHL. HIGHLIGHTSO_LIDendrites of bushy neurons degenerate during aging with reduced complexity C_LIO_LIBushy dendrites receive AN input primarily from SGNs with medium/high threshold C_LIO_LIDendritic synapses show similar total volume but increased density during aging C_LIO_LIDendritic degeneration in bushy neurons and altered AN input may underlie ARHL C_LI

neuroscience↗

bioRxiv2022

Core Defense Hotspots within Pseudomonas aeruginosa are a consistent and rich source of anti-phage defense systems

Bacteria use a diverse arsenal of anti-phage immune systems, including CRISPR-Cas and restriction enzymes. Identifying the full defense repertoire of a given species is still challenging, however. Here, we developed a computational tool to broadly identify anti-phage systems, which was applied to >180,000 genomes available on NCBI, revealing Pseudomonas aeruginosa to possess the most diverse anti-phage arsenal of any species with >200 sequenced genomes. Using network analysis to identify the common neighbors of anti-phage systems, we surprisingly identified two highly conserved core defense hotspot loci (cDHS1 and cDHS2). Across more than 1,000 P. aeruginosa strains, cDHS1 is up to 224 kb (mean: 34 kb) with varied arrangements of at least 31 immune systems while cDHS2 has 24 distinct systems (mean: 15.4 kb). cDHS1/2 are present in most P. aeruginosa isolates, in contrast to highly variable mobile DHSs. Most cDHS genes are of unknown function potentially representing new anti-phage systems, which we validated by identifying a novel anti-phage system (Shango) commonly encoded in cDHS1. Identification of core gene markers that flank immune islands could be a simple approach for immune system discovery and may represent popular landing spots for diverse MGEs carrying anti-phage systems.

genomics↗

bioRxiv2022

Differential bending stiffness of the bacterial flagellar hook under counterclockwise and clockwise rotations

The bacterial hook, as a universal joint coupling rotation of the flagellar motor and the filament, is an important component of the flagellum that propels the bacteria to swim. The mechanical properties of the hook are essential for the flagellum to achieve normal functions. In multi-flagellated bacteria such as Escherichia coli, the hook must be compliant so that it can bend for the filaments to form a coherently rotating bundle to generate the thrust when the motor rotates counterclockwise (CCW), yet it also must be rigid so that the bundle can disrupt for the bacteria to tumble to change swimming direction when the motor rotates clockwise (CW). Here, by combining an elastic rod model with high-resolution bead assay to accurately measure the bending stiffness of the hook under CCW or CW rotation in vivo, we elucidate how the hook accomplishes this dual functionality: the hook stiffens under CW rotation, with bending stiffness under CW rotation twice as large as that under CCW rotation. This enables a robust run-and-tumble swimming motility for multi-flagellated bacteria.

biophysics↗

bioRxiv2022

STAMarker: Determining spatial domain-specific variable genes with saliency maps in deep learning

Spatial transcriptomics characterizes gene expression profiles while retaining the information of the spatial context, providing an unprecedented opportunity to understand cellular systems. One of the essential tasks in such data analysis is to determine spatially variable genes (SVGs), which demonstrate spatial expression patterns. Existing methods only consider genes individually and fail to model the inter-dependence of genes. To this end, we present an analytic tool STAMarker for robustly determining spatial domain-specific SVGs with saliency maps in deep learning. STAMarker is a three-stage ensemble framework consisting of graphattention autoencoders, multilayer perceptron (MLP) classifiers, and saliency map computation by the backpropagated gradient. We illustrate the effectiveness of STAMarker and compare it with three competing methods on four spatial transcriptomic data generated by various platforms. STAMarker considers all genes at once and is more robust when the dataset is very sparse. STAMarker could identify spatial domain-specific SVGs for characterizing spatial domains and enable in-depth analysis of the region of interest in the tissue section.

bioinformatics↗

bioRxiv2022

Syncytin-mediated open-ended membrane tubular connections facilitate the intercellular transfer of cargos including Cas9 protein

Much attention has been focused on the possibility that cytoplasmic proteins and RNA may be conveyed between cells in extracellular vesicles (EVs) and tunneling nanotube (TNT) structures. Here, we set up two quantitative delivery reporters to study cargo transfer between cells. We found that EVs are internalized by reporter cells but do not efficiently deliver functional Cas9 protein to the nucleus. In contrast, donor and acceptor cells co-cultured to permit cell contact resulted in a highly effective transfer. Among our tested donor and acceptor cell pairs, HEK293T and MDA-MB-231 recorded optimal intercellular transfer. Depolymerization of F-actin greatly decreased Cas9 transfer whereas inhibitors of endocytosis or knock-down of genes implicated in this process had little effect on transfer. Imaging results suggest that intercellular transfer of cargos occurred through open-ended membrane tubular connections. In contrast, cultures consisting only of HEK293T cells form close-ended tubular connections ineffective in cargo transfer. Depletion of human endogenous fusogens, syncytins, especially syncytin-2 in MDA-MB-231 cells, significantly reduced Cas9 transfer. Full-length mouse syncytin, but not truncated mutants, rescued the effect of depletion of human syncytins on Cas9 transfer. Mouse syncytin overexpression in HEK293T cells partially facilitated Cas9 transfer among HEK293T cells. These findings suggest that syncytin may serve as the fusogen responsible for the formation of an open-ended connection between cells.

cell biology↗

bioRxiv2022

Flux estimation analysis systematically characterizes the metabolic shifts of the central metabolism pathway in human cancer.

Glucose and glutamine are major carbon and energy sources that promote the fast proliferation of cancer cells. Metabolic shifs observed on cell line or mouse models may not reflect the general metabolic shifts in real human cancer tissue. In this study, we conducted a computational characterization of the flux distribution and variations of the central energy metabolism and key branches in a pan-cancer analysis, including glycolytic pathway, production of lactate, TCA cycle, nucleic acids synthesis, glutaminolysis, glutaminate, glutamine and glutathione metabolism, amino acids synthesis, in 11 cancer subtypes and 9 matched adjacent normal tissue types, by using TCGA tissue transcriptomics data. Our analysis confirms the increased influx in glucose uptake and glycolysis and decreased upper part of TCA cycle, i.e., Warbug effect in the analyzed cancer types. However, consistently increased lactate production and second half of TCA cycle were only seen in certain cancer types. More interestingly, we did not see cancer tissues have highly shifted glutaminolysis compared to their adjacent normal controls. A systems biology model of metabolic shifts in through cancer and tissue types is further developed and analyzed. We observed that (1) normal tissues have distinct metabolic phenotypes, (2) cancer types have drastically different metabolic shifts compared to their adjacent normal controls, and (3) the different shifts happened to tissue specific metabolic phenotypes result in a converged metabolic phenotype through cancer types and cancer progression. This study strongly suggests the possibility to have a unified framework for studies of cancer-inducing stressors, adaptive metabolic reprogramming, and cancerous behaviors.

bioinformatics↗

bioRxiv2022

Uncovering transcriptional regulatory network during regeneration for boosting wheat transformation

Genetic transformation is important for gene functional study and crop breeding. Though it is available in many plant species, the transformation efficiency in wheat is generally low, which greatly restricts the genetic manipulation in wheat. Here, we use multi-omic analysis strategy to uncover core transcriptional regulatory network (TRN) driving wheat shoot regeneration and identify key factors that boost the transformation efficiency. RNA-seq, ATAC-seq and CUT&Tag were used to profile the transcriptome and chromatin dynamic during regeneration process from immature embryo of wheat variety Fielder. Sequential expression of gene clusters that mediating cell fate transition during regeneration is induced by auxin signaling, in coordination with changes of chromatin accessibility, H3K27me3 and H3K4me3 status. The TRN driving wheat shoot regeneration was built-up and 446 key transcriptional factors (TFs) occupied the core of network were identified, including functionally tested regeneration factors in other species. We further compared the regeneration process between wheat and Arabidopsis and found that DNA binding with one finger (DOF) TFs show distinct patterns in two species. Furthermore, we found that TaDOF5.6 (TraesCS6A02G274000) and TaDOF3.4 (TraesCS2B02G592600) can significantly improve the transformation efficiency of different wheat varieties. Thus, our data uncovers the molecular regulatory insights for wheat shoot regeneration process and provides potential novel targets for improving transformation efficiency in wheat.

developmental biology↗

bioRxiv2022

R-loop editing by DNA cytosine deaminase APOBEC3B determines the activity of estrogen receptor enhancers

Estrogen receptor (ER) activation results in the formation of DNA double strand breaks (DSB), which promote genomic instability and tumour heterogeneity in ER-positive breast cancers. The single-stranded DNA (ssDNA) cytosine deaminase APOBEC3B (A3B) regulates ER activity by inducing DSB at ER enhancers. To delineate how A3B recognises its substrates and unveil the underlying mechanism leading to the formation of ER-induced DSB, we sampled A3B-mediated deamination sites using whole genome sequencing in a human breast cancer cell model lacking base excision repair function. Our genome-wide analysis revealed that C>U conversions carried out by A3B in R-loop structures are processed into DSB in the vicinity of ER promoters or enhancers. A mechanism which required both the processing of A3B-editing sites and R-loops by distinct DNA damage repair mechanisms. In addition, using BioID-enabled mass-spectroscopy proteomics, we identified TDRD3 as a key A3B-binding partner directing the activity of A3B to ER-induced R-loops. This study suggests a function for A3B in sustaining tumour evolution as an adaptive response at the transcriptional and epigenetic level and supports A3B as a promising target to control ER activity in cancer.

cancer biology↗

bioRxiv2022

Identification of RNA-based cell-type markers for the stem cell manufacture system with a statistical scoring function

Cell-type biomarkers are useful in stem cell manufacture to monitor cell purification, cell quantity, and quality. However, the study on cell-type markers, specifically for stem cell manufacture, is limited. The emerging questions are which RNA transcripts can serve as biomarkers during stem cell culture, and what method can efficiently and accurately discover these biomarkers. We developed a scoring function system to identify RNA biomarkers with RNA-seq data. We applied the method to two data sets, one for extracellular RNAs (ex-RNAs) and the other for intracellular microRNAs (miRNAs). The data set have RNA-seq data of ex-RNAs from cell culture media for six different types of cells, including human embryonic stem cells. To get the RNA-seq data from intracellular miRNAs, we cultured three types of cells: human embryonic stem cells (H9), neural stem cells (NSC), hESC-derived endothelial cells (EC) and conducted small RNA-seq to their intracellular miRNAs. Using these data, we identified a set of ex-RNAs/smRNAs as candidates of biomarkers for different types of cells for cell manufacture. We also used deep-learning based prediction methods and simulated data to validate these discovered biomarkers.

bioinformatics↗