Biology subjects

Tang, X.

Publications and source records attributed to Tang, X..

At least 145 records · Page 8Linked to original sources

bioRxiv2021

Correlational networking guides the discovery of cryptic proteases for lanthipeptide maturation

Bacterial natural product biosynthetic genes, canonically clustered, have been increasingly found to rely on cryptic enzymes encoded elsewhere in the genome for completion of biosynthesis. The study and application of lanthipeptides are frequently hindered by cryptic proteases required for final maturation. Here, we establish a global correlation network bridging the gap between lanthipeptide precursors and cryptic proteases. Applying our analysis to 161,954 bacterial genomes, we establish 5,209 correlations between precursors and cryptic proteases, with 91 prioritized. We use network predictions and co-expression analysis to reveal a previously missing protease for the maturation of class I lanthipeptide paenilan. We further discover widely distributed bacterial M16B metallopeptidases of previously unclear biological function as a new family of lanthipeptide proteases. We show the involvement of a pair of bifunctional M16B proteases in the production of novel class III lanthipeptides with high substrate specificity. Together, these results demonstrate the strength of our correlational networking approach to the discovery of cryptic lanthipeptide proteases and potentially other missing enzymes for natural products biosynthesis. Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

biochemistry↗

bioRxiv2021

All-optical mechanobiology interrogation of YAP in human cancer and normal cells using a novel multi-functional system

Long-term multi-functional imaging and analysis of live cells require streamlined functional coordination of various hardware and software platforms. However, manual control of various equipment produced by different manufacturers is labor-intensive and time-consuming, potentially decreasing the accuracy, reproducibility, and quality of acquired data. Therefore, an all-in-one and user-programmable system that enables automatic, multi-functional, and long-term image acquisition and is compatible with most fluorescent microscopy platforms can desirably benefit the scientific community. In this paper, we introduce the full operating protocols of utilizing a novel integrated software system that consists of (1) a home-built software program, titled "Automatic Multi-functional Integration Program (AMFIP)", which enables automatic multi-channel imaging acquisition, and (2) a suite of quantitative imaging analysis and cell traction computation packages. We applied this integrated system to reveal the previously unknown relationship between the spatial-temporal distribution of mechano-sensitive Yes-associated protein (YAP) and the cell mechanics, including cell spreading and traction, in human normal cells (B2B) and lung cancer cells (PC9). Leveraging our systems capability of multi-channel control and readout, we found: (1) B2B normal cells and PC9 cancer cells show distinct YAP expression, traction, and cell dynamics relationship during cell spreading and migration processes; and (2) PC9 cancer cells apply noticeable peri-nuclear forces on substrates. In summary, this paper presents a detailed stepwise protocol on how to utilize an integrated user-programmable system that enables automatic multi-functional imaging and analysis to elucidate YAP mechanosensitivity. These tools open the possibility for detailed explorations of multifaceted signaling dynamics in the context of cell physiology and pathology. SUMMARYThis paper presents a detailed stepwise protocol on how to utilize an integrated user-programmable system that enables all-optical electro-mechanobiology interrogation to elucidate YAP mechanosensitivity.

biophysics↗

bioRxiv2021

Comparative phylogenomic analysis reveals evolutionary genomic changes and novel toxin families in endophytic Liberibacter pathogens

Liberibacter pathogens are the causative agents of several severe crop diseases worldwide, including citrus Huanglongbing and potato Zebra Chip. These bacteria are endophytic and non-culturable, which makes experimental approaches challenging and highlights the need for bioinformatic analysis in advancing our understanding about Liberibacter pathogenesis. Here, we performed an in-depth comparative phylogenomic analysis of the Liberibacter pathogens and their free-living, nonpathogenic, ancestral species, aiming to identify the major genomic changes and determinants associated with their evolutionary transitions in living habitats and pathogenicity. We found that prophage loci represent the most variable regions among Liberibacter genomes. Using gene neighborhood analysis and phylogenetic classification, we systematically recovered, annotated, and classified all prophage loci into four types, including one previously unrecognized group. We showed that these prophages originated through independent gene transfers at different evolutionary stages of Liberibacter and only the SC-type prophage was associated with the emergence of the pathogens. Using ortholog clustering, we vigorously identified two additional sets of genomic genes, which were either lost or gained in the ancestor of the pathogens. Consistent with the habitat change, the lost genes were enriched for biosynthesis of cellular building blocks. Importantly, among the gained genes, we uncovered several previously unrecognized toxins, including a novel class of polymorphic toxins, a YdjM phospholipase toxin, and a secreted EEP protein. Our results substantially extend the knowledge on the evolutionary events and potential determinants leading to the emergence of endophytic, pathogenic Liberibacter species and will facilitate the design of functional experiments and the development of new detection and blockage methods of these pathogens. ImportanceLiberibacter pathogens are associated with several severe crop diseases, including citrus Huanglongbing, the most destructive disease to the citrus industry. Currently, no effective cure or treatments are available, and no resistant citrus variety has been found. The fact that these obligate endophytic pathogens are not culturable has made it extremely challenging to experimentally uncover from the whole genome the genes/proteins important to Liberibacter pathogenesis. Further, earlier bioinformatics studies failed to identify the key genomic determinants, such as toxins and effector proteins, that underlie the pathogenicity of the bacteria. In this study, an in-depth comparative genomic analysis of Liberibacter pathogens together with their ancestral non-pathogenic species identified the prophage loci and several novel toxins that are evolutionarily associated with the emergence of the pathogens. These results shed new lights on the disease mechanism of Liberibacter pathogens and will facilitate the development of new detection and blockage methods targeting the toxins.

microbiology↗

bioRxiv2021

Maternal Piwi Regulates Primordial Germ Cell Development to Ensure the Fertility of Female Progeny in Drosophila

In many animals, germline development is initiated by proteins and RNAs that are expressed maternally. PIWI proteins and their associated small noncoding PIWI-interacting RNAs (piRNAs), which guide PIWI to target RNAs by base-pairing, are among the maternal components deposited into the germline of the early embryo in Drosophila. Piwi has been extensively studied in the adult ovary and testis, where it is required for transposon suppression, germline stem cell self-renewal, and fertility. Consequently, loss of Piwi in the adult ovary using piwi-null alleles or knockdown from early oogenesis results in complete sterility, limiting investigation into possible embryonic functions of maternal Piwi. In this study, we show that the maternal Piwi protein persists in the embryonic germline through gonad coalescence, suggesting that maternal Piwi can regulate germline development beyond early embryogenesis. Using a maternal knockdown strategy, we find that maternal Piwi is required for the fertility and normal gonad morphology of female, but not male, progeny. Following maternal piwi knockdown, transposons were mildly derepressed in the early embryo but were fully repressed in the ovaries of adult progeny. Furthermore, the maternal piRNA pool was diminished, reducing the capacity of the PIWI/piRNA complex to target zygotic genes during embryogenesis. Examination of embryonic germ cell proliferation and ovarian gene expression showed that the germline of female progeny was partially masculinized by maternal piwi knockdown. Our study reveals a novel role for maternal Piwi in the germline development of female progeny and suggests that the PIWI/piRNA pathway is involved in germline sex determination in Drosophila.

genetics↗

bioRxiv2021

In situ electro-sequencing in three-dimensional tissues

Pairwise mapping of single-cell gene expression and electrophysiology in intact three-dimensional (3D) tissues is crucial for studying electrogenic organs (e.g., brain and heart)1-5. Here, we introduce in situ electro-sequencing (electro-seq), combining soft bioelectronics with in situ RNA sequencing to stably map millisecond-timescale cellular electrophysiology and simultaneously profile a large number of genes at single-cell level across 3D tissues. We applied in situ electro-seq to 3D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) patches, precisely registering the CM gene expression with electrophysiology at single-cell level, enabling multimodal in situ analysis. Such multimodal data integration substantially improved the dissection of cell types and the reconstruction of developmental trajectory from spatially heterogeneous tissues. Using machine learning (ML)-based cross-modal analysis, in situ electro-seq identified the gene-to-electrophysiology relationship over the time course of cardiac maturation. Further leveraging such a relationship to train a coupled autoencoder, we demonstrated the prediction of single-cell gene expression profile evolution using long-term electrical measurement from the same cardiac patch or 3D millimeter-scale cardiac organoids. As exemplified by cardiac tissue maturation, in situ electro-seq will be broadly applicable to create spatiotemporal multimodal maps and predictive models in electrogenic organs, allowing discovery of cell types and gene programs responsible for electrophysiological function and dysfunction.

genomics↗

bioRxiv2021

Single-molecule imaging of chromatin remodelers reveals role of ATPase in promoting fast kinetics of target search and dissociation from chromatin

Conserved ATP-dependent chromatin remodelers establish and maintain genome-wide chromatin architectures of regulatory DNA during cellular lifespan, but the temporal interactions between remodelers and chromatin targets have been obscure. We performed live-cell single-molecule tracking for RSC, SWI/SNF, CHD1, ISW1, ISW2, and INO80 remodeling complexes in budding yeast and detected hyperkinetic behaviors for chromatin-bound molecules that frequently transition to the free state for all complexes. Chromatin-bound remodelers display notably higher diffusion than nucleosomal histones, and strikingly fast dissociation kinetics with 4-7 s mean residence times. These enhanced dynamics require ATP binding or hydrolysis by the catalytic ATPase, uncovering an additional function to its established role in nucleosome remodeling. Kinetic simulations show that multiple remodelers can repeatedly occupy the same promoter region on a timescale of minutes, implicating an unending tug-of-war that controls a temporally shifting window of accessibility for the transcription initiation machinery.

molecular biology↗

bioRxiv2021

Barcoded Competitive Clone-Initiating Cell (BC-CIC) Analysis Reveals Differences in Ovarian Cancer Cell Genotype and Niche Specific Clonal Fitness During Growth and Metastasis In Vivo

During oncogenesis, pathogenic clones develop which contain cells capable of spreading throughout the body, ultimately compromising vital organ functions and physiology. Understanding how metastatic clones develop and spread is critical for improving cancer treatments. However, our understanding of these processes has been hampered by a paucity of quantitative methodologies to comprehensively map, track and characterize such clones. To address this shortcoming, we have developed a DNA barcoding and next-generation sequencing based system-wide clonal tracking technology integrated with a computational data analysis pipeline called Clone-Initiating Cell (CIC) Calculator. The CIC Calculator interfaces with the CIC Morbus Mandala (CIC-MM) plot, a novel tool to visually comprehend and detect four distinct categories that explains their complex relationships with various tissues/organ sites. Further, we describe machine learning approaches to study CIC number, frequency, and estimate clone size and distribution demonstrating distinct growth patterns, and their inter-relationships and their routes of metastatic spread at clonal resolution. We demonstrate these methodologies, using our novel multifunctional lentiviral barcode libraries, and specifically barcoded tubal-ovarian metastatic OVCAR5 cell lines (engineered to express varying levels of metastasis promoting LRRC15 gene) and co-injected cells in a competitive CIC assay into tubal or ovarian sites in highly immunodeficient NSG mice. DNA was isolated from primary tumors, omental/bowel metastasis and system-wide anatomical site/organs. Amplicon sequencing libraries were constructed with spike-in-control barcodes (serving as internal calibration controls) to estimate absolute clone sizes. The computational pipeline CIC Calculator was then used to deconvolute and filter the data, set stringent thresholds, and generate high-quality information on CIC numbers and frequencies, clone sizes, linkages across sites and classify clones based on their extent of metastatic activity. Using of CIC-MM plot, statistical models and machine learning approaches, we generated high-resolution clonal maps of metastasis for each animal. The information generated included clone types and system-wide metastasis, similar and dissimilar clonal patterns of dominance at heterotopic sites and their routes of metastases. The data revealed previously unknown influences of cellular genotype and their implanted sites on selecting certain clones with specific system-wide clonal patterns, and identified rare LRRC15 expressor clones (classified as CIC.Toti) predisposed to exploit all sites, albeit at varying degrees of dominance. The genomic technology and computational methodology described here are tissue-agnostic. They enable rapid adoption for an investigation into various stages of system-wide metastasis and growth of transplantable malignant cells at the highest clonal resolution.

cancer biology↗

bioRxiv2021

Automatic Multi-functional Integration Program (AMFIP) towards All-optical Mechanobiology Interrogation

Automatic operations of multi-functional and time-lapse live-cell imaging are necessary for biomedical studies of active, multi-faceted, and long-term biological phenomena. To achieve automatic control, most existing solutions often require the purchase of extra software programs and hardware that rely on the manufacturers own specifications. However, these software programs are usually non-user-programmable and unaffordable for many laboratories. Manager is a widely used open-source software platform for controlling many optoelectronic instruments. Due to limited development since its introduction, Manager lacks compatibility with some of the latest microscopy equipment. To address this unmet need, we have developed a novel software-based automation program, titled Automatic Multi-functional Integration Program (AMFIP), as a new Java-based and hardware-independent plugin for Manager. Without extra hardware, AMFIP enables the functional synchronization of Manager, the Nikon NIS-Elements platform, and other 3rd party software to achieve automatic operations of most commercially available microscopy systems, including but not limited to Nikon. AMFIP provides a user-friendly and programmable graphical user interface (GUI), opening the door to expanding the customizability for many hardware and software. Users can customize AMFIP according to their own specific experimental requirements and hardware environments. To verify AMFIPs performance, we applied it to elucidate the relationship between cell spreading and spatial-temporal cellular expression of Yes-associated protein (YAP), a mechanosensitive protein that shuttles between cytoplasm and nucleus upon mechanical stimulation, in an epithelial cell line. We found that the ratio of YAP expression in nucleus and cytoplasm decreases as the spreading area of cells increases, suggesting that the accumulation of YAP in the nucleus decreases throughout the cell spreading processes. In summary, AMFIP provides a new open-source and charge-free solution to integrate multiple hardware and software to satisfy the need of automatic imaging operations in the scientific community.

bioengineering↗

bioRxiv2021

Clustering-based time series analysis on insulin response in the blood-brain barrier

BackgroundCritical functions of the blood-brain barrier (BBB), including cerebral blood flow and vascular response, are regulated by insulin signaling pathways. Therefore, endothelial insulin resistance could lead to vascular dysfunction, which is associated with neurodegenerative diseases such as Alzheimers disease (AD). ObjectiveThe objective of the current study is to map the dynamics of insulin-responsive pathways in polarized human cerebral microvascular endothelial cells (hCMEC/D3) cell monolayers, a widely used BBB cell culture model, to identify molecular mechanisms underlying BBB dysfunction in AD. MethodsRNA-Sequencing (RNA-Seq) was performed on hCMEC/D3 cell monolayers with and without insulin treatment at various time points. The Short Time-series Expression Miner (STEM) method was used to identify clusters of genes with distinct and representative patterns. Functional annotation and pathway analysis of the genes from top clusters were conducted using the Webgestalt and Ingenuity Pathway Analysis (IPA) software, respectively. ResultsQuantitative expression differences of 19,971 genes between the insulin-treated and control monolayers at five-time points were determined. STEM software identified 11 clusters with 3061 genes across that displayed various temporal patterns. Gene ontology enrichment analysis performed using the top 5 clusters demonstrated that these genes were enriched in various biological processes associated with AD pathophysiology. The IPA analyses revealed that signaling pathways exacerbating AD pathology such as inflammation were downregulated after insulin treatment (clusters 1 to 3). In contrast, pathways attenuating AD pathology were upregulated, including synaptogenesis and BBB repairment (clusters 4 and 5). ConclusionsThese findings unravel the dynamics of insulin action on the BBB endothelium and inform about downstream signaling cascades that potentially regulate neurovascular unit (NVU) functions that are disrupted in AD.

bioinformatics↗

bioRxiv2021

Efficient Inhibition of SARS-CoV-2 Using Chimeric Antisense Oligonucleotides through RNase L Activation

There is an urgent need for effective antiviral drugs to alleviate the current COVID-19 pandemic. Here, we rationally designed and developed chimeric antisense oligonucleotides to degrade envelope and spike RNAs of SARS-CoV-2. Each oligonucleotide comprises a 3 antisense sequence for target recognition and a 5-phosphorylated 2-5 poly(A)4 for guided ribonuclease L (RNase L) activation. Since RNase L can potently cleave single strand RNA during innate antiviral response, the improved degradation efficiency of chimeric oligonucleotides was twice as much as classic antisense oligonucleotides in Vero cells, for both SARS-CoV-2 RNA targets. In pseudovirus infection models, one of chimeric oligonucleotides targeting spike RNA achieved potent and broad-spectrum inhibition of both SARS-CoV-2 and its recently reported N501Y and/or {Delta}H69/{Delta}V70 mutants. These results showed that the constructed chimeric oligonucleotides could efficiently degrade pathogenic RNA of SARS-CoV-2 facilitated by immune activation, showing promising potentials as antiviral nucleic acid drugs for COVID-19.

microbiology↗

bioRxiv2021

Intrinsic cardiac adrenergic cell contributes to septic cardiomyopathy

Occurring independently of cardiac sympathetic nervous system, the intrinsic cardiac adrenergic (ICA) cells have been identified as an important regulator in both of developing and adult cardiac physiological and pathological processes. However, its role in septic cardiomyopathy remains unknown. Herein, we report that lipopolysaccharide (LPS) dose- and time-dependently increased norepinephrine (NE) release from ICA cells, which aggravates myocardial TNF- production and dysfunction. Inhibition of NE synthesis in ICA cells alleviated LPS-elicited cardiac dysfunction as well as TNF- production in Langendorff perfusing hearts. Mechanistically, ICA cell expressed Toll-like receptor 4 (TLR4), activated by LPS, to increase the expression of tyrosine hydroxylase, a key enzyme responsible for NE biosynthesis, via AP-1 binding to its promoter. Surprisingly, LPS-TLR4 signaling triggered no TNF- production in ICA cells due to the elevated Nfkbia and Tnfaip6 expression. In LPS-treated co-culture of ICA cells and cardiomyocytes, the raised NE from ICA cells activated cardiomyocyte {beta}1-adrenergic receptor ({beta}1-AR), driving Ca2+/calmodulin-dependent protein kinase II (CaMKII) to increase the activities of NF-{kappa}B and mitogen-activated protein kinase pathways, which were mimicked by dobutamine. Our findings reveal a cell type-specific TLR4 function triggering NE synthesis, but not TNF- production in inflammatory pathogenesis, and identify ICA cell-derived NE as a paracrine signal in the cross talk among different cardiac cells to enhance myocardial injury during LPS challenge, suggesting that targeting ICA cell-derived NE may be a potential therapeutic strategy for septic cardiomyopathy.

immunology↗

bioRxiv2021

ClusterMap: multi-scale clustering analysis of spatial gene expression

Quantifying RNAs in their spatial context is crucial to understanding gene expression and regulation in complex tissues. In situ transcriptomic methods generate spatially resolved RNA profiles in intact tissues. However, there is a lack of a unified computational framework for integrative analysis of in situ transcriptomic data. Here, we present an unsupervised and annotation-free framework, termed ClusterMap, which incorporates physical proximity and gene identity of RNAs, formulates the task as a point pattern analysis problem, and thus defines biologically meaningful structures and groups. Specifically, ClusterMap precisely clusters RNAs into subcellular structures, cell bodies, and tissue regions in both two- and three-dimensional space, and consistently performs on diverse tissue types, including mouse brain, placenta, gut, and human cardiac organoids. We demonstrate ClusterMap to be broadly applicable to various in situ transcriptomic measurements to uncover gene expression patterns, cell-cell interactions, and tissue organization principles from high-dimensional transcriptomic images.

genomics↗

bioRxiv2021

Matrix-Bound Growth Factors are Released upon Cartilage Compression by an Aggrecan-Dependent Sodium Flux that is Lost in Osteoarthritis

Articular cartilage is a dense extracellular matrix-rich tissue that degrades following chronic mechanical stress, resulting in osteoarthritis (OA). The tissue has low intrinsic repair especially in aged and osteoarthritic joints. Here we describe three pro-regenerative factors; fibroblast growth factor 2 (FGF2), connective tissue growth factor, bound to transforming growth factor-beta (CTGF-TGF{beta}), and hepatoma-derived growth factor (HDGF), that are rapidly released from the pericellular matrix (PCM) of articular cartilage upon mechanical injury. All three growth factors bound heparan sulfate, and were displaced by exogenous NaCl. We hypothesised that sodium, sequestered within the aggrecan-rich matrix, was freed by injurious compression, thereby enhancing the bioavailability of pericellular growth factors. Indeed, growth factor release was abrogated when cartilage aggrecan was depleted by IL-1 treatment, and in severely damaged human osteoarthritic cartilage. A flux in free matrix sodium upon mechanical compression of cartilage was visualised by 23Na magnetic resonance imaging (MRI) just below the articular surface. This corresponded to a region of reduced tissue stiffness, measured by scanning acoustic microscopy and second harmonic generation microscopy, and where Smad2/3 was phosphorylated upon cyclic compression. Our results describe a novel intrinsic repair mechanism, controlled by matrix stiffness and mediated by the free sodium concentration, in which heparan sulfate-bound growth factors are released from cartilage upon injurious load. They identify aggrecan as a depot for sequestered sodium, explaining why osteoarthritic tissue loses its ability to repair. Treatments that restore matrix sodium to allow appropriate release of growth factors upon load are predicted to enable intrinsic cartilage repair in osteoarthritis. Significance StatementOsteoarthritis is the most prevalent musculoskeletal disease, affecting 250 million people worldwide 1. We identify a novel intrinsic repair response in cartilage, mediated by aggrecan-dependent sodium flux, and dependent upon matrix stiffness, which results in the release of a cocktail of pro-regenerative growth factors after injury. Loss of aggrecan in late-stage osteoarthritis prevents growth factor release and likely contributes to disease progression. Treatments that restore matrix sodium in osteoarthritis may recover the intrinsic repair response to improve disease outcome.

cell biology↗

bioRxiv2021

Spike mutations decrease SARS-CoV-2 sensitivity to neutralizing antibodies but not ACE2-Ig in vitro

The emergence of SARS-CoV-2 variants poses greater challenges to the control of COVID-19 pandemic. Here, we parallelly investigated three important characteristics of seven SARS-CoV-2 variants, including two mink-associated variants, the B.1.617.1 variant, and the four WHO-designated variants of concerns (B.1.1.7, B.1.351, P.1, and B.1.617.2). We first investigated the ability of these variants to bind and use animal ACE2 orthologs as entry receptor. We found that, in contrast to a prototype variant, the B.1.1.7, B.1.351, and P.1 variants had significantly enhanced affinities to cattle, pig, and mouse ACE2 proteins, suggesting increased susceptibility of these species to these SARS-CoV-2 variants. We then evaluated in vitro neutralization sensitivity of these variants to four monoclonal antibodies in clinical use. We observed that all the variants were partially or completely resistant against at least one of the four tested antibodies, with B.1.351 and P.1 showing significant resistance to three of them. As ACE2-Ig is a broad-spectrum anti-SARS-CoV-2 drug candidate, we then evaluated in vitro neutralization sensitivity of these variants to eight ACE2-Ig constructs previously described in three different studies. All the SARS-CoV-2 variants were efficiently neutralized by these ACE2-Ig constructs. Interestingly, compared to the prototype variant, most tested variants including the variants of concern B.1.1.7, B.1.351, P.1, and B.1.617.2 showed significantly increased (up to [~]15-fold) neutralization sensitivity to ACE2-Ig constructs that are not heavily mutated in the spike-binding interface of the soluble ACE2 domain, suggesting that SARS-CoV-2 evolves toward better utilizing ACE2, and that ACE2-Ig is an attractive drug candidate for coping with SARS-CoV-2 mutations.

microbiology↗

bioRxiv2021

Large field of view-spatially resolved transcriptomics at nanoscale resolution

Spatially resolved transcriptomic technologies are promising tools to study cell fate decisions in a physical microenvironment, which is fundamental for enhancing our knowledge of mammalian development. However, the imbalance between resolution, transcript capture and field of view of current methodologies precludes their systematic application to analyze relatively large and three-dimensional mid- and late-gestation mammalian embryos. Here, we combined DNA nanoball (DNB) patterned arrays and tissue RNA capture to create SpaTial Enhanced REsolution Omics-sequencing (Stereo-seq). This approach allows transcriptomic profiling of large histological sections with high resolution and sensitivity. We have applied Stereo-seq to study the kinetics and directionality of transcriptional variation in a time course of mouse organogenesis. We used this information to gain insight into the molecular basis of regional specification, neuronal migration and differentiation in the developing brain. Furthermore, we mapped the expression of a panel of developmental disease-related loci on our global transcriptomic maps to define the spatiotemporal windows of tissue vulnerability. Our panoramic atlas constitutes an essential resource to investigate longstanding questions concerning normal and abnormal mammalian development.

cell biology↗

bioRxiv2020

Spatio-Temporal Coordination Of Transcription Preinitiation Complex Assembly In Live Cells

Transcription initiation by RNA polymerase II (Pol II) requires preinitiation complex (PIC) assembly at gene promoters. In the dynamic nucleus where thousands of promoters are broadly distributed in chromatin, it is unclear how ten individual components converge on any target to establish the PIC. Here, we use live-cell, single-molecule tracking in S. cerevisiae to document subdiffusive, constrained exploration of the nucleoplasm by PIC components and Mediators key functions in guiding this process. On chromatin, TBP, Mediator, and Pol II instruct assembly of a short-lived PIC, which occurs infrequently but efficiently at an average promoter where initiation-coupled disassembly may occur within a few seconds. Moreover, PIC exclusion by nucleosome encroachment underscores regulated promoter accessibility by chromatin remodeling. Thus, coordinated nuclear exploration and recruitment to accessible targets underlies dynamic PIC establishment in yeast. Collectively, our study provides a global spatio-temporal model for transcription initiation in live cells.

molecular biology↗

bioRxiv2020

Leveraging selective hippocampal vulnerability among Alzheimer's disease subtypes reveals a novel tau binding partner SERPINA5

Selective vulnerability is a central concept to the myriad of devastating neurodegenerative disorders. Although hippocampus and cortex are selectively vulnerable in Alzheimers disease (AD), the degree of involvement lies along a spectrum that we previously defined as AD subtypes revealing distinct clinical correlates. To operationalize heterogeneity of disease spectrum, we classified corticolimbic patterns of neurofibrillary tangles to capture extreme and representative phenotypes. We combined bulk RNA sequencing with digital pathology to examine hippocampal vulnerability in AD. Using a multidisciplinary approach, we uncovered disease-relevant hippocampal gene expression changes. Biological relevance was prioritized using machine learning and several levels of human validation. This resulted in five genes highly predictive of neuropathologically diagnosed AD: SERPINA5, RYBP, SLC38A2, FEM1B, and PYDC1. Deeper investigation revealed SERPINA5 to be a novel tau binding partner that may represent a "tipping point" in the dynamic maturity of neurofibrillary tangles. Our study highlights the importance of embracing heterogeneity of the human brain to yield promising gene candidates as exampled by SERPINA5.

neuroscience↗

bioRxiv2020

TIPS: Trajectory Inference of Pathway Significance through Pseudotime Comparison for Functional Assessment of single-cell RNAseq Data

Recent advances in bioinformatics analyses have led to the development of novel tools enabling the capture and trajectory mapping of single-cell RNA sequencing (scRNAseq) data. However, there is a lack of methods to assess the contributions of biological pathways and transcription factors to an overall developmental trajectory mapped from scRNAseq data. In this manuscript, we present a simplified approach for trajectory inference of pathway significance (TIPS) that leverages existing knowledgebases of functional pathways and transcription factor targets to enable further mechanistic insights into a biological process. TIPS returns both the key pathways whose changes are associated with the process of interest, as well as the individual genes that best reflect these changes. TIPS also provides insight into the relative timing of pathway changes, as well as a suite of visualizations to enable simplified data interpretation of scRNAseq libraries generated using a wide range of techniques. The TIPS package can be run through either a web server, or downloaded as a user-friendly GUI run in R, and may serve as a useful tool to help biologists perform deeper functional analyses and visualization of their single-cell and/or large cohort RNAseq data.

bioinformatics↗