Biology subjects

Nair, S.

Publications and source records attributed to Nair, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Design, development, and evaluation of gene therapeutics specific to KSHV-associated diseases

Kaposis sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposis sarcoma (KS) and two human lymphoproliferative diseases: primary effusion lymphoma and AIDS-related multicentric Castlemans disease. KSHV-encoded latency-associated nuclear antigen (LANA) is expressed in KSHV-infected cancer cells and is responsible for maintaining viral genomes in infected cells. Thus, LANA is an attractive target for therapeutic intervention for KSHV-associated diseases. Here, we devised a cancer gene therapy vector using the adeno-associated virus (AAV), which capitalizes the LANAs function to maintain terminal repeat (TR) containing circular genome in latently infected cells and the TRs enhancer function for KSHV inducible gene promoters. By including two TR copies with a lytic inducible gene promoter (TR2-OriP), we prepared an AAV vector, which expresses an engineered thymidine kinase (TK) selectively in KSHV-infected cells. Ganciclovir (GCV), an anti-herpesvirus drug, effectively eradicated multiple KSHV-infected cells that include iPSC-derived epithelial colony-forming cells, but not non-KSHV-infected counterparts in the presence of AAV8-TR2-OriP-TK. In addition, AAV8-TR2-OriP-TK prevents KSHV virion production from reactivated cells, spreading KSHV infections from reactivated cells. Anti-cancer drugs, known to reactivate KSHV, stimulated TK expression from the vector and, therefore, synergized with AAV8 TR2-OriP-TK to induce KSHV-infected cancer cell death. Finally, the AAV8-TR2-OriP-TK with GCV completely diminished KSHV-infected cancer cells in the xenograft tumor model. The new cancer gene therapeutics should augment the current clinical protocol for KS.

microbiology↗

bioRxiv2025

Pooled long-read sequencing for structural variant characterization in schistosome populations

Pooled sequencing provides a rapid cost-effective approach to assess genetic variation segregating within populations of organisms. However, such studies are typically limited to single nucleotide variants and small indels ([≤] 50bp), and have not been used for structural variants (SVs; >50bp) which impact large portions of most genomes and may significantly impact phenotype. Here, we examined SVs circulating in five laboratory populations of the human parasite Schistosoma mansoni by generating long-read sequences from pools of worms (92 -152 per population). We were able identify and genotype 17,446 SVs, representing 6.5% of the genome despite challenges in identifying low frequency variants. SVs included deletions (n=8,525), duplications (n=131), insertions (n=8,410), inversions (n=311), and translocations (n=69) and were enriched in repeat regions. More than half (59%) of the SVs were shared between [≥]4 populations, but 12% were found in only one of the five populations. Within this subset, we identified 168 population-specific SVs that were at-or-near fixation (>95% alternate allele frequency) in one population but missing (<5%) in the other four populations. Five of these variants impact the coding sequence of 6 genes. We also identified 8 SVs with extreme allele frequency differences between populations within quantitative trait loci for biomedically important pathogen phenotypes (drug resistance, larval stage production) identified in prior genetic mapping studies. These results demonstrate that long-read sequence data from pooled individuals is a viable method to quickly catalogue SVs circulating within populations. Furthermore, some of these variants may be responsible for, or linked to, regions experiencing, population-specific directional selection. Significance StatementStructural variants (SVs) are large genomic variants that are frequently overlooked despite being the largest source of genetic variation within a population. This is because large SVs are expensive and difficult to genotype relative to single nucleotide variants or small indels, so are typically overlooked in population studies. This study attempts to solve these problems by using pooled samples and long-read sequencing to survey SVs circulating in five laboratory populations of the human parasite, Schistosoma mansoni. We were able to identify 17,446 SVs that impact 6.5% of the genome. A number of these SVs may be linked to population-specific adaptations. We also found 8 SVs that were associated with known parasitic traits from previous studies. This work highlights the value of long-read sequencing of pooled samples to document genetic diversity and provides a new method for exploring the role of SVs in parasite evolution and pathogenicity.

genomics↗

bioRxiv2024

ChromBPNet: bias factorized, base-resolution deep learning models of chromatin accessibility reveal cis-regulatory sequence syntax, transcription factor footprints and regulatory variants

Despite extensive mapping of cis-regulatory elements (cREs) across cellular contexts with chromatin accessibility assays, the sequence syntax and genetic variants that regulate transcription factor (TF) binding and chromatin accessibility at context-specific cREs remain elusive. We introduce ChromBPNet, a deep learning DNA sequence model of base-resolution accessibility profiles that detects, learns and deconvolves assay-specific enzyme biases from regulatory sequence determinants of accessibility, enabling robust discovery of compact TF motif lexicons, cooperative motif syntax and precision footprints across assays and sequencing depths. Extensive benchmarks show that ChromBPNet, despite its lightweight design, is competitive with much larger contemporary models at predicting variant effects on chromatin accessibility, pioneer TF binding and reporter activity across assays, cell contexts and ancestry, while providing interpretation of disrupted regulatory syntax. ChromBPNet also helps prioritize and interpret regulatory variants that influence complex traits and rare diseases, thereby providing a powerful lens to decode regulatory DNA and genetic variation.

genetics↗

bioRxiv2024

PDBe tools for an in-depth analysis of small molecules in the Protein Data Bank

The Protein Data Bank (PDB) is the primary global repository for experimentally determined 3D structures of biological macromolecules and their complexes with ligands, proteins, and nucleic acids. PDB contains over 47,000 unique small molecules bound to the macromolecules. Despite the extensive data available, the complexity of small molecule data in the PDB necessitates specialised tools for effective analysis and visualisation. PDBe has developed a number of tools, including PDBe CCDUtils (https://github.com/PDBeurope/ccdutils) for accessing and enriching ligand data, PDBe Arpeggio (https://github.com/PDBeurope/arpeggio) for analysing interactions between ligands and macromolecules, and PDBe RelLig (https://github.com/PDBeurope/rellig) for identifying the functional roles of ligands (such as reactants, cofactors, or drug-like molecules) within protein-ligand complexes. Furthermore, the enhanced ligand annotations and data generated by these tools are presented in a comprehensive view on the novel PDBe-KB ligand pages, providing a holistic view of small molecules that enables the establishment of their biological contexts (Example page for Imatinib: https://wwwdev.ebi.ac.uk/pdbe-srv/pdbechem/chemicalCompound/show/STI). By improving the standardisation of ligand identification, adding various annotations, and offering advanced visualisation capabilities, these tools help researchers navigate the complexities of small molecules and their roles in biological systems, facilitating mechanistic understanding of biological functions. The ongoing enhancements to these resources are designed to support the scientific community in gaining valuable insights into ligands and their applications across various fields, including drug discovery, molecular biology, systems biology, structural biology, and pharmacology.

bioinformatics↗

bioRxiv2024

Decoding sequence determinants of gene expression in diverse cellular and disease states

Sequence-to-function models that predict gene expression from genomic DNA sequence have proven valuable for many biological tasks, including understanding cis-regulatory syntax and interpreting non-coding genetic variants. However, current state-of-the-art models have been trained largely on bulk expression profiles from healthy tissues or cell lines, and have not learned the properties of precise cell types and states that are captured in large-scale single-cell transcriptomic datasets. Thus, they lack the ability to perform these tasks at the resolution of specific cell types or states across diverse tissue and disease contexts. To address this gap, we present Decima, a model that predicts the cell type- and condition- specific expression of a gene from its surrounding DNA sequence. Decima is trained on single-cell or single-nucleus RNA sequencing data from over 22 million cells, and successfully predicts the cell type-specific expression of unseen genes based on their sequence alone. Here, we demonstrate Decimas ability to reveal the cis-regulatory mechanisms driving cell type-specific gene expression and its changes in disease, to predict non-coding variant effects at cell type resolution, and to design regulatory DNA elements with precisely tuned, context-specific functions.

genomics↗

bioRxiv2024

Typhi Mykrobe: fast and accurate lineage identification and antimicrobial resistance genotyping directly from sequence reads for the typhoid fever agent Salmonella Typhi

BackgroundTyphoid fever results from systemic infection with Salmonella enterica serovar Typhi (Typhi) and causes 10 million illnesses annually. Disease control relies on prevention (water, sanitation, and hygiene interventions or vaccination) and effective antimicrobial treatment. Antimicrobial resistant (AMR) Typhi lineages have emerged and become established in many parts of the world. Knowledge of local pathogen populations informed by genomic surveillance, including of lineages (defined by the GenoTyphi scheme) and AMR determinants, is increasingly used to inform local treatment guidelines and to inform vaccination strategy. Current tools for genotyping Typhi require multiple read alignment or assembly steps and have not been validated for analysis of data generated with Oxford Nanopore Technologies (ONT) long-read sequencing devices. Here, we introduce Typhi Mykrobe, a command line software tool for rapid genotyping of Typhi lineages, AMR determinants, and plasmid replicons direct from sequencing reads. ResultsWe validated Typhi Mykrobe lineage genotyping by comparison with the current standard read mapping-based approach and demonstrated 99.8% concordance across nearly 13,000 genomes sequenced with Illumina platforms. For the few isolates with discordant calls, we show that Typhi Mykrobe results are better supported by the evidence from raw sequence read data than the results generated using the mapping-based approach. We also demonstrate 99.9% concordance for detection of AMR determinants compared with the current standard assembly-based approach, with similar results for plasmid marker detection. Typhi Mykrobe predicts clinical resistance categorisation (S/I/R) for eight drug classes, and we show strong agreement with phenotypic categorisations generated from reference laboratory minimum inhibitory concentration (MIC) data for n=1,572 Illumina-sequenced isolates (>99% agreement within one doubling dilution). We show strong concordance (>96% for genotype and >98% for AMR and plasmid) between calls made from ONT reads and those made from Illumina reads for isolates sequenced on both platforms (n =93 genomes). Typhi Mykrobe takes less than a minute per sample and is available at https://github.com/typhoidgenomics/genotyphi. ConclusionsTyphi Mykrobe provides rapid and sensitive genotyping of Typhi genomes direct from Illumina and ONT reads, although lower accuracy was observed for R9 ONT data. It demonstrated accurate assignment of GenoTyphi lineage, detection of AMR determinants and prediction of corresponding AMR phenotypes, and identification of plasmid replicons.

microbiology↗

bioRxiv2024

gReLU: A comprehensive framework for DNA sequence modeling and design

Deep learning models are increasingly being used to perform a variety of tasks on DNA sequences, such as predicting tissue- and cell type-specific sequence activity, deriving cis-regulatory rules, predicting non-coding variant effects, and designing synthetic regulatory sequences. However, these models require specialized knowledge to build, train and interpret correctly. In addition, the field is hampered by the lack of interoperability between models and software built by different groups. Here we present gReLU, a comprehensive software framework that enables users to easily execute advanced sequence modeling pipelines, including data preprocessing, model training, hyperparameter tuning, evaluation, interpretation, variant effect prediction, and design of novel regulatory elements. The software is accompanied by a model zoo containing state-of-the-art pre-trained models that can easily be downloaded, applied, and fine-tuned. This framework and resources will accelerate research across the field of DNA sequence modeling and enable the effective design of synthetic regulatory elements.

genomics↗

bioRxiv2024

Consumption of human-relevant levels of sucrose-water rewires macronutrient uptake and utilization mechanisms in a tissue specific manner

Consumption of sugar-sweetened beverages (SSBs) have been linked to metabolic dysfunction, obesity, diabetes and enhanced risk of cardiovascular diseases across all age-groups globally. Decades of work that have provided insights into pathophysiological manifestations of sucrose overfeeding have employed paradigms that rarely mimic human consumption of SSBs. Thus, our understanding of multi-organ cross-talk and molecular and/or cellular mechanisms, which operate across scales and drive physiological derangement is still poor. By employing a paradigm of sucrose water feeding in mice that closely resembles chronic SSB consumption in humans (10% sucrose in water), we have unraveled hitherto unknown tissue-specific mechanistic underpinnings, which contribute towards perturbed physiology. Our findings illustrate that systemic impaired glucose homeostasis, mediated by hepatic gluconeogenesis and insulin resistance, does not involve altered gene expression programs in the liver. We have discovered the pivotal role of the small intestine, which in conjunction with liver and muscles, drives dyshomeostasis. Importantly, we have uncovered rewiring of molecular mechanisms in the proximal intestine that is either causal or consequential to systemic ill-effects of chronic sucrose water consumption including dysfunction of liver and muscle mitochondria. Tissue-specific molecular signatures, which we have unveiled, clearly indicate that inefficient utilization of glucose is exacerbated by enhanced uptake by the gut. Besides providing systems-wide mechanistic insights, we propose that consumption of SSBs causes intestinal molecular addiction for deregulated absorption of hexose-sugars, and drives diseases such as diabetes and obesity.

physiology↗

bioRxiv2024

Withania somnifera root extract (LongeFera-TM) confers beneficial effects on health and lifespan of the model worm Caenorhabditis elegans

Withania somnifera is among the most widely prescribed medicinal plants in traditional Indian medicine. Hydroalcoholic extract of the roots of this plant was investigated for its effects on the overall health and lifespan of the model worm Caenorhabditis elegans. Extract-exposed worms registered longer lifespan, higher fertility, better motility and metabolic activity. Whole transcriptome analysis of the extract-treated worms revealed the differential expression of the genes associated with lifespan extension, eggshell assembly and integrity, progeny formation, yolk lipoproteins, collagen synthesis, cuticle molting, etc. This extract seems to exert its beneficial effect on C. elegans partly by triggering remodeling of the developmentally programmed apical extracellular matrix (aECM). Differential expression of certain important genes (cpg-2, cpg-3, sqt- 1, dpy-4, dpy-13, and col-17) was confirmed through PCR assay too. Some of the differently expressed genes (gfat-2, unc-68, dpy-4, dpy-13, col-109, col-169, and rmd-1) in worms experiencing pro-health effect of the extract were found through cooccurrence analysis to have their homologous counterpart in humans. Our results validate the potential of W. somnifera extract as a phytopharmaceutical.

molecular biology↗

bioRxiv2024

Transcriptomic comparison of in vitro models of the human placenta

Studying the human placenta through in vitro cell culture methods is necessary due to limited access and amenability of human placental tissue to certain experimental methods as well as distinct anatomical and physiological differences between animal and human placentas. Selecting an in vitro culture model of the human placenta is challenging due to representation of different trophoblast cell types with distinct biological roles and limited comparative studies that define key characteristics of these models. Therefore, the aim of this research was to create a comprehensive transcriptomic comparison of common in vitro models of the human placenta compared to bulk placental tissue from the CANDLE and GAPPS cohorts (N=1083). We performed differential gene expression analysis on publicly available RNA sequencing data from 6 common in vitro models of the human placenta (HTR-8/SVneo, BeWo, JEG-3, JAR, Primary Trophoblasts, and Villous Explants) and compared to CANDLE and GAPPS bulk placental tissue or cytotrophoblast, syncytiotrophoblast, and extravillous trophoblast cell types derived from bulk placental tissue. All in vitro placental models had a substantial number of differentially expressed genes (DEGs, FDR<0.01) compared to the CANDLE and GAPPS placentas (Average DEGs=10,873), and the individual trophoblast cell types (Average DEGs=5,346), indicating that there are vast differences in gene expression compared to bulk and cell-type specific human placental tissue. Hierarchical clustering identified 53 gene clusters with distinct expression profiles across placental models, with 22 clusters enriched for specific KEGG pathways, 7 clusters enriched for high-expression placental genes, and 7 clusters enriched for absorption, distribution, metabolism, and excretion genes. In vitro placental models were classified by fetal sex based on expression of Y-chromosome genes that identified HTR-8/SVneo cells as being of female origin, while JEG-3, JAR, and BeWo cells are of male origin. Overall, none of the models were a close approximation of the transcriptome of bulk human placental tissue, highlighting the challenges with model selection. To enable researchers to select appropriate models, we have compiled data on differential gene expression, clustering, and fetal sex into an accessible web application: "Comparative Transcriptomic Placental Model Atlas (CTPMA)" which can be utilized by researchers to make informed decisions about their selection of in vitro placental models.

developmental biology↗

bioRxiv2024

ProSAAS is Preferentially Secreted from Neurons During Homeostatic Scaling and Reduces Amyloid Plaque Size in the 5xFAD Mouse Hippocampus

The accumulation of {beta}-amyloid in Alzheimers disease greatly impacts neuronal health and synaptic function. To maintain network stability in the face of altered synaptic activity, neurons engage a feedback mechanism termed homeostatic scaling; however, this process is thought to be disrupted during disease progression. Previous proteomics studies have shown that one of the most highly regulated proteins in cell culture models of homeostatic scaling is the small secretory chaperone proSAAS. Our prior work has shown that proSAAS exhibits anti-aggregant behavior against alpha synuclein and {beta}-amyloid fibrillation in vitro, and is upregulated in cell models of proteostatic stress. However, the specific role that this protein might play in homeostatic scaling, and its anti-aggregant role in Alzheimers progression, is not clear. To learn more about the role of proSAAS in maintaining hippocampal proteostasis, we compared its expression in a primary neuron model of homeostatic scaling to other synaptic components using Western blotting and qPCR, revealing that proSAAS protein responses to homeostatic up- and down-regulation were significantly higher than those of two other synaptic vesicle components, 7B2 and carboxypeptidase E. However, proSAAS mRNA expression was static, suggesting translational control (and/or reduced degradation). ProSAAS was readily released upon depolarization of differentiated hippocampal cultures, supporting its synaptic localization. Immunohistochemical analysis demonstrated abundant proSAAS within the mossy fiber layer of the hippocampus in both wild-type and 5xFAD mice; in the latter, proSAAS was also concentrated around amyloid plaques. Interestingly, overexpression of proSAAS in the CA1 region via stereotaxic injection of proSAAS-encoding AAV2/1 significantly decreased amyloid plaque burden in 5xFAD mice. We hypothesize that dynamic changes in proSAAS expression play a critical role in hippocampal proteostatic processes, both in the context of normal homeostatic plasticity and in the control of protein aggregation during Alzheimers disease progression.

neuroscience↗

bioRxiv2024

Nuclear compression-mediated DNA damage drives ATR-dependent Lamin expression and mouse ESC differentiation

Embryonic stem cells (ESCs) which are susceptible to DNA damage depend on a robust and highly efficient DNA damage response (DDR) mechanism for their survival. However, the implications of physical force-mediated DNA damage on ESC fate remains unclear. We show that stiffness-dependent spreading of mouse ESCs (mESCs) induces DNA damage through nuclear compression, with DNA damage causing differentiation through early induction of Lamin A/C expression. Interestingly, differentiation is associated with rescue of DNA damage and activation of the DDR factor ATR. While ATR is typically known to play roles in DDR pathway, its role during stiffness-mediated nuclear compression and mESC differentiation is unknown. Nuclear enrichment of activated ATR on stiff substrates and reduction of Lamin A/C expression upon ATR inhibition suggests that mESC differentiation is driven by nuclear compression-mediated DNA damage and involves ATR-dependent modulation of Lamin A/C.

cell biology↗

bioRxiv2024

Presence of phage-plasmids in multiple serovars of Salmonella enterica

Evidence is accumulating in the literature that the horizontal spread of antimicrobial resistance (AMR) genes mediated by bacteriophages and bacteriophage-like plasmid (phage-plasmid) elements is much more common than previously envisioned. For instance, we recently identified and characterised a circular P1-like phage-plasmid harbouring a blaCTX-M-15 gene conferring extended-spectrum beta-lactamase (ESBL) resistance in Salmonella enterica serovar Typhi. As the prevalence and epidemiological relevance of such mechanisms has never been systematically assessed in Enterobacterales, in this study we carried out a follow-up retrospective analysis of UK Salmonella isolates previously sequenced as part of routine surveillance protocols between 2016 and 2021. Using a high-throughput bioinformatics pipeline we screened 47,784 isolates for the presence of the P1 lytic replication gene repL, identifying 226 positive isolates from 25 serovars and demonstrating that phage-plasmid elements are more frequent than previously thought. The affinity for phage-plasmids appears highly serovar-dependent, with several serovars being more likely hosts than others; most of the positive isolates (170/226) belonged to S. Typhimurium ST34 and ST19. The phage-plasmids ranged between 85.8-98.2kb in size, with an average length of 92.1kb; detailed analysis indicated a high amount of diversity in gene content and genomic architecture. 132 phage-plasmids had the p0111 plasmid replication type, and 94 the IncY type; phylogenetic analysis indicated that both horizontal and vertical gene transmission mechanisms are likely to be involved in phage-plasmid propagation. Finally, phage-plasmids were present in isolates that were resistant and non-resistant to antimicrobials. In addition to providing a first comprehensive view of the presence of phage-plasmids in Salmonella, our work highlights the need for a better surveillance and understanding of phage-plasmids as AMR carriers, especially through their characterisation with long-read sequencing. Data SummaryAll of the FASTQ files examined in this study have been uploaded to the Sequence Read Archive under BioProject PRJNA248792. Accessions of individual isolates which were found to contain phage plasmids are listed in Supplementary Table S1. Impact StatementBacteriophage-like plasmids are increasingly being recognised as important mobile elements in many species of bacteria, particularly due to their involvement in the transmission of antimicrobial resistance (AMR); however, few studies of their overall prevalence in clinical datasets have been undertaken to date. In this study we have performed the first large-scale surveillance of human disease-associated Salmonella genomes for the presence of P1-like phage-plasmids, showing that they are more common than previously thought. Furthermore, we highlight how valuable information about the evolution and transmission of phage-plasmids in Salmonella and other Enterobacterales can be revealed by linking phage-plasmid prevalence and genetic diversity to epidemiologically relevant metadata such as S. enterica serovar, outbreak clusters, time, and geography. Our work shows the ability to use sequencing data and scalable bioinformatics workflows for the detection and characterisation of these extrachromosomal elements, highlights the importance of screening for novel mechanisms of AMR transmission, and provides a foundation for further surveillance studies of phage-plasmid prevalence.

microbiology↗

bioRxiv2024

The preoptic Kisspeptin/nNOS/GnRH (KiNG) neuronal network regulates rhythmic LH release through a dual activation-inhibition mechanism

Gonadotropin-releasing hormone (GnRH) neurons are the final common target of a complex network of cells cooperating for the central control of reproduction. The balance between excitatory and inhibitory transsynaptic and non-synaptic inputs is crucial for the maintenance of the GnRH rhythms: the pulse and the surge. The precise mechanisms behind this remain under debate. In this work, we challenge the hypothesis that excitatory and inhibitory inputs from kisspeptin and neuronal nitric oxide (NO) synthase (nNOS)-expressing neurons orchestrates GnRH release, in a microcircuit that we call the Kisspeptin/nNOS/GnRH (KiNG) neuronal network. Our work specifically focuses on the role of nNOS neurons located in the organum vasculosum laminae terminalis (OV) and the median preoptic nucleus (MePO). nNOS and kisspeptin neurons interact anatomically and functionally, with the kisspeptin receptor (Kiss1r) being differentially regulated in nNOS-expressing neurons across the female estrous cycle. Using a novel viral tool allowing for the measurement of NO/cGMP levels with exquisite sensitivity, we demonstrate that kisspeptin is able to induce NO-dependent cGMP production in the OV/MePO, including in GnRH neurons in vivo. Using electrophysiological, genetic, chemogenetic and pharmacologic approaches, we reveal that NO production from nNOS neurons in the OV/MePO is needed to fine-tune the GnRH/LH response to kisspeptin, and specifically to turn off GnRH release, thus generating pulses. Our findings provide valuable insights into the tripartite KiNG neuronal network governing the regulation of the GnRH/LH pulse and surge.

neuroscience↗

bioRxiv2023

CRISPR-Cas13d screens identify KILR a breast cancer risk-associated lncRNA that regulates DNA replication and repair.

Long noncoding RNAs (lncRNAs) have surpassed the number of protein-coding genes, yet the majority have no known function. We previously discovered >800 lncRNAs at regions identified by breast cancer genome-wide association studies (GWAS). Here, we performed a pooled CRISPR-Cas13d RNA knockdown screen to identify which of these lncRNAs altered cell proliferation. We found that KILR, a lncRNA that functions as a tumor suppressor, safeguards breast cells against uncontrolled proliferation. The half-life of KILR is significantly reduced by the risk haplotype, revealing an alternative mechanism by which variants alter cancer risk. We showed that KILR sequesters RPA1, a subunit of the RPA complex, required for DNA replication and repair. Reduced KILR expression promotes cell proliferation by increasing the available pool of RPA1 and the speed of DNA replication. Our findings confirm lncRNAs as mediators of breast cancer risk, emphasize the need to annotate noncoding transcripts in relevant cell types when investigating GWAS variants and provide a scalable platform for mapping phenotypes associated with lncRNAs.

cancer biology↗

bioRxiv2023

Transcription factor stoichiometry, motif affinity and syntax regulate single-cell chromatin dynamics during fibroblast reprogramming to pluripotency

The concentration and stoichiometry of transcription factors (TFs) determine cellular identity and can be manipulated to drive cell state transitions. Understanding how changes in TF concentration regulate chromatin state and expression across cell state transitions remains a challenge. We investigated this relationship by profiling chromatin accessibility and gene expression at single-cell resolution across a densely sampled time course of reprogramming human fibroblasts to induced pluripotent stem cells via ectopic expression of OCT4, SOX2, KLF4, and MYC (OSKM). Using deep learning sequence models of base-resolution chromatin accessibility profiles across cell states, we deciphered predictive transcription factor (TF) motif syntax in regulatory elements, inferred affinity- and concentration-dependent dynamics of TF footprints, linked peaks to putative target genes, and elucidated rewiring of cis-regulatory networks. Our models reveal that early in reprogramming, OSK, at supraphysiological concentrations, rapidly open transient regulatory elements by occupying non-canonical low-affinity binding sites. As OSK concentration falls, the accessibility of these transient elements decays as a function of motif affinity. We find that these OSK-dependent transient elements sequester the somatic TF AP-1. This redistribution is strongly associated with the silencing of fibroblast-specific genes within individual nuclei. Together, our integrated single-cell resource and models reveal insights into the cis-regulatory code of reprogramming at unprecedented resolution. We establish a quantitative, predictive framework that links TF stoichiometry, motif syntax, and somatic silencing to provide new perspectives on the control of cell identity by TFs during fate transitions.

genetics↗

bioRxiv2023

Extramacrochaetae regulates Notch signaling in the Drosophila eye through non-apoptotic caspase activity

Many cell fate decisions are determined transcriptionally. Accordingly, some fate specification is prevented by Inhibitor of DNA binding (Id) proteins that interfere with DNA binding by master regulatory transcription factors. We show that the Drosophila Id protein Extra macrochaetae (Emc) also affects developmental decisions by regulating caspase activity. Emc, which prevents proneural bHLH transcription factors from specifying neural cell fate, also prevents homodimerization of another bHLH protein, Daughterless (Da), and thereby maintains expression of the Death-Associated Inhibitor of Apoptosis (diap1) gene. Accordingly, we found that multiple effects of emc mutations on cell growth and on eye development were all caused by activation of caspases. These effects included acceleration of the morphogenetic furrow, failure of R7 photoreceptor cell specification, and delayed differentiation of non-neuronal cone cells. Within emc mutant clones, Notch signaling was elevated in the morphogenetic furrow, increasing morphogenetic furrow speed. This was associated with caspase-dependent increase in levels of Delta protein, the transmembrane ligand for Notch. Posterior to the morphogenetic furrow, elevated Delta cis-inhibited Notch signaling that was required for R7 specification and cone cell differentiation. Growth inhibition of emc mutant clones in wing imaginal discs also depended on caspases. Thus, emc mutations reveal the importance of restraining caspase activity even in non-apoptotic cells to prevent abnormal development, in the Drosophila eye through effects on Notch signaling.

developmental biology↗

bioRxiv2023

The Implant-Induced Foreign Body Response is Limited by CD13-Dependent Regulation of Ubiquitination of Fusogenic Proteins

Implanted medical devices from artificial heart valves, arthroscopic joints to implantable sensors often induce a Foreign Body Response (FBR), a form of chronic inflammation resulting from the inflammatory reaction to a persistent foreign stimulus. The FBR is characterized by a subset of multinucleated giant cells (MGCs) formed by macrophage fusion, the Foreign Body Giant cells (FBGCs), accompanied by inflammatory cytokines, matrix deposition and eventually, deleterious fibrotic implant encapsulation. Despite efforts to improve biocompatibility, implant-induced FBR persists, compromising the utility of devices and making efforts to control the FBR imperative for long-term function. Controlling macrophage fusion in FBGC formation presents a logical target to prevent implant failure, but the actual contribution of FBGCs to FBR-induced damage is controversial. CD13 is a molecular scaffold and in vitro induction of CD13KO bone-marrow progenitors generates many more MGCs than WT, suggesting CD13 regulates macrophage fusion. Moreover, in the mesh implant model of FBR, CD13KO mice produced significantly more peri-implant FBGCs with enhanced TGF{beta} expression and increased collagen deposition vs. WT. Pre-fusion, increased protrusion and microprojection formation accompanies hyperfusion in the absence of CD13. Expression of fusogenic proteins driving cell-cell fusion was aberrantly sustained at high levels in CD13KO MGCs, which we show is due to a novel CD13 function, regulating ubiquitin/proteasomal protein degradation. By controlling protein degradation, CD13 becomes a physiologic brake preventing aberrant macrophage fusion and may be a novel therapeutic target to improve success of implanted medical devices. Furthermore, our data directly implicates FBGCs in the detrimental fibrosis that characterizes the FBR.

cell biology↗