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Biology subjects

Parameswaran, S.

Publications and source records attributed to Parameswaran, S..

At least 19 recordsLinked to original sources

Systematic investigation of Epstein-Barr virus transcriptional regulator interactions with the human genome

We systematically investigate interactions between Epstein-Barr virus (EBV) transcriptional regulators (vTRs) and the human genome. Starting with 16 known and candidate vTRs, we identify nine whose introduction into human cells results in substantial alterations to host gene expression. Genome-scale determination of vTR genomic binding events and alterations to chromatin accessibility reveals a detailed map of EBVs functional interactions with the human genome, including >100,000 vTR binding events impacting almost a quarter of all human genes. BMRF1 emerges as a potent regulator, impacting >7,000 genes and altering >37,000 chromatin regions. Our results provide new evidence that EBV RTA interacts with and stabilizes the binding of human RBPJ. Network analysis reveals that many human genes are targeted by multiple EBV vTRs, highlighting the vast coordinated impact of EBV on human gene expression. This study provides a valuable, extensive resource for examining EBV-induced alterations to human gene regulation, with data available on multiple platforms.

genomics↗

Leveraging Basecaller's Move Table to Generate a Lightweight k-mer Model

Nanopore sequencing by Oxford Nanopore Technologies (ONT) enables direct analysis of DNA and RNA by capturing raw electrical signals. Different nanopore chemistries have varied k-mer lengths, current levels, and standard deviations, which are stored in k-mer models. Particularly in cases where official models are lacking or unsuitable for specific sequencing conditions, tailored k-mer models are crucial to ensure precise signal-to-sequence alignment and interpretation. The process of transforming raw signals into nucleotide sequences, known as basecalling, is a fundamental step in nanopore sequencing. In this study, we leverage the basecallers move table to create a lightweight denovo k-mer model for RNA004 chemistry. We showcase the effectiveness of our custom k-mer model through high alignment rates (97.48%) compared to larger default models. Additionally, our 5-mer model exhibits similar performance as the default 9-mer models in m6A methylation detection.

bioinformatics↗

Inter-cellular mRNA Transfer Alters Human Pluripotent Stem Cell State

Inter-cellular transmission of mRNA is being explored in mammalian species using immortal cell lines (1-3). Here, we uncover an inter-cellular mRNA transfer phenomenon that allows for the adaptation and reprogramming of human primed pluripotent stem cells (hPSCs). This process is induced by the direct cell contact-mediated coculture with mouse embryonic stem cells (mESCs) under the condition impermissible for human primed PSC culture. Mouse-derived mRNA contents are transmitted into adapted hPSCs only in the coculture. Transfer-specific mRNA analysis show the enrichment for divergent biological pathways involving transcription/translational machinery and stress-coping mechanisms, wherein such transfer is diminished when direct cell contacts are lost. After 5 days of mESC culture, surface marker analysis, and global gene profiling confirmed that mRNA transfer-prone hPSC efficiently gains a naive-like state. Furthermore, transfer-specific knockdown experiments targeting mouse-specific transcription factor-coding mRNAs in hPSC show that mouse-derived Tfcp2l1, Tfap2c, and Klf4 are indispensable for human naive-like conversion. Thus, inter-species mRNA transfer triggers cellular reprogramming in mammalian cells. Our results support that episodic mRNA transfer can occur in cell cooperative and competitive processes(4), which provides a fresh perspective on understanding the roles of mRNA mobility for intra- and inter-species cellular communications.

cell biology↗

Human cytomegalovirus extensively re-organizes the human genome, diminishing TEAD1 transcription factor activity

Human cytomegalovirus (HCMV) infects up to 80% of the worlds population. Here, we show that HCMV infection leads to widespread changes in human chromatin accessibility and chromatin looping, with hundreds of thousands of genomic regions affected 48 hours after infection. Integrative analyses reveal HCMV-induced perturbation of Hippo signaling through drastic reduction of TEAD1 transcription factor activity. We confirm extensive concordant loss of TEAD1 binding, active H3K27ac histone marks, and chromatin looping interactions upon infection. Our data position TEAD1 at the top of a hierarchy involving multiple altered important developmental pathways. HCMV infection reduces TEAD1 activity through four distinct mechanisms: closing of TEAD1-bound chromatin, reduction of YAP1 and phosphorylated YAP1 levels, reduction of TEAD1 transcript and protein levels, and alteration of TEAD1 exon-6 usage. Altered TEAD1-based mechanisms are highly enriched at genetic risk loci associated with eye and ear development, providing mechanistic insight into HCMVs established roles in these processes.

genomics↗

A single cell atlas of the mouse seminal vesicle

During mammalian reproduction, sperm are delivered to the female reproductive tract bathed in a complex medium known as seminal fluid, which plays key roles in signaling to the female reproductive tract and in nourishing sperm for their onwards journey. Along with minor contributions from the prostate and the epididymis, the majority of seminal fluid is produced by a somewhat understudied organ known as the seminal vesicle. Here, we report the first single-cell RNA-seq atlas of the mouse seminal vesicle, generated using tissues obtained from 23 mice of varying ages, exposed to a range of dietary challenges. We define the transcriptome of the secretory cells in this tissue, identifying a relatively homogeneous population of the epithelial cells which are responsible for producing the majority of seminal fluid. We also define the immune cell populations - including large populations of macrophages, dendritic cells, T cells, and NKT cells - which have the potential to play roles in producing various immune mediators present in seminal plasma. Together, our data provide a resource for understanding the composition of an understudied reproductive tissue with potential implications for paternal control of offspring development and metabolism.

genomics↗

Interactive visualisation of raw nanopore signal data with Squigualiser

Nanopore sequencing measures ionic current during the translocation of DNA, RNA or protein molecules through a nanoscale protein pore. This raw current signal data can be basecalled into sequence information and has the potential to identify other diverse molecular features, such as base modifications, secondary structures, etc. Despite the unique properties and potential utility of nanopore signal data, there are currently limited options available for signal data visualisation. To address this, we have developed Squigualiser, a toolkit for intuitive, interactive visualisation of sequence-aligned signal data, which currently supports both DNA and RNA sequencing data from Oxford Nanopore Technologies (ONT) instruments. A series of methodological innovations enable efficient alignment of raw signal data to a reference genome/transcriptome with single-base resolution. Squigualiser generates an interactive signal browser view (HTML file), in which the user can navigate across a genome/transcriptome region and customise the display. Multiple independent reads are integrated into a signal pileup format and different datasets can be displayed as parallel tracks to facilitate their comparison. Squigualiser provides the most sophisticated framework for nanopore signal data visualisation to date and will catalyse new advances in signal analysis. We provide Squigualiser as an open-source tool for the nanopore community: https://github.com/hiruna72/squigualiser

bioinformatics↗

Accessible chromatin maps of inflammatory bowel disease intestine nominate cell-type mediators of genetic disease risk

Inflammatory Bowel Disease (IBD) is a chronic autoinflammatory disorder with rising incidence in pediatrics. TNFa inhibition (TNFi) is the first-line biologic therapy in children, but many do not achieve mucosal healing. Identifying which patients will benefit from TNFi and the underlying nonresponse mechanisms is critical. We built a novel resource: whole genome sequencing linked to multiome-seq (single-nuclei transcriptome and chromatin accessibility) of intestinal biopsies from a cohort of children with IBD, whose TNFi response was defined by mucosal healing. Our study uncovers links between IBD genetic risk and TNFi response. First, classifiers integrating genetic data with clinical variables identified the IBD polygenic risk score as a top predictor of TNFi response. Second, multiome-seq analysis implicated IBD risk variants in persistent cytokine signaling in monocytes, macrophage and fibroblasts of nonresponders. These data reveal genetic mechanisms of treatment response in pediatric IBD and suggest alternative therapeutic approaches for TNFi nonresponders.

genomics↗

The unique function of Runx1 in skeletal muscle differentiation and regeneration is mediated by an ETS interaction domain

The conserved Runt-related (RUNX) transcription factor family are well-known master regulators of developmental and regenerative processes. Runx1 and Runx2 are both expressed in satellite cells (SC) and skeletal myotubes. Conditional deletion of Runx1 in adult SC negatively impacted self-renewal and impaired skeletal muscle maintenance. Runx1- deficient SC retain Runx2 expression but cannot support muscle regeneration in response to injury. To determine the unique molecular functions of Runx1 that cannot be compensated by Runx2 we deleted Runx1 in C2C12 that retain Runx2 expression and established that myoblasts differentiation was blocked in vitro due in part to ectopic expression of Mef2c, a target repressed by Runx1. Structure-function analysis demonstrated that the Ets-interacting MID/EID region of Runx1, absent from Runx2, is critical to regulating myoblasts proliferation, differentiation, and fusion. Analysis of in-house and published ChIP-seq datasets from Runx1 (T-cells, muscle) versus Runx2 (preosteoblasts) dependent tissue identified enrichment for a Ets:Runx composite site in Runx1-dependent tissues. Comparing ATACseq datasets from WT and Runx1KO C2C12 cells showed that the Ets:Runx composite motif was enriched in peaks open exclusively in WT cells compared to peaks unique to Runx1KO cells. Thus, engagement of a set of targets by the RUNX1/ETS complex define the non-redundant functions of Runx1.

developmental biology↗

Lineage tracing of newly accrued nuclei in skeletal myofibers uncovers distinct transcripts and interplay between nuclear populations

Multinucleated skeletal muscle cells have an obligatory need to acquire additional nuclei through fusion with activated skeletal muscle stem cells when responding to both developmental and adaptive growth stimuli. A fundamental question in skeletal muscle biology has been the reason underlying this need for new nuclei in syncytial cells that already harbor hundreds of nuclei. To begin to answer this long-standing question, we utilized nuclear RNA-sequencing approaches and developed a lineage tracing strategy capable of defining the transcriptional state of recently fused nuclei and distinguishing this state from that of pre-existing nuclei. Our findings reveal the presence of conserved markers of newly fused nuclei both during development and after a hypertrophic stimulus in the adult. However, newly fused nuclei also exhibit divergent gene expression that is determined by the myogenic environment to which they fuse. Moreover, accrual of new nuclei through fusion is required for nuclei already resident in adult myofibers to mount a normal transcriptional response to a load-inducing stimulus. We propose a model of mutual regulation in the control of skeletal muscle development and adaptations, where newly fused and pre-existing myonuclear populations influence each other to maintain optimal functional growth.

cell biology↗

minimap2-fpga: Integrating hardware-accelerated chaining for efficient end-to-end long-read sequence mapping

minimap2 is the gold-standard software for reference-based sequence mapping in third-generation long-read sequencing. While minimap2 is relatively fast, further speedup is desirable, especially when processing a multitude of large datasets. In this work, we present minimap2-fpga, a hardware-accelerated version of minimap2 that speeds up the mapping process by integrating an FPGA kernel optimised for chaining. We demonstrate speed-ups in end-to-end run-time for data from both Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio). minimap2-fpga is up to 79% and 53% faster than minimap2 for [~] 30x ONT and [~] 50x PacBio datasets respectively, when mapping without base-level alignment. When mapping with base-level alignment, minimap2-fpga is up to 62% and 10% faster than minimap2 for [~] 30x ONT and [~] 50x PacBio datasets respectively. The accuracy is near-identical to that of original minimap2 for both ONT and PacBio data, when mapping both with and without base-level alignment. minimap2-fpga is supported on Intel FPGA-based systems (evaluations performed on an on-premise system) and Xilinx FPGA-based systems (evaluations performed on a cloud system). We also provide a well-documented library for the FPGA-accelerated chaining kernel to be used by future researchers developing sequence alignment software with limited hardware background.

bioinformatics↗

High-resolution promoter interaction analysis in Type 3 Innate Lymphoid Cells implicates Batten Disease gene CLN3 in Crohn's Disease aetiology

Innate lymphoid cells (ILCs) are rare, tissue-resident innate lymphocytes that functionally mirror CD4+ T helper cell lineages but lack antigen receptors. Type 3 ILCs (ILC3s) are enriched in the gut, airways, and mucosal lymphoid tissues, where they regulate inflammation and promote barrier integrity. To define the regulatory architecture of primary human ILC3s, we map promoter-anchored chromosomal contacts using high-resolution, low-input Promoter Capture Hi-C (PCHi-C) in these cells alongside CD4+ T cells. By combining statistical detection with a PCHi-C-adapted Activity-by-Contact approach, we link promoters to distal regulatory elements, identifying hundreds of ILC3-specific contacts. We use these maps to connect genome-wide association study (GWAS) risk variants for Crohns disease to target genes using multiCOGS, a Bayesian framework that integrates PCHi-C with summary-statistic imputation and multivariate fine-mapping. This analysis highlights both known and unanticipated candidates, including CLN3, a causal gene for the neurodevelopmental Batten disease. Using a mouse ILC3-like cell line, we show that Cln3 is downregulated upon cytokine stimulation, and Cln3 overexpression alters stimulation-induced transcriptional programmes and cytokine secretion. Extending this approach, we generate a catalogue of ILC3-linked risk genes for five additional autoimmune conditions and show that they are enriched for regulators of the ILC3 inflammatory response identified in a CRISPR interference screen. Together, these findings illuminate long-range gene control in ILC3s and prioritise known and newly implicated autoimmune risk genes with potential roles in this clinically important cell type.

genetics↗

Flexible and efficient handling of nanopore sequencing signal data with slow5tools

BackgroundNanopore sequencing is an emerging technology that is being rapidly adopted in research and clinical genomics. We recently developed SLOW5, a new file format for storage and analysis of raw data from nanopore sequencing experiments. SLOW5 is a community-centric, open source format that offers considerable performance benefits over the existing nanopore data format, known as FAST5. Here we introduce slow5tools, a simple, intuitive toolkit for handling nanopore raw signal data in SLOW5 format. ResultsSlow5tools enables lossless FAST5-to-SLOW5 and SLOW5-to-FAST5 data conversion, and a range of tools for structuring, indexing, viewing and querying SLOW5 files. Slow5tools uses multi-threading, multi-processing and other engineering strategies to achieve fast data conversion and manipulation, including live FAST5-to-SLOW5 conversion during sequencing. We outline a series of examples and benchmarking experiments to illustrate slow5tools usage, and describe the engineering principles underpinning its high performance. ConclusionSlow5tools is an essential toolkit for handling nanopore signal data, which was developed to support adoption of SLOW5 by the nanopore community. Slow5tools is written in C/C++ with minimal dependencies and is freely available as an open-source program under an MIT licence: https://github.com/hasindu2008/slow5tools.

bioinformatics↗

maxATAC: genome-scale transcription-factor binding prediction from ATAC-seq with deep neural networks

Transcription factors read the genome, fundamentally connecting DNA sequence to gene expression across diverse cell types. Determining how, where, and when TFs bind chromatin will advance our understanding of gene regulatory networks and cellular behavior. The 2017 ENCODE-DREAM in vivo Transcription-Factor Binding Site (TFBS) Prediction Challenge highlighted the value of chromatin accessibility data to TFBS prediction, establishing state-of-the- art methods for TFBS prediction from DNase-seq. However, the more recent Assay-for- Transposase-Accessible-Chromatin (ATAC)-seq has surpassed DNase-seq as the most widely- used chromatin accessibility profiling method. Furthermore, ATAC-seq is the only such technique available at single-cell resolution from standard commercial platforms. While ATAC-seq datasets grow exponentially, suboptimal motif scanning is unfortunately the most common method for TFBS prediction from ATAC-seq. To enable community access to state-of-the-art TFBS prediction from ATAC-seq, we (1) curated an extensive benchmark dataset (127 TFs) for ATAC-seq model training and (2) built "maxATAC", a suite of user-friendly, deep neural network models for genome-wide TFBS prediction from ATAC-seq in any cell type. With models available for 127 human TFs, maxATAC is the first collection of high-performance TFBS prediction models for ATAC-seq. maxATAC performance extends to primary cells and single-cell ATAC-seq, enabling improved TFBS prediction in vivo. We demonstrate maxATACs capabilities by identifying TFBS associated with allele-dependent chromatin accessibility at atopic dermatitis genetic risk loci. Author SummaryProteins called transcription factors interpret the genome, reading both DNA sequence and chromatin state, to orchestrate gene expression across the diversity of human cell types. In any given cell type, most chromatin is "inaccessible", and only those parts of the genetic code needed or likely to be needed soon are "accessible" for transcription factor binding to affect gene expression and cellular behavior. Hundreds of transcription factors are expressed in a given cell type and context (e.g., age, disease), and knowledge of their context-specific DNA binding sites is key to uncovering how transcription factors regulate cellular behaviors in health or disease. However, experimentally profiling the >1,600 human transcription factors across all cell types and contexts is infeasible. We built a suite of computational models "maxATAC" to predict transcription factor binding from a measurement of accessible chromatin, ATAC-seq. Importantly, ATAC-seq is feasible even at single-cell resolution. Thus, this data type, in combination with maxATAC, can be used to infer transcription factor binding sites in directly-relevant cell types isolated from physiological and disease settings, enabling insights into disease mechanisms, including how genetic variants and cellular context impact transcription factor binding, gene expression patterns and disease risk.

bioinformatics↗

Epigenetic and Transcriptional Dysregulation in CD4+ T cells of Patients with Atopic Dermatitis

Atopic dermatitis (AD) is one of the most common skin disorders in children. Disease etiology involves genetic and environmental factors, with the 29 independent AD risk loci enriched for risk allele-dependent gene expression in the skin and CD4+ T cell compartments. We investigated epigenetic mechanisms that may account for genetic susceptibility in CD4+ T cells. To understand gene regulatory activity differences in peripheral blood T cells in AD, we measured chromatin accessibility (ATAC-seq), NFKB1 binding (ChIP-seq), and gene expression (RNA-seq) in stimulated CD4+ T cells from subjects with active moderate-to-severe AD and age-matched, non-allergic controls. Open chromatin regions in stimulated CD4+ T cells were highly enriched for AD genetic risk variants, with almost half of AD risk loci overlapping with AD-dependent ATAC-seq peaks. AD-specific open chromatin regions were strongly enriched for NF{kappa}B DNA binding motifs. ChIP-seq identified hundreds of NFKB1-occupied genomic loci that were AD-specific or Control-specific. As expected, the AD-specific ChIP-seq peaks were strongly enriched for NF{kappa}B DNA binding motifs. Surprisingly, Control-specific NKFB1 ChIP-seq peaks were not enriched for NFKB1 motifs, instead containing motifs for other classes of human TFs, suggesting a mechanism involving altered indirect NFKB1 binding. Using DNA sequencing data, we identified 63 instances of genotype-dependent chromatin accessibility at 36 AD risk variants (30% of AD risk loci) that could lead to genotype-dependent expression at these loci. We propose that CD4+ T cells respond to stimulation in an AD-specific manner, resulting in disease and genotype-dependent chromatin accessibility involving NFKB binding. AUTHOR SUMMARYStimulated CD4+ T cells from patients with atopic dermatitis have disease-dependent regulation of how gene expression is regulated. This regulation is disease dependent and the way the DNA is accessible and the transcription factor NFKB1 binds is enriched for genetic risk variants. Clinically, the CD4+ T cells in the peripheral blood of patients with AD respond to stimulation in a disease and genotype-dependent manner.

genomics↗

SLOW5: a new file format enables massive acceleration of nanopore sequencing data analysis

Nanopore sequencing is an emerging genomic technology with great potential. However, the storage and analysis of nanopore sequencing data have become major bottlenecks preventing more widespread adoption in research and clinical genomics. Here, we elucidate an inherent limitation in the file format used to store raw nanopore data - known as FAST5 - that prevents efficient analysis on high-performance computing (HPC) systems. To overcome this, we have developed SLOW5, an alternative file format that permits efficient parallelisation and, thereby, acceleration of nanopore data analysis. For example, we show that using SLOW5 format, instead of FAST5, reduces the time and cost of genome-wide DNA methylation profiling by an order of magnitude on common HPC systems, and delivers consistent improvements on a wide range of different architectures. With a simple, accessible file structure and a ~25% reduction in size compared to FAST5, SLOW5 format will deliver substantial benefits to all areas of the nanopore community.

genomics↗

Topoisomerase 1 inhibition therapy protects against SARS-CoV-2-induced inflammation and death in animal models.

The ongoing pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is currently affecting millions of lives worldwide. Large retrospective studies indicate that an elevated level of inflammatory cytokines and pro-inflammatory factors are associated with both increased disease severity and mortality. Here, using multidimensional epigenetic, transcriptional, in vitro and in vivo analyses, we report that Topoisomerase 1 (Top1) inhibition suppresses lethal inflammation induced by SARS-CoV-2. Therapeutic treatment with two doses of Topotecan (TPT), a FDA-approved Top1 inhibitor, suppresses infection-induced inflammation in hamsters. TPT treatment as late as four days post-infection reduces morbidity and rescues mortality in a transgenic mouse model. These results support the potential of Top1 inhibition as an effective host-directed therapy against severe SARS-CoV-2 infection. TPT and its derivatives are inexpensive clinical-grade inhibitors available in most countries. Clinical trials are needed to evaluate the efficacy of repurposing Top1 inhibitors for COVID-19 in humans.

microbiology↗

Runx1 Shapes the Chromatin Landscape Via a Cascade of Direct and Indirect Targets

Runt-related transcription factor 1 (Runx1) can act as both an activator and a repressor. Here we show that CRISPR-mediated deletion of Runx1 in an embryonic kidney-derived cell (mK4) results in large-scale genome-wide changes to chromatin accessibility and gene expression. Open chromatin regions near down-regulated loci are enriched for Runx sites, remain bound by Runx2, but lose chromatin accessibility and expression in Runx1 knockout cells. Unexpectedly, regions near upregulated genes are depleted of Runx sites and are instead enriched for Zeb transcription factor binding sites. Re-expressing Zeb2 in Runx1 knockout cells restores suppression. These data confirm that Runx1 activity is uniquely needed to maintain open chromatin at many loci, and demonstrate that genome-scale derepression is an indirect consequence of losing Runx1-dependent Zeb expression.

genomics↗

Epstein-Barr virus nuclear antigen 2 (EBNA2) extensively rewires the human chromatin landscape at autoimmune risk loci

The interplay between environmental and genetic factors plays a key role in the development of many autoimmune diseases. In particular, the Epstein-Barr virus (EBV) is an established contributor to multiple sclerosis, lupus, and other disorders. Previously, we demonstrated that the EBV nuclear antigen 2 (EBNA2) transactivating protein occupies up to half of the risk loci for a set of seven autoimmune disorders. To further examine the mechanistic roles played by EBNA2 at these loci on a genome-wide scale, we globally examined gene expression, chromatin accessibility, chromatin looping, and EBNA2 binding, in a B cell line that was 1) uninfected, 2) infected with a strain of EBV lacking EBNA2, or 3) infected with a strain that expresses EBNA2. We identified >400 EBNA2-dependent differentially expressed human genes and >5,000 EBNA2 binding events in the human genome. ATAC-seq analysis revealed >2,000 regions in the human genome with EBNA2-dependent chromatin accessibility, and HiChIP-seq data revealed >1,700 regions where EBNA2 altered chromatin looping interactions. Importantly, autoimmune genetic risk loci were highly enriched at the sites of these EBNA2-dependent chromatin-altering events. We present examples of autoimmune risk genotype-dependent EBNA2 events, nominating genetic risk mechanisms for autoimmune risk loci such as ZMIZ1. Taken together, our results reveal important interactions between host genetic variation and EBNA2-driven disease mechanisms. Further, our study highlights a critical role for EBNA2 in rewiring human gene regulatory programs through rearrangement of the chromatin landscape and nominates these interactions as components of genetic mechanisms that influence the risk of multiple autoimmune diseases.

genomics↗