Biology subjects

Lareau, C. A.

Publications and source records attributed to Lareau, C. A..

At least 19 recordsLinked to original sources

bioRxiv2026

A dish-to-biobank framework links β-cell nutrient-stress programs to genetic and dietary risk for Type 2 Diabetes

Type 2 diabetes (T2D) arises from genetic susceptibility and chronic metabolic stress, but whether these converge on shared molecular programs in human populations remains unclear. Here, we develop a dish-to-biobank framework linking controlled {beta}-cell perturbation to population-scale disease genetics through the circulating plasma proteome, and apply it to T2D. scRNA-seq of human stem cell-derived islets under factorial glucose and palmitate exposure identifies their combination (glucolipotoxicity) as the condition eliciting the strongest SC-{beta} cell transcriptional response, with glucolipotoxicity-upregulated genes uniquely enriched for T2D heritability, monogenic diabetes genes, and rare-variant burden signals. CRISPR knockout of {beta}-cell identity regulators PAX6 and PDX1 aligns with this program, establishing convergence of environmental and genetic perturbations on a shared disease-relevant state. We then used the plasma proteome as an accessible population-scale readout of these experimentally defined {beta}-cell stress programs, scoring 45,956 UK Biobank White British participants. We define heritable stress signatures that associate with refined carbohydrate and saturated fat intake, and undergo trans-tissue genetic regulation, with a subset of variants showing diet-dependent effects. Together, these findings establish glucolipotoxicity as a genetically anchored model of {beta}-cell dysfunction and provide a generalizable framework for linking controlled cellular perturbations to human disease genetics at population scale.

genetics↗

bioRxiv2026

Resolving human neuronal herpesvirus reactivation via petabase-scale association studies

Mounting evidence implicates herpesvirus reactivation in the etiology of Alzheimers disease, yet we lack a refined molecular characterization of pathogenesis in neurodegeneration. Here, we mine over 10 petabytes of human sequencing data for viral transcripts, identifying recurrent herpes simplex virus 1 (HSV-1) reactivation in healthy but not pathological post-mortem human brain tissue. Integrative single-nucleus analyses resolve direct evidence of HSV-1 expression in RORB+ glutamatergic neurons, implicating viral reactivation in a neuronal population progressively lost during dementia.

genomics↗

bioRxiv2026

Scalable genotyping in fixed transcriptomes resolves clonal heterogeneity via single-cell sequencing

Despite the promise of single-cell transcriptomics for understanding cell states in heterogeneous populations, widely used platforms have limited ability to link transcriptional states to somatic mutations within the same cells. Here, we introduce Genotyping in Fixed Transcriptomes (GIFT) for the simultaneous detection of large numbers of targeted genetic variants with whole transcriptome profiles in single cells. The core innovation of GIFT is a rationally designed gapfilling reaction between adjacent single-stranded DNA (ssDNA) probes that barcodes native transcript sequence to enable highly-specific targeted mutation detection. GIFT achieves greater than 99% genotyping accuracy and flexible capture of hundreds of mutations per cell, including in formalin-fixed, paraffin-embedded (FFPE) tissue, enabling clonal lineage tracing in heterogeneous settings. We demonstrate the unique scalability of GIFT by profiling more than 700,000 cells from 35 donors with myeloproliferative neoplasms (MPN), revealing mutation-dependent hematopoietic responses to systemic inflammation associated with the characteristic JAK2V617 mutation, including an allelic dose gradient of interferon-associated transcriptional programs and priming of hematopoietic stem cells that develop into divergent disease states. The technical advantages of GIFT enable direct resolution of genotype-to-phenotype relationships via clonal tracing with comprehensive cell-state measurements at single-cell resolution.

genomics↗

bioRxiv2025

Clonal lineage tracing of innate immune cells in human cancer

Innate immune cells constitute the majority of the tumor microenvironment (TME), where they mediate both natural anti-tumor immunity and immunotherapy responses. While single-cell T- and B-cell receptor sequencing has provided fundamental insights into the clonal dynamics of human adaptive immunity, the lack of appropriate tools has precluded similar analysis of innate immune cells. Here, we describe a method that leverages somatic mitochondrial DNA (mtDNA) mutations to reconstruct clonal lineage relationships between single cells across cell types in native human tissues. We jointly sequenced single-cell transposase-accessible chromatin and mtDNA to profile n=124,958 cells from matched tumor, non-involved lung tissue (NILT), and peripheral blood of early-stage non-small cell lung cancer (NSCLC) patients, as well as n=93,757 cells from matched tumor and peripheral blood of ovarian cancer patients. Single-cell concomitant profiling of lineage and cell states of thousands of immune cells resolved clonality across cell types, tissue sites, and malignancies. Clonal tracing of innate immune cells demonstrates that TME-resident myeloid subsets, including macrophages and type 3 dendritic cells (DC3), are clonally linked to both circulating and tissue-infiltrating monocytes. Further, we identify distinct DC-biased and macrophage-biased myeloid clones, enriched in the tumor and NILT, respectively, and find that their circulating monocyte precursors exhibit distinct epigenetic profiles, suggesting that myeloid differentiation fate may be predetermined before TME infiltration. These results delineate the clonal pathways of intratumoral myeloid cell recruitment and differentiation in human cancer and suggest that remodeling of the tumor myeloid compartment may be peripherally programmed.

genomics↗

bioRxiv2025

Population-scale sequencing resolves correlates and determinants of latent Epstein-Barr Virus infection

Epstein-Barr Virus (EBV) is an endemic herpesvirus implicated in autoimmunity, cancer, and neurological disorders. Though primary infection typically resolves with subclinical symptoms, long-term complications can arise due to immune dysregulation or viral latency, in which EBV DNA is detectable in blood for decades. Despite the ubiquity of this virus, we have an incomplete understanding of the highly variable responses to EBV that range from asymptomatic infection to a trigger for severe disease. Here, we demonstrate that existing whole genome sequencing (WGS) data contains ample non-human DNA sequences to reconstruct a molecular biomarker of latent EBV infection consistent with orthogonal phenotypes, including viral serology. Using the UK Biobank (n = 490,560) and All of Us (n = 245,394), we uncover reproducible complex trait associations that nominate latent blood-derived EBV DNA as a respiratory, autoimmune, and cardiovascular disease biomarker. Further, we evaluate the genetic determinants of persistent EBV DNA via genome-wide and exome-wide association studies, uncovering protein-altering variants from 147 genes. Single-cell and pathway-scale enrichment analyses implicate variable antigen processing and presentation as a primary genetic determinant of latent EBV persistence, with gene programs expressed in B cells and antigen-presenting cells. Using predicted viral epitope presentation affinities, we implicate genetic variation in MHC class II as a key modulator of EBV DNA persistence. Our analyses demonstrate how existing WGS data can derive novel molecular biomarkers, which may generalize to dozens of viruses comprising the blood virome1.

genetics↗

bioRxiv2024

Artifacts in single-cell mitochondrial DNA mutation analyses misinform phylogenetic inference

Sequencing mitochondrial DNA (mtDNA) variants from single cells has resolved clonality and lineage in native human samples and clinical specimens. Prior work established that heteroplasmic mtDNA variants can be used to delineate clonality in hematopoiesis, but they have limited ability to reconstruct cellular phylogenies. However, a recent report by Weng et al. challenges the current paradigm by describing an unprecedented number of shared mtDNA variants between cells that reportedly resolve high-resolution phylogenetic trees. We re-examined the claims of Weng et al., and identified two major points of concern regarding this unprecedented connectedness. First, shared variants between cells are disproportionately detected in a single molecule per cell, and second, these variants are enriched 10-20-fold at the edges of mtDNA molecules, reminiscent of artifacts reported in other sequencing approaches. Further, our analyses show that pruning low support and likely artificial mtDNA variants removes nearly all of the reported phylogenetic structure. Thus, we strongly caution against using mtDNA variant calling workflows that rely on minimal evidence, including the computational pipeline introduced in Weng et al., as variants with high connectedness and low evidence are likely artifacts that lead to the construction of false phylogenies.

genomics↗

bioRxiv2024

Functional dissection of complex and molecular trait variants at single nucleotide resolution

Identifying the causal variants and mechanisms that drive complex traits and diseases remains a core problem in human genetics. The majority of these variants have individually weak effects and lie in non-coding gene-regulatory elements where we lack a complete understanding of how single nucleotide alterations modulate transcriptional processes to affect human phenotypes. To address this, we measured the activity of 221,412 trait-associated variants that had been statistically fine-mapped using a Massively Parallel Reporter Assay (MPRA) in 5 diverse cell-types. We show that MPRA is able to discriminate between likely causal variants and controls, identifying 12,025 regulatory variants with high precision. Although the effects of these variants largely agree with orthogonal measures of function, only 69% can plausibly be explained by the disruption of a known transcription factor (TF) binding motif. We dissect the mechanisms of 136 variants using saturation mutagenesis and assign impacted TFs for 91% of variants without a clear canonical mechanism. Finally, we provide evidence that epistasis is prevalent for variants in close proximity and identify multiple functional variants on the same haplotype at a small, but important, subset of trait-associated loci. Overall, our study provides a systematic functional characterization of likely causal common variants underlying complex and molecular human traits, enabling new insights into the regulatory grammar underlying disease risk.

genetics↗

bioRxiv2024

Transcript-specific enrichment enables profiling rare cell states via scRNA-seq

Single-cell genomics technologies have accelerated our understanding of cell-state heterogeneity in diverse contexts. Although single-cell RNA sequencing (scRNA-seq) identifies many rare populations of interest that express specific marker transcript combinations, traditional flow sorting limits our ability to enrich these populations for further profiling, including requiring cell surface markers with high-fidelity antibodies. Additionally, many single-cell studies require the isolation of nuclei from tissue, eliminating the ability to enrich learned rare cell states based on extranuclear protein markers. To address these limitations, we describe Programmable Enrichment via RNA Flow-FISH by sequencing (PERFF-seq), a scalable assay that enables scRNA-seq profiling of subpopulations from complex cellular mixtures defined by the presence or absence of specific RNA transcripts. Across immune populations (n = 141,227 cells) and fresh-frozen and formalin-fixed paraffin-embedded brain tissue (n = 29,522 nuclei), we demonstrate the sorting logic that can be used to enrich for cell populations via RNA-based cytometry followed by high-throughput scRNA-seq. Our approach provides a rational, programmable method for studying rare populations identified by one or more marker transcripts.

genomics↗

bioRxiv2024

Regulation of immune signal integration and memory by inflammation-induced chromosome conformation

3-dimensional (3D) genome conformation is central to gene expression regulation, yet our understanding of its contribution to rapid transcriptional responses, signal integration, and memory in immune cells is limited. Here, we study the molecular regulation of the inflammatory response in primary macrophages using integrated transcriptomic, epigenomic, and chromosome conformation data, including base pair-resolution Micro-Capture C. We demonstrate that interleukin-4 (IL-4) primes the inflammatory response in macrophages by stably rewiring 3D genome conformation, juxtaposing endotoxin-, interferon-gamma-, and dexamethasone-responsive enhancers in close proximity to their cognate gene promoters. CRISPR-based perturbations of enhancer-promoter contacts or CCCTC-binding factor (CTCF) boundary elements demonstrated that IL-4-driven conformation changes are indispensable for enhanced and synergistic endotoxin-induced transcriptional responses, as well as transcriptional memory following stimulus removal. Moreover, transcriptional memory mediated by changes in chromosome conformation often occurred in the absence of changes in chromatin accessibility or histone modifications. Collectively, these findings demonstrate that rapid and memory transcriptional responses to immunological stimuli are encoded in the 3D genome.

genomics↗

bioRxiv2023

Multi-modal skin atlas associates a multicellular immune-stromal community with altered cornification and T cell expansion in atopic dermatitis

In healthy skin, a cutaneous immune system maintains the balance between tolerance towards innocuous environmental antigens and immune responses against pathological agents. In atopic dermatitis (AD), barrier and immune dysfunction result in chronic tissue inflammation. Our understanding of the skin tissue ecosystem in AD remains incomplete with regard to the hallmarks of pathological barrier formation, and cellular state and clonal composition of disease-promoting cells. Here, we generated a multi-modal cell census of 310,691 cells spanning 86 cell subsets from whole skin tissue of 19 adult individuals, including non-lesional and lesional skin from 11 AD patients, and integrated it with 396,321 cells from four studies into a comprehensive human skin cell atlas in health and disease. Reconstruction of human keratinocyte differentiation from basal to cornified layers revealed a disrupted cornification trajectory in AD. This disrupted epithelial differentiation was associated with signals from a unique immune and stromal multicellular community comprised of MMP12+ dendritic cells (DCs), mature migratory DCs, cycling ILCs, NK cells, inflammatory CCL19+ IL4I1+ fibroblasts, and clonally expanded IL13+IL22+IL26+ T cells with overlapping type 2 and type 17 characteristics. Cell subsets within this immune and stromal multicellular community were connected by multiple inter-cellular positive feedback loops predicted to impact community assembly and maintenance. AD GWAS gene expression was enriched both in disrupted cornified keratinocytes and in cell subsets from the lesional immune and stromal multicellular community including IL13+IL22+IL26+ T cells and ILCs, suggesting that epithelial or immune dysfunction in the context of the observed cellular communication network can initiate and then converge towards AD. Our work highlights specific, disease-associated cell subsets and interactions as potential targets in progression and resolution of chronic inflammation.

molecular biology↗

bioRxiv2023

Codon affinity in mitochondrial DNA shapes evolutionary and somatic fitness

Summary ParagraphSomatic variation contributes to biological heterogeneity by modulating cellular proclivity to differentiate, expand, adapt, or die. While large-scale sequencing efforts have revealed the foundational role of somatic variants to drive human tumor evolution, our understanding of the contribution of mutations to modulate cellular fitness in non-malignant contexts remains understudied. Here, we identify a mosaic synonymous variant (m.7076A>G) in the mitochondrial DNA (mtDNA) encoded cytochrome c-oxidase subunit 1 gene (MT-CO1, p.Gly391=), which was present at homoplasmy in 47% of immune cells from a healthy donor. Using single-cell multi-omics, we discover highly specific selection against the m.7076G mutant allele in the CD8+ effector memory T cell compartment in vivo, reminiscent of selection observed for pathogenic mtDNA alleles1, 2 and indicative of lineage-specific metabolic requirements. While the wildtype m.7076A allele is translated via Watson-Crick-Franklin base-pairing, the anticodon diversity of the mitochondrial transfer RNA pool is limited, requiring wobble-dependent translation of the m.7076G mutant allele. Notably, mitochondrial ribosome profiling revealed altered codon-anticodon affinity at the wobble position as evidenced by stalled translation of the synonymous m.7076G mutant allele encoding for glycine. Generalizing this observation, we provide a new ontogeny of the 8,482 synonymous variants in the human mitochondrial genome that enables interpretation of functional mtDNA variation. Specifically, via inter- and intra-species evolutionary analyses, population-level complex trait associations, and the occurrence of germline and somatic mtDNA mutations from large-scale sequencing studies, we demonstrate that synonymous variation impacting codon:anticodon affinity is actively evolving across the entire mitochondrial genome and has broad functional and phenotypic effects. In summary, our results introduce a new ontogeny for mitochondrial genetic variation and support a model where organismal principles can be discerned from somatic evolution via single-cell genomics.

genetics↗

bioRxiv2022

Single-cell multi-omics reveals dynamics of purifying selection of pathogenic mitochondrial DNA across human immune cells

Cells experience intrinsic and extrinsic pressures that affect their proclivity to expand and persist in vivo. In congenital disorders caused by loss-of-function mutations in mitochondrial DNA (mtDNA), metabolic vulnerabilities may result in cell-type specific phenotypes and depletion of pathogenic alleles, contributing to purifying selection. However, the impact of pathogenic mtDNA mutations on the cellular hematopoietic landscape is not well understood. Here, we establish a multi-omics approach to quantify deletions in mtDNA alongside cell state features in single cells derived from Pearson syndrome patients. We resolve the interdependence between pathogenic mtDNA and lineage, including purifying selection against deletions in effector/memory CD8 T-cell populations and recent thymic emigrants and dynamics in other hematopoietic populations. Our mapping of lineage-specific purifying selection dynamics in primary cells from patients carrying pathogenic heteroplasmy provides a new perspective on recurrent clinical phenotypes in mitochondrial disorders, including cancer and infection, with potential broader relevance to age-related immune dysfunction.

genetics↗

bioRxiv2022

Latent human herpesvirus 6 is reactivated in chimeric antigen receptor T cells

Cell therapies have yielded durable clinical benefits for patients with cancer, but the risks associated with the development of therapies from manipulated human cells are still being understood. For example, we currently lack a comprehensive understanding of the mechanisms of neurotoxicity observed in patients receiving T cell therapies, including recent reports of encephalitis caused by human herpesvirus 6 (HHV-6) reactivation1. Here, via petabase-scale viral RNA data mining, we examine the landscape of human latent viral reactivation and demonstrate that HHV-6B can become reactivated in human CD4+ T cells in standard in vitro cultures. Using single-cell sequencing, we identify a rare population of HHV-6 super-expressors (~1 in 300-10,000 cells) that possess high viral transcriptional activity in chimeric antigen receptor (CAR) T cell culture before spreading to infect other cells in vitro. Through the analysis of single-cell sequencing data from patients receiving cell therapy products that are FDA-approved2 or used in clinical studies3,4, we identify the presence of CAR+, HHV-6 super-expressor T cells in vivo. Together, our study implicates cell therapy products as a source of lytic HHV-6 reported in clinical trials1,5-7 and has broad implications for the design, production, and monitoring of cell therapies.

immunology↗

bioRxiv2021

Single-Cell Multiomics Defines Tolerogenic Extrathymic Aire-Expressing Populations with Unique Homology to Thymic Epithelium

The Autoimmune Regulator (Aire) gene, well defined for its role in medullary thymic epithelial cells (mTECs) and immune self-tolerance, is also expressed in extrathymic Aire-expressing cells (eTACs) in the secondary lymphoid organs. eTACs have been shown to be hematopoietic antigen presenting cells (APCs) and potent inducers of immune tolerance (1-3). However, the precise identity and function of these cells remain unclear. Here, we use high-dimensional single-cell multiomics and functional approaches to define eTACs at the transcriptional, genomic, and proteomic level. We find that eTACs consist of two similar cell types: CCR7+ Aire-expressing migratory dendritic cells (AmDCs) and a unique Aire-hi population co-expressing Aire and RAR-related orphan receptor gamma-t (ROR{gamma}t). The latter, which have significant transcriptional and genomic homology to migratory dendritic cells (migDCs) and mTECs, we term Janus cells (JCs). All eTACs, and JCs in particular, have a highly accessible chromatin structure and high levels of broad gene expression, including tissue-specific antigens, as well as remarkable transcriptional and genomic homology to thymic medullary epithelium. As in the thymus, Aire expression in eTACs is also dependent on RANK-RANK-ligand interactions. Furthermore, lineage-tracing shows that JCs are not precursors to the majority of AmDCs. Finally, self-antigen expression by eTACs is sufficient to mediate negative selection of T cells escaping thymic selection and can prevent autoimmune diabetes in non-obese diabetic mice. This transcriptional, genomic, and functional symmetry between a hematopoietic Aire-expressing population in the periphery and an epithelial Aire-expressing population in the thymus suggests that a core biological program may influence self-tolerance and self-representation across the spectrum of immune development.

immunology↗

bioRxiv2021

Transient dendritic cell activation diversifies the T cell response to acute infection

The precise timing of T cell priming during infection remains unclear. Here, we mapped the cellular dynamics of all immune lineages during acute infection with Listeria monocytogenes (Lm). We identified highly transient DC activation 2 days post-infection that functions as a critical time window for priming effector T cells. Regulation of this transient state was mediated by DC extrinsic IFN{gamma} provided by lymphocytes. Furthermore, antigen-specific T cells that arrive late to the site of priming and miss peak DC activation acquire only memory T cell fates. This temporal regulation of fate is recapitulated by CD8+ DCs ex vivo, suggesting that shifts in activation state of a single antigen presenting cell population alter T cell fates. These results uncover a novel mechanism for temporal regulation of T cell differentiation during a dynamic immune response to acute infection. One-Sentence SummaryThe immune system generates a balanced protective response through rapidly shifting activation states and recruitment of new cells into an ongoing inflammatory landscape.

immunology↗

bioRxiv2021

Functional Inference of Gene Regulation using Single-Cell Multi-Omics

Cells require coordinated control over gene expression when responding to environmental stimuli. Here, we apply scATAC-seq and scRNA-seq in resting and stimulated human blood cells. Collectively, we generate [~]91,000 single-cell profiles, allowing us to probe the cis-regulatory landscape of immunological response across cell types, stimuli and time. Advancing tools to integrate multi-omic data, we develop FigR - a framework to computationally pair scATAC-seq with scRNA-seq cells, connect distal cis-regulatory elements to genes, and infer gene regulatory networks (GRNs) to identify candidate TF regulators. Utilizing these paired multi-omic data, we define Domains of Regulatory Chromatin (DORCs) of immune stimulation and find that cells alter chromatin accessibility prior to production of gene expression at time scales of minutes. Further, the construction of the stimulation GRN elucidates TF activity at disease-associated DORCs. Overall, FigR enables the elucidation of regulatory interactions across single-cell data, providing new opportunities to understand the function of cells within tissues.

genomics↗

bioRxiv2021

Mitochondrial variant enrichment from high-throughput single-cell RNA-seq resolves clonal populations

Reconstructing lineage relationships in complex tissues can reveal mechanisms underlying development and disease. Recent methods combine single-cell transcriptomics with mitochondrial DNA variant detection to establish lineage relationships in primary human cells, but are not scalable to interrogate complex tissues. To overcome this limitation, here we develop a technology for high-confidence detection of mitochondrial mutations from high-throughput single-cell RNA-sequencing. We use the new method to identify skewed immune cell expansions in primary human clonal hematopoiesis.

genomics↗

bioRxiv2020

Single-cell multimodal profiling of proteins and chromatin accessibility using PHAGE-ATAC

Multi-modal measurements of single cell profiles are a powerful tool for characterizing cell states and regulatory mechanisms. While current methods allow profiling of RNA along with either chromatin or protein levels, connecting chromatin state to protein levels remains a barrier. Here, we developed PHAGE-ATAC, a method that uses engineered camelid single-domain antibody ( nanobody)-displaying phages for simultaneous single-cell measurement of surface proteins, chromatin accessibility profiles, and mtDNA-based clonal tracing through a massively parallel droplet-based assay of single-cell transposase-accessible chromatin with sequencing (ATAC-seq). We demonstrate PHAGE-ATAC for multimodal analysis in primary human immune cells and for sample multiplexing. Finally, we construct a synthetic high-complexity phage library for selection of novel antigen-specific nanobodies that bind cells of particular molecular profiles, opening a new avenue for protein detection, cell characterization and screening with single-cell genomics.

genomics↗