Biology subjects

Johnson, I.

Publications and source records attributed to Johnson, I..

12 recordsLinked to original sources

bioRxiv2026

Low-heteroplasmy mitochondrial DNA mutations improve clonal reconstruction of human cells

Reconstructing clonal relationships among human cells is fundamental to understanding development, aging, and disease. Somatic mitochondrial DNA (mtDNA) mutations act as endogenous single-cell barcodes measurable alongside cell-state profiles, but lineage tracing has traditionally focused on high-heteroplasmy variants, which are easier to detect but few and potentially shaped by selection. Whether the more abundant lower-heteroplasmy variants encode bona fide lineage information has not been tested against an independent clonal reference. Using lentiviral barcoding of human hematopoietic cells to establish ground-truth clone identities, we show that after stringent molecule-level error filtering, mutation calls below 10% per-cell heteroplasmy account for roughly half of all lineage-informative calls. Retaining the full heteroplasmy spectrum approximately doubled the clonal-assignment area under the precision-recall curve relative to a >10% cutoff, and single-molecule-supported calls improved recovery when retained collectively. These findings establish lower-heteroplasmy mtDNA mutations as an abundant, bona fide record of clonal history, substantially expanding the clonal resolution attainable in human tissues without genetic engineering.

genomics↗

bioRxiv2026

Circadian Clock Programming of Anticipatory Antiviral Immunity Gates Enteric Virus Infection Susceptibility

Susceptibility to viral infection varies widely but is not fully explained by genetics, immune status, or exposure level. We show that time of day strongly influences infection outcome, with up to 100-fold differences in enteric viral burden depending on infection timing. This temporal gating is abolished in mice lacking a functional circadian clock. We identify the antiviral transcription factor IRF1 as a direct target of the circadian transcription factor BMAL1, resulting in rhythmic expression of a basal antiviral gene program prior to infection. Loss of IRF1 eliminates this program and abrogates time-of-day-dependent differences in viral replication. This circuit operates within intestinal myeloid cells, establishing a preexisting antiviral state. These findings indicate that the circadian clock programs host susceptibility in the intestine, before infection occurs.

immunology↗

bioRxiv2026

Modeling mitochondrial inheritance enables high-precision single-cell lineage tracing in humans

Somatic mutations in mitochondrial DNA (mtDNA) provide natural barcodes that enable engineering-free lineage tracing in human tissues, but the complex dynamics of mtDNA inheritance across cell divisions and incomplete sampling of mtDNA introduce uncertainty in reconstructed lineages. Here, we present MitoDrift, a probabilistic framework that integrates Wright-Fisher drift dynamics with sparse single-cell measurements to produce confidence-refined lineage trees enriched for accurate clonal relationships. Validation with gold-standard lentiviral barcoding and whole-genome sequencing demonstrates that MitoDrift outperforms existing tree reconstruction methods in precision while maintaining high clonal recovery, enabling robust analyses linking lineage to cell state. Applying MitoDrift to human hematopoiesis reveals an age-associated decline in clonal diversity with differential impact across cell types and identifies heritable regulatory programs in hematopoietic stem cells in vivo, linking AP-1/stress-associated programs to clonal expansions. In multiple myeloma, MitoDrift captures therapy-associated clonal remodeling undetectable by copy number analysis, revealing phenotypic transitions and linking gene regulatory programs to differential drug sensitivity. Collectively, MitoDrift enables high-precision lineage tracing at scale and establishes quantitative lineage-state analysis in primary human tissues, linking clonal history to transcriptional and epigenetic programs in tissue homeostasis, aging, and disease.

genomics↗

bioRxiv2025

Regenerative agriculture effects on biomass, drought resilience and 14C-photosynthate allocation in wheat drilled into ley compared to disc or ploughed arable soil

Regenerative agriculture practices including leys and no-tillage facilitate biological reassembly of soil aggregates, increasing water, carbon and nutrient storage, but how this effects crop biomass, photosynthate partitioning, and drought resilience is unclear. To address this, we took monoliths growing semi-dwarf and taller wheat genotypes from 3-year plots that were ploughed or disc cultivated, or direct-drilled into a grass-clover ley and applied 35 kg N ha-1. Half the monoliths received a spring drought, then all were watered and the wheat 14CO2 pulse-labelled at stem elongation. The ley soil had lower bulk density, stored more water, and the taller wheat genotype maintained 8-fold higher proportion of water-stable macroaggregates despite unexpectedly having the smaller root biomass. Yields on the ley soil (3.74 t ha-1) were 77% -123% higher than on ploughed and disc cultivated soils, and unaffected by genotype or drought, despite its >70% reduction in root biomass. Of the 14C initially retained in wheat, 72% was in shoots, with root allocation decreasing by 75-90% in droughted ley soil, and at harvest soil retained <1% of the 14C, with significantly lower values for taller wheat, and ley. We conclude that soil health regeneration in the ley enhanced wheat yields, but reduced photosynthate allocation to root biomass under drought. Although taller wheat maintained better macroaggregation in ley soil, this was not explained by root biomass or photosynthate allocation and unexpectedly failed to increase soil 14C sequestration. We find no evidence that ley regeneration of macroaggregation enhances soil C sequestration under wheat, despite higher yields.

plant biology↗

bioRxiv2025

An inducible tethered function assay reveals differential regulatory effects for the RNA-binding protein YBX3 and its domains.

RNA-binding protein (RBP) regulation is widespread across biology from development to learning and memory. Often RBPs contain multiple modular domains, which contribute to distinct RNA- binding activity or interactions necessary for regulation. However, assays to determine specific regulatory activity of individual domains are limited. The tethered function assay (TFA) provides a direct method to assess the functional activity of RBPs and their domains. The assay consists of two key components: (i) a reporter plasmid that encodes an mRNA, such as luciferase or GFP, engineered with high-affinity binding sites for an exogenous RNA-binding factor, and (ii) an effector construct that expresses a chimera of the RBP fused to the RNA-binding factor. Co- transfection of these constructs allows for measurement of reporter activity as a quantitative readout for RBP regulatory function. We developed an inducible TFA (iTFA) system through generation of a stable inducible mammalian cell line to express a reporter mRNA encoding nanoluciferase (nLuc) with six high-affinity MS2 binding sites within its 3 untranslated region. These cells can be transfected with a single plasmid that expresses an RBP fused to the MS2 coat protein. This approach enhances efficiency and reproducibility through reduction of transfection burden to a single plasmid and simplified normalization. We used this platform to dissect the individual and combined regulatory effects of YBX3 domains, a modular RBP with differential regulatory activity. The iTFA system provides a streamlined, tunable platform for functional analysis of RBPs that facilitates rapid interrogation of RBP or domain-specific activities in mammalian cells.

molecular biology↗

bioRxiv2024

Studies of infused megakaryocytes into mice support a ''catch-and-release'' model of pulmonary-centric thrombopoiesis

Many aspects of thrombopoiesis, the release of platelets from megakaryocytes (Mks), remain under debate, including where this process occurs. Murine lung in situ-microscopy studies suggested that a significant fraction of circulating platelets was released from lung-entrapped, marrow-derived Mks. We now confirm these that endogenous murine (m) Mks are immobilized in the lungs. Intravenously infused in vitro-differentiated, mMks and human (h) Mks are similarly entrapped, followed by shedding of their cytoplasm over [~]30 minutes with a peak number of released platelets occurring 1.5-4 hours later. However, while infused Mks from both species shed large intrapulmonary cytoplasmic fragments that underwent further processing into platelet-sized fragments, the two differed: many mMks escaped from and then recycled back to the lungs, while most hMks were enucleated upon first intrapulmonary passage. Infused immature hMks, inflammatory hMks, umbilical cord-blood-derived hMks and immortalized Mk progenitor cell (imMKCL)-derived hMks were also immobilized in the lung of recipient mice, and released their cytoplasm, but did so to different degrees. Pharmacologically-treated hMks support that membrane stiffness was key to pulmonary thrombopoiesis. Intraarterial infused hMks resulted in few Mks being immobilized in tissues other than the lungs and was accompanied by a blunted and delayed rise in circulating human platelets. These studies demonstrate that the lung entraps and processes both circulating Mks and released large cytoplasmic fragments consistent with a prior lung/heart murine study and support a pulmonary-centric "catch-and-release" model of thrombopoiesis. Thrombopoiesis is a drawn-out process with the majority of cytoplasmic processing of Mks occurring in the pulmonary bed. Key PointsO_LIInfused in vitro-differentiated megakaryocytes synchronously release cytoplasmic fragments selectively in the pulmonary bed. C_LIO_LILarge, released megakaryocyte fragments recycle to the lungs, undergo further fission, terminally forming platelets. C_LI

cell biology↗

bioRxiv2024

Aberrant TNF signaling in pancreatic lymph nodes of patients with Type 1 Diabetes

The therapeutic landscape for Type 1 Diabetes (T1D) is rapidly changing as ongoing clinical trials aim to delay beta-cell loss by inhibiting proinflammatory cytokines. However, the precise timing and cellular contexts of cytokine dysregulation remains unknown. We generated the largest existing measurement of gene expression and chromatin accessibility in [~]1 million immune cells from the pancreatic lymph nodes and spleens of 34 T1D and non-diabetic organ donors. Our study revealed heightened gene activity of the tumor necrosis factor (TNF) pathway and subsequent chromatin remodeling in central memory CD4+ T cells residing in the pancreatic lymph nodes of T1D and non-diabetic islet-autoantibody positive donors. These findings, validated in mice, offer a mechanism underlying the efficacy of TNF inhibitors, currently undergoing clinical trials to delay T1D onset.

immunology↗

bioRxiv2022

A single amino acid transporter controls the uptake of priming-inducing beta-amino acids and the associated trade-off between induced resistance and plant growth.

Selected beta-amino acids, such as beta-aminobutyric acid (BABA) and R-beta-homoserine (RBH), can prime plants for resistance against broad-spectrum diseases. Here, we describe a genome-wide screen of fully annotated Arabidopsis T-DNA insertion lines for impaired in RBH-induced immunity (iri) against the downy mildew pathogen Hyaloperonospora arabidopsidis, yielding 104 lines that were partially affected and 4 lines that were completely impaired in RBH-induced resistance. The iri1-1 mutant phenotype could be confirmed by an independent T-DNA insertion in the same gene, encoding the high-affinity amino acid transporter LHT1. Using uptake experiments with IRI1/LHT1-expressing yeast cells and mass spectrometry-based quantification of RBH and BABA in leaves of mutant and over-expression lines of IRI1/LHT1, we demonstrate that IRI1/LHT1 acts as the main transporter for cellular uptake and systemic distribution of RBH and BABA. Subsequent characterisation of mutant and over-expression lines of IRI1/LHT1 for induced resistance and growth responses revealed that the level of IRI1/LHT1 expression determines the trade-off between induced resistance and plant growth by RBH and BABA.

plant biology↗

bioRxiv2020

Enhanced specificity of high sensitivity somatic variant profiling in cell-free DNA via paired normal sequencing: design, validation, and clinical experience of the MSK-ACCESS liquid biopsy assay

Circulating cell-free DNA (cfDNA) from blood plasma of cancer patients can be used to interrogate somatic tumor alterations non-invasively or when adequate tissue is unavailable. We have developed and clinically implemented MSK-ACCESS (Analysis of Circulating cfDNA to Evaluate Somatic Status), an NGS assay for detection of very low frequency somatic alterations in select exons and introns of 129 genes. Analytical validation demonstrated 92% sensitivity in de-novo mutation calling down to 0.5% allele frequency and 98% for a priori mutation profiling. To evaluate the performance and utility of MSK-ACCESS, we report results from the first 681 prospective blood samples (617 patients) that underwent clinical analysis to guide patient management. Somatic mutations, copy number, and/or structural variants were detected in 73% of the samples, and 56% of these circulating-tumor DNA (ctDNA) positive samples had clinically actionable alterations. The utilization of matched white blood cell sequencing allowed retention of somatic alterations while filtering out over 10,000 germline and clonal hematopoiesis variants, thereby greatly enhancing the specificity of the assay. Taken together, our experience illustrates the importance of analyzing a matched normal sample when interpreting cfDNA results and highlights the potential of cfDNA profiling to guide treatment selection, monitor treatment response, and identify mechanisms of treatment resistance.

genomics↗

bioRxiv2019

Anisotropic ESCRT-III architecture governs helical membrane tube formation

ESCRT-III proteins assemble into ubiquitous membrane-remodeling polymers during many cellular processes. Here we describe the structure of helical membrane tubes that are scaffolded by bundled ESCRT-III filaments. Cryo-ET reveals how the shape of the helical membrane tube arises from the assembly of distinct bundles of protein filaments that bind the membrane with different mean curvatures. Cryo-EM reveals how one of these ESCRT-III filaments engages the membrane tube through a novel interface. Mathematical modeling of the helical membrane tube suggests how its shape emerges from differences in membrane binding energy, positional rigidity, and membrane tension. Altogether, our findings support a model in which increasing the rigidity of ESCRT-III filaments through the assembly of multi-strands triggers buckling of the membrane.\n\nOne Sentence SummaryESCRT-III heteropolymers deform membranes into helical tubes.

biochemistry↗

bioRxiv2017

Fast and Accurate Genomic Analyses using Genome Graphs

The human reference genome serves as the foundation for genomics by providing a scaffold for alignment of sequencing reads, but currently only reflects a single consensus haplotype, which impairs read alignment and downstream analysis accuracy. Reference genome structures incorporating known genetic variation have been shown to improve the accuracy of genomic analyses, but have so far remained computationally prohibitive for routine large-scale use. Here we present a graph genome implementation that enables read alignment across 2,800 diploid genomes encompassing 12.6 million SNPs and 4.0 million indels. Our Graph Genome Pipeline requires 6.5 hours to process a 30x coverage WGS sample on a system with 36 CPU cores compared with 11 hours required by the GATK Best Practices pipeline. Using complementary benchmarking experiments based on real and simulated data, we show that using a graph genome reference improves read mapping sensitivity and produces a 0.5% increase in variant calling recall, or about 20,000 additional variants being detected per sample, while variant calling specificity is unaffected. Structural variations (SVs) incorporated into a graph genome can be genotyped accurately under a unified framework. Finally, we show that iterative augmentation of graph genomes yields incremental gains in variant calling accuracy. Our implementation is a significant advance towards fulfilling the promise of graph genomes to radically enhance the scalability and accuracy of genomic analyses.

bioinformatics↗

bioRxiv2017

Conserved lipid and small molecule modulation of COQ8 reveals regulation of the ancient UbiB family

Human COQ8A (ADCK3) and Saccharomyces cerevisiae Coq8p (collectively COQ8) are UbiB family proteins essential for mitochondrial coenzyme Q (CoQ) biosynthesis. However, the biochemical activity of COQ8 and its direct role in CoQ production remain unclear, in part due to lack of known endogenous regulators of COQ8 function and of effective small molecules for probing its activity in vivo. Here we demonstrate that COQ8 possesses evolutionarily conserved ATPase activity that is activated by binding to membranes containing cardiolipin and by phenolic compounds that resemble CoQ pathway intermediates. We further create an analog-sensitive version of Coq8p and reveal that acute chemical inhibition of its endogenous activity in yeast is sufficient to cause respiratory deficiency concomitant with CoQ depletion. Collectively, this work defines lipid and small molecule modulators of an ancient family of atypical kinase-like proteins and establishes a chemical genetic system for further exploring the mechanistic role of COQ8 in CoQ biosynthesis.

biochemistry↗