Biology subjects

Humphreys, D. T.

Publications and source records attributed to Humphreys, D. T..

8 recordsLinked to original sources

bioRxiv2026

A scalable MNase-seq framework for reproducible nucleosome profiling across pluripotent stem cell and cardiomyocyte models

Micrococcal nuclease (MNase) digestion is widely used to profile chromatin accessibility and nucleosome footprinting. However, its application is often limited by sensitivity to reaction conditions, high cell input requirements, and the lack of standardized protocols across cell types. Here we developed a robust MNase workflow encompassing buffer composition, DNA purification chemistry, fixation and decrosslinking parameters, cell input scalability, and an in-house yeast spike-in for quantitative normalization. We validated this unified framework across human induced pluripotent stem cells (hiPSCs), hiPSC-derived cardiomyocytes at multiple differentiation stages, primary murine embryonic cardiac cells, and adult mouse cardiomyocytes, and demonstrated comparable digestion efficiencies and kinetics despite marked differences in cellular architecture and chromatin organization. Genome-wide MNase-seq in hiPSCs, combined with the nucMACC bioinformatic pipeline, resolved concentration-dependent nucleosomal occupancy and precise nucleosome positioning at pluripotency-related regulatory elements. This modular, end-to-end, and scalable workflow provides a standardized platform for reproducible MNase-based chromatin profiling across diverse in vitro and in vivo models. TEASERA unified, rapid, and scalable MNase-seq workflow for reproducible mononucleosomal and subnucleosomal profiling from stem cells to adult cardiomyocytes. HIGHLIGHTSO_LISystematic MNase optimization across buffer, DNA purification, and cell input variables C_LIO_LIUnified workflow validated in hiPSCs, hiPSC-CMs, embryonic, and adult cardiomyocytes C_LIO_LIFixed-cell protocol enables weeks of storage without loss of DNA quality C_LIO_LICost-effective yeast spike-in ensures quantitative normalization for MNase-seq C_LIO_LIGenome-wide analyses confirm robust and precise nucleosome positioning at regulatory elements C_LI

genomics↗

bioRxiv2024

Aging limits stemness and tumorigenesis in the lung by reprogramming iron homeostasis

Aging is associated with a decline in the number and fitness of adult stem cells1-4. Aging-associated loss of stemness is posited to suppress tumorigenesis5,6, but this hypothesis has not been tested in vivo. Here, using physiologically aged autochthonous genetically engineered mouse models and primary cells7,8, we demonstrate aging suppresses lung cancer initiation and progression by degrading stemness of the alveolar cell of origin. This phenotype is underpinned by aging-associated induction of the transcription factor NUPR1 and its downstream target lipocalin-2 in the cell of origin in mice and humans, leading to a functional iron insufficiency in the aged cells. Genetic inactivation of the NUPR1--lipocalin-2 axis or iron supplementation rescue stemness and promote tumorigenic potential of aged alveolar cells. Conversely, targeting the NUPR1-- lipocalin-2 axis is detrimental to young alveolar cells via induction of ferroptosis. We find that aging-associated DNA hypomethylation at specific enhancer sites associates with elevated NUPR1 expression, which is recapitulated in young alveolar cells by inhibition of DNA methylation. We uncover that aging drives a functional iron insufficiency, which leads to loss of stemness and tumorigenesis, but promotes resistance to ferroptosis. These findings have significant implications for the therapeutic modulation of cellular iron homeostasis in regenerative medicine and in cancer prevention. Furthermore, our findings are consistent with a model whereby most human cancers initiate in young individuals, revealing a critical window for such cancer prevention efforts.

cancer biology↗

bioRxiv2024

Impaired yolk sac NAD metabolism disrupts murine embryogenesis with relevance to human birth defects

Congenital malformations can originate from numerous genetic or non-genetic factors but in most cases the causes are unknown. Genetic disruption of nicotinamide adenine dinucleotide (NAD) de novo synthesis causes multiple malformations, collectively termed Congenital NAD Deficiency Disorder (CNDD), highlighting the necessity of this pathway during embryogenesis. Previous work in mice shows that NAD deficiency perturbs embryonic development specifically when organs are forming. While the pathway is predominantly active in the liver postnatally, the site of activity prior to and during organogenesis is unknown. Here, we used a mouse model of human CNDD and assessed pathway functionality in embryonic livers and extraembryonic tissues via gene expression, enzyme activity and metabolic analyses. We found that the extra-embryonic visceral yolk sac endoderm exclusively synthesises NAD de novo during early organogenesis before the embryonic liver takes over this function. Under CNDD-inducing conditions, visceral yolk sacs had reduced NAD levels and altered NAD-related metabolic profiles, affecting embryo metabolism. Expression of requisite pathway genes is conserved in the equivalent yolk sac cell type in humans. Our findings show that visceral yolk sac-mediated NAD de novo synthesis activity is essential for mouse embryogenesis and its perturbation causes CNDD. As mouse and human yolk sacs are functionally homologous, our data improve the understanding of human congenital malformation causation.

developmental biology↗

bioRxiv2024

A Bag-Of-Motif Model Captures Cell States at Distal Regulatory Sequences

Deciphering the intricate regulatory code governing cell-type-specific gene expression is a fundamental goal in genetics. Current methods struggle to capture the complex interplay between gene distal regulatory sequences and cell context. We developed a computational approach, BOM (Bag-of-Motifs), which represents cis-regulatory sequences by the type and number of TF binding motifs it contains, irrespective of motif order, orientation, and spacing. This simple yet powerful representation allows BOM to efficiently capture the complexity of cell-type-specific information encoded within these sequences. We apply BOM to mouse, human, and zebrafish distal regulatory regions, demonstrating remarkable accuracy. Notably, the method outperforms more complex deep learning models at the same task using fewer parameters. BOM can also uncover cross-species sequence similarities unrecognized by genome alignments. We experimentally validate our in silico predictions using enhancer reporter assay, showing that motifs with the most significant explanatory power are sequence determinants of cell-type specific enhancer activity. BOM offers a novel systematic framework for studying cell-type or condition-specific cis-regulatory sequences. Using BOM, we demonstrate the existence of a highly predictive sequence code at distal regulatory regions in mammals driven by TF binding motifs.

bioinformatics↗

bioRxiv2023

Single cell sequencing data identify distinct B cell and fibroblast populations in stricturing Crohns disease

We used human full thickness Crohns disease (CD) small bowel resection specimens and single cell RNA sequencing to identify potential therapeutic targets for stricturing (S)CD. Using an unbiased approach, 16 cell lineages were assigned within 14,539 sequenced cells from patient-matched SCD and non-stricturing (NSCD) preparations. SCD and NSCD contained identical cell types. Amongst immune cells, B cells and plasma cells were selectively increased in SCD samples. B cell subsets suggested formation of tertiary lymphoid tissue in SCD and compared with NSCD there was an increase in IgG, and a decrease in IgA plasma cells, consistent with their potential role in CD fibrosis. Two Lumican-positive fibroblast subtypes were identified and subclassified based on expression of selectively enriched genes as fibroblast clusters (C)12 and C9. Cells within these clusters expressed the profibrotic genes Decorin (C12) and JUN (C9). C9 cells expressed ACTA2; ECM genes COL4A1, COL4A2, COL15A1, COL6A3, COL18A1 and ADAMDEC1; LAMB1 and GREM1. GO and KEGG Biological terms showed extracellular matrix, stricture organisation and regulation of WNT pathway genes are associated with the C12 and C9 gene sets. Trajectory and differential gene analysis of C12 and C9 identified four sub-clusters. Intra sub-cluster gene analysis detected co-regulated gene modules that aligned along predicted pseudotime trajectories and identified CXCL14 and ADAMDEC1 as key module markers. Our findings support further investigation of fibroblast heterogeneity and interactions with local and circulating immune cells at earlier time points in fibrosis progression. Breaking these interactions by targeting one or other population may improve therapeutic management for SCD.

genomics↗

bioRxiv2022

Quantitative trait and transcriptome analysis of genetic complexity underpinning cardiac interatrial septation in mice using an advanced intercross line

Unlike single-gene mutations leading to Mendelian conditions, common human diseases are likely emergent phenomena arising from multilayer, multiscale and highly interconnected interactions. Atrial and ventricular septal defects are the most common forms of cardiac congenital anomalies in humans. Atrial septal defects (ASD) show an open communication between left and right atria postnatally, potentially resulting in serious hemodynamic consequences if untreated. A milder form of atrial septal defect, patent foramen ovale (PFO), exists in about one quarter of the human population, strongly associated with ischaemic stroke and migraine. The anatomic liabilities and genetic and molecular basis of atrial septal defects remain unclear. Here, we advance our previous analysis of atrial septal variation through quantitative trait locus (QTL) mapping of an advanced intercross line (AIL) established between the inbred QSi5 and 129T2/SvEms mouse strains, that show extremes of septal phenotypes. Analysis resolved 37 unique septal QTL with high overlap between QTL for distinct septal traits. Whole genome sequencing of parental strains identified high confidence candidate deleterious variants, including in known human congenital heart disease genes, whereas transcriptome analysis of developing septa revealed networks involving ribosome, nucleosome, mitochondrial and extracellular matrix biosynthesis underlying septal variation. Analysis of variant architecture across different gene features, including enhancers and promoters, provided evidence for involvement of non-coding as well as protein coding variants. Our study provides the first high resolution picture of genetic complexity and network liability underlying common congenital heart disease, with relevance to human ASD and PFO.

genetics↗

bioRxiv2021

A standardized method to purify cardiomyocytes from individual mouse hearts of any age

Primary cardiomyocytes are invaluable for understanding postnatal heart development. However, a universal method to obtain freshly purified cardiomyocytes without using different agedependent isolation procedures and cell culture, is lacking. Here, we report the development of a standardised method that allows rapid isolation and purification of high-quality cardiomyocytes from individual neonatal through to adult C57BL/6J murine hearts. Langendorff retrograde perfusion, which is currently limited to adult hearts, was adapted for use in neonatal and infant hearts by developing an easier in situ aortic cannulation technique. Tissue digestion conditions were optimised to achieve efficient digestion of hearts of all ages in a comparable timeframe (<14 min). This resulted in a high yield (1.56-2.2x106 cells/heart) and viability (~70-100%) of cardiomyocytes post-isolation. An immunomagnetic cell separation step was then applied to yield highly purified cardiomyocytes (~95%) as confirmed by immunocytochemistry, flow cytometry, and qRT-PCR. For cell-type specific studies, cardiomyocyte DNA, RNA, and protein could be extracted in sufficient yields to conduct molecular experiments. We generated transcriptomic datasets for neonatal cardiomyocytes from individual hearts, for the first time, which revealed nine sex-specific genes (FDR<0.05) encoded on the sex chromosomes. Finally, we also developed an in situ fixation protocol that preserved the native cytoarchitecture of cardiomyocytes (~94% rodshaped post-isolation), and used it to evaluate cell morphology during cardiomyocyte maturation, as well as capture spindle-shaped neonatal cells undergoing cytokinesis. Together, these procedures allow molecular and morphological profiling of high-quality cardiomyocytes from individual hearts of any postnatal age.

cell biology↗

bioRxiv2018

Ularcirc: Visualisation and enhanced analysis of circular RNAs via back and canonical forward splicing

Circular RNAs (circRNAs) are a unique class of transcripts that can only be identified from sequence alignments spanning discordant junctions, commonly referred to as backsplice junctions (BSJ). The challenges of detecting a BSJ from short read high throughput sequencing (HTS) data has steered software development to focus primarily on algorithmic methods to accurately capture BSJs. Here we present Ularcirc, the first software tool that provides a complete circRNA workflow from detection, integrated visualization, quality filtering of BSJ and forward splicing junctions (FSJ), through to sequence retrieval and downstream functional analysis. More importantly, Ularcirc uses an innovative method to filter out false positive circRNAs coined read alignment distribution (RAD) score which allows detection of circRNAs independent of gene annotations. We used Ularcirc to characterise circRNAs from public and in-house generated data sets and demonstrate how to discover (i) novel splicing patterns of parental transcripts, (ii) internal splicing patterns of circRNA, and (iii) the complexity of BSJ formation. Furthermore, we identify circRNAs that have potential open reading frames longer than their linear sequence. Finally, we have identified and validated the presence of a novel class of circRNA generated from ApoA4 transcripts whose BSJ derive from multiple sites within coding exons. Ularcirc can be accessed via https://github.com/VCCRI/Ularcirc.

bioinformatics↗