Biology subjects

Knowles, J. W.

Publications and source records attributed to Knowles, J. W..

9 recordsLinked to original sources

bioRxiv2026

A positively selected microRNA controls a reversible aging program in striated muscle

Aging is the primary risk factor for most chronic diseases and is characterized in striated muscle by progressive functional decline, mitochondrial dysfunction, and chronic inflammation. The miR-128-1 locus resides within a positively selected haplotype on chromosome 2q21.3 associated with variation in grip strength, pulmonary function, and cardiometabolic traits in humans. Here, we show that antisense oligonucleotide-mediated inhibition of miR-128-3p restores muscle mass and function in aged mice, improves cardiac function while limiting adverse remodeling following myocardial infarction, and ameliorates skeletal and cardiac muscle pathology in mouse and pig models of Duchenne muscular dystrophy. Across these contexts, miR-128-3p inhibition induces a conserved transcriptional response characterized by activation of mitochondrial programs and suppression of inflammatory and fibrotic signaling, resembling the effects of established longevity interventions. These findings identify miR-128-3p as a regulator of a conserved aging-associated program and establish its inhibition as a strategy to restore tissue function across aging-related muscle pathologies.

molecular biology↗

bioRxiv2025

Genetics of growth rate in induced pluripotent stem cells

Human induced pluripotent stem cells (iPSCs) have transformed biomedical research by enabling the generation of diverse cell types from accessible somatic tissues. However, certain fundamental biological properties, such as the genetic and epigenetic determinants of iPSC proliferation, remain poorly characterized. We measured the growth of iPSC lines derived from 602 unique donors using high-throughput time-lapse imaging, quantified proliferation through a growth Area-Under-the-Curve (gAUC) phenotype, and correlated gAUC with the gene expression and genotype of the cell lines. We identified 3,091 genes associated with gAUC, many of which are well established regulators of cell proliferation. We also found that rare deleterious variants in WDR54 were associated with reduced iPSC growth and that WDR54 was differentially expressed with respect to gAUC. Although no common variants showed a genome-wide association with gAUC, iPSC lines from monozygotic twins were highly correlated, and common genetic variation explained approximately 71-75% of the variance in iPSC growth rates. These results indicate a complex genetic architecture of iPSC growth rates, where rare, large-effect variants in important growth regulators, including WDR54, are layered onto a highly polygenic background. These findings have important implications for the design of pooled iPSC-based studies and disease models, which may be confounded by intrinsic growth differences.

developmental biology↗

bioRxiv2024

Loss of RREB1 reduces adipogenesis and improves insulin sensitivity in mouse and human adipocytes

There are multiple independent genetic signals at the Ras-responsive element binding protein 1 (RREB1) locus associated with type 2 diabetes risk, fasting glucose, ectopic fat, height, and bone mineral density. We have previously shown that loss of RREB1 in pancreatic beta cells reduces insulin content and impairs islet cell development and function. However, RREB1 is a widely expressed transcription factor and the metabolic impact of RREB1 loss in vivo remains unknown. Here, we show that male and female global heterozygous knockout (Rreb1+/-) mice have reduced body length, weight, and fat mass on high-fat diet. Rreb1+/- mice have sex- and diet-specific decreases in adipose tissue and adipocyte size; male mice on high-fat diet had larger gonadal adipocytes, while males on standard chow and females on high-fat diet had smaller, more insulin sensitive subcutaneous adipocytes. Mouse and human precursor cells lacking RREB1 have decreased adipogenic gene expression and activated transcription of genes associated with osteoblast differentiation, which was associated with Rreb1+/- mice having increased bone mineral density in vivo. Finally, human carriers of RREB1 T2D protective alleles have smaller adipocytes, consistent with RREB1 loss-of-function reducing diabetes risk.

physiology↗

bioRxiv2023

Single-cell transcriptome dataset of human and mouse in vitro adipogenesis models

Adipogenesis is a process in which fat-specific progenitor cells (preadipocytes) differentiate into adipocytes that carry out the key metabolic functions of the adipose tissue, including glucose uptake, energy storage, and adipokine secretion. Several cell lines are routinely used to study the molecular regulation of adipogenesis, in particular the immortalized mouse 3T3-L1 line and the primary human Simpson-Golabi-Behmel syndrome (SGBS) line. However, the cell-to-cell variability of transcriptional changes prior to and during adipogenesis in these models is not well understood. Here, we present a single-cell RNA-Sequencing (scRNA-Seq) dataset collected before and during adipogenic differentiation of 3T3-L1 and SGBS cells. To minimize the effects of experimental variation, we mixed 3T3-L1 and SGBS cells and used computational analysis to demultiplex transcriptomes of mouse and human cells. In both models, adipogenesis results in the appearance of three cell clusters, corresponding to preadipocytes, early and mature adipocytes. These data provide a groundwork for comparative studies on human and mouse adipogenesis, as well as on cell-to-cell variability in gene expression during this process.

cell biology↗

bioRxiv2022

X chromosome dosage drives statin-induced dysglycemia and mitochondrial dysfunction

Statin drugs lower blood cholesterol levels for cardiovascular disease prevention. Women are more likely than men to experience adverse statin effects, particularly new-onset diabetes (NOD) and muscle weakness. We determined that female mice are more susceptible than males to glucose intolerance, fasting hyperglycemia, and muscle weakness after short-term statin treatment. Lipidomic, transcriptomic, and biochemical analyses identified reduced docosahexaenoic acid (DHA) levels, and impaired redox tone and mitochondrial respiration specifically in statin-treated female mice. Statin adverse effects could be prevented in females by complementation with a source of DHA. Statin adverse effects segregated with XX chromosome complement, and specifically dosage of the Kdm5c gene, which regulates fatty acid gene expression and has differential expression levels in females and males. In humans, we found that women experience more severe reductions than men in DHA levels after short-term statin administration, and that DHA reduction was correlated with increases in fasting glucose levels. Furthermore, induced pluripotent stem cells derived from women, but not men, who developed NOD exhibited impaired mitochondrial function when treated with statin. Overall, our studies identify biochemical mechanisms, biomarkers, and a genetic risk factor for susceptibility to statin adverse effects, and point to DHA supplementation as a preventive co-therapy.

physiology↗

bioRxiv2022

CROP-Seq: a single-cell CRISPRi platform for characterizing candidate genes relevant to metabolic disorders in human adipocytes

ObjectiveCROP-Seq combines gene silencing using CRISPR interference (CRISPRi) with single-cell RNA sequencing (scRNA-Seq) to conduct a functional reverse genetic screen of novel gene targets associated with adipocyte differentiation or function, with single-cell transcriptomes as the readout. MethodsWe created a human preadipocyte SGBS cell line with stable expression of KRAB-dCas9 for CRISPRi-mediated gene knock-down. This line was transduced with a lentiviral library of sgRNAs targeting 6 genes of interest (3 sgRNAs / gene, 18 sgRNAs), 6 positive control genes (3 sgRNAs / gene, 18 sgRNAs), and non-targeting control sgRNAs (4 sgRNAs). Transduced cells were selected and differentiated, and individual cells were captured using microfluidics at day 0, 4 and 8 of adipogenic differentiation. Next, expression and sgRNA libraries were created and sequenced. Bioinformatic analysis of resulting scRNA-Seq expression data was used to determine the effects of gene knock-down and the dysregulated pathways, and to predict cellular phenotypes. ResultsSingle-cell transcriptomes obtained from SGBS cells following CRISPRi recapitulate different states of differentiation from preadipocytes to adipocytes. We confirmed successful knock-down of targeted genes. Transcriptome-wide changes were observed for all targeted genes, with over 400 differentially expressed genes identified per gene at least at one timepoint. Knock-down of known adipogenesis regulators PPARG and CEBPB inhibited adipogenesis. Gene set enrichment analyses revealed molecular processes for adipose tissue differentiation and function for novel genes. MAFF knock-down led to a downregulation of transcriptional response to proinflammatory cytokine TNF- in preadipocytes. TIPARP knock-down resulted in an increase in the expression of a beiging marker UCP1 at D8 of adipogenesis. ConclusionsThe CROP-Seq system in SGBS cells can determine the consequences of target gene knock-down at the transcriptome level. This powerful, hypothesis-free tool can identify novel regulators of adipogenesis, preadipocyte and adipocyte function associated with metabolic disease. HighlightsO_LICRISPR interference screen coupled with single-cell RNA sequencing (CROP-Seq) C_LIO_LIParallel screening of 12 genes in human SGBS adipocytes and preadipocytes C_LIO_LIUncovered novel regulators of adipogenesis and adipocyte function C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/497796v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1ee34b6org.highwire.dtl.DTLVardef@1c6ba8aorg.highwire.dtl.DTLVardef@fa89org.highwire.dtl.DTLVardef@403966_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2022

G protein-coupled receptor 151 regulates glucose metabolism and hepatic gluconeogenesis

Human genetics has been instrumental in identification of genetic variants linked to type 2 diabetes (T2D). Recently a rare, putative loss-of-function mutation in the orphan G-protein coupled receptor 151 (GPR151) was found to be associated with lower odds ratio for T2D, but the mechanism behind this association has remained elusive. Here for the first time we show that Gpr151 is a fasting- and glucagon-responsive hepatic gene which regulates hepatic gluconeogenesis. Gpr151 ablation in mice leads to suppression of hepatic gluconeogenesis genes and reduced hepatic glucose production in response to pyruvate. Importantly, the restoration of hepatic Gpr151 levels in the Gpr151 knockout mice reverses the reduced hepatic glucose production. Our findings establish a previously unknown role of Gpr151 in the liver and provides an explanation to the lowered T2D risk in individuals with nonsynonymous mutations in GPR151. This study highlights the therapeutic potential of targeting GPR151 for treatment of metabolic disease.

physiology↗

bioRxiv2021

An integrated approach to identify environmental modulators of genetic risk factors for complex traits

Complex traits and diseases can be influenced by both genetics and environment. However, given the large number of environmental stimuli and power challenges for gene-by-environment testing, it remains a critical challenge to identify and prioritize specific disease-relevant environmental exposures. We propose a novel framework for leveraging signals from transcriptional responses to environmental perturbations to identify disease-relevant perturbations that can modulate genetic risk for complex traits and inform the functions of genetic variants associated with complex traits. We perturbed human skeletal muscle, fat, and liver relevant cell lines with 21 perturbations affecting insulin resistance, glucose homeostasis, and metabolic regulation in humans and identified thousands of environmentally responsive genes. By combining these data with GWAS from 31 distinct polygenic traits, we show that heritability of multiple traits is enriched in regions surrounding genes responsive to specific perturbations and, further, that environmentally responsive genes are enriched for associations with specific diseases and phenotypes from the GWAS catalogue. Overall, we demonstrate the advantages of large-scale characterization of transcriptional changes in diversely stimulated and pathologically relevant cells to identify disease-relevant perturbations.

genomics↗

bioRxiv2017

Large-scale phenome-wide association study of PCSK9 loss-of-function variants demonstrates protection against ischemic stroke

PCSK9 inhibitors are a potent new therapy for hypercholesterolemia and have been shown to decrease risk of coronary heart disease. Although short-term clinical trial results have not demonstrated major adverse effects, long-term data will not be available for some time. Genetic studies in large well-phenotyped biobanks offer a unique opportunity to predict drug effects and provide context for the evaluation of future clinical trial outcomes. We tested association of the PCSK9 loss-of-function variant rsll591147 (R46L) in a hypothesis-driven 11 phenotype set and a hypothesis-generating 278 phenotype set in 337,536 individuals of British ancestry in the United Kingdom Biobank (UKB), with independent discovery (n = 225K) and replication (n = 112K). In addition to the known association with lipid levels (OR 0.63) and coronary heart disease (OR 0.73), the T allele of rs11591147 showed a protective effect on ischemic stroke (OR 0.61, p = 0.002) but not hemorrhagic stroke in the hypothesis-driven screen. We did not observe an association with type 2 diabetes, cataracts, heart failure, atrial fibrillation, and cognitive dysfunction. In the phenome-wide screen, the variant was associated with a reduction in metabolic disorders, ischemic heart disease, coronary artery bypass graft operations, percutaneous coronary interventions and history of angina. A single variant analysis of UKB data using TreeWAS, a Bayesian analysis framework to study genetic associations leveraging phenotype correlations, also showed evidence of association with cerebral infarction and vascular occlusion. This result represents the first genetic evidence in a large cohort for the protective effect of PCSK9 inhibition on ischemic stroke, and corroborates exploratory evidence from clinical trials. PCSK9 inhibition was not associated with variables other than those related to low density lipoprotein cholesterol and atherosclerosis, suggesting that other effects are either small or absent.

genetics↗