Biology subjects

Williams, K. J.

Publications and source records attributed to Williams, K. J..

At least 19 recordsLinked to original sources

bioRxiv2026

POGZ safeguards neuronal gene chromatin architecture and transcription

Disruption of chromatin organization is a common pathogenic mechanism in neurodevelopmental disorders, yet how changes in 3D genome architecture relate to transcriptional dysfunction in the developing brain remains unclear. POGZ, a transposase-derived chromatin regulator mutated in White-Sutton syndrome and autism spectrum disorder (ASD) has been linked to both heterochromatin and accessible regulatory DNA, but its in vivo function in the brain is unresolved. Using immunoprecipitation-mass spectrometry in embryonic day 13.5 (E13.5) mouse cortex, we identify the H3K9 methyltransferases G9a/GLP as principal POGZ interactors, placing POGZ within the core H3K9 methylation machinery in vivo. We find that POGZ loss in embryonic mouse cortex drives bidirectional, megabase-scale redistribution of H3K9me3, with ectopic losses and gains over discrete neuronal gene loci. In developing Pogz-/-cortex, regions with H3K9me3 gains are repositioned to the nuclear lamina and exhibit strengthened B-compartment scores by Micro-C, locus-restricted erosion of TAD architecture, weakened boundary insulation, and reduced CTCF occupancy. Within these domains, nascent RNA synthesis at neuronal genes is markedly diminished. These results identify POGZ as a G9a/GLP-associated chromatin regulator that protects neurodevelopmental gene domains from heterochromatinization and perinuclear sequestering, preserving 3D architecture and transcription during cortical development. Major PointsO_LIPOGZ interacts with the G9a/GLP H3K9 methyltransferase complex in the developing mouse cortex, identified by IP-mass spectrometry. C_LIO_LILoss of POGZ drives megabase-scale redistribution of H3K9me3 and increases compartmentalization and nuclear-lamina association of neurodevelopmental gene loci, shown by ChIP-seq, Micro-C, and DNA FISH. C_LIO_LIMicro-C reveals disrupted TAD architecture at specific neuronal gene loci in Pogz-/-.PRO-seq shows POGZ is required to maintain nascent RNA synthesis of neurodevelopmental genes within disrupted chromatin domains. C_LI

neuroscience↗

bioRxiv2026

SCD-dependent lipid metabolism licenses alternative macrophage activation and macrophage plasticity

Lipid metabolic reprogramming accompanies macrophage activation, yet our understanding of why macrophages profoundly reshape their lipid composition remains unclear. Here, we identify stearoyl-CoA desaturase (SCD) as a lipid-metabolic checkpoint required for the acquisition of the alternatively activated macrophage (AAM) cell state. We show that SCD maintains lipid desaturation balance by ensuring the conversion of newly synthesized saturated long-fatty acids (SFAs) into monounsaturated fatty acids (MUFAs). Critically, disruption of SCD resulted in the rapid accumulation of saturated phosphatidic acid (PA) and hyperactivation of mTORC1, which perturbed the acquisition of stable AAM epigenetic and transcriptional programs. Notably, inhibition of upstream lipogenic enzymes (e.g., ACC and FASN) did not impair AAM polarization, underscoring that an imbalance between newly synthesized saturated and monounsaturated fatty acids, not the loss of de novo lipogenesis, determines AAM identity. Unexpectedly, the loss of SCD redirected IL-4-activated macrophages into an aberrant cell state poised for pro-inflammatory responses, indicating that disruption of this lipid metabolic checkpoint leads to misinterpretation of anti-inflammatory instructional cues. Rescue of SCD-deficient macrophages via mTORC1 inhibition confirmed a causal role for the SCD-PA-mTORC1 axis in controlling macrophage plasticity. Finally, we find that SCD deficiency and resultant PA accumulation attenuated Toxoplasma gondii-driven AAM polarization and reduced pathogen burden. Together, these data establish the SCD-PA lipid signaling axis as a critical metabolic regulator of macrophage plasticity with direct consequences for host defense.

immunology↗

bioRxiv2025

Regional Vulnerability of Cardiac Chambers to Radiotherapy: A Multi-Omics Perspective

The heart is highly vulnerable to radiotherapy (RT)-induced injury, leading to molecular and structural remodeling collectively termed radiation-induced cardiac toxicity (RICT). Although several biological pathways have been implicated, the regional, cardiac-specific molecular responses to radiation exposure remain incompletely understood. Here, a multi-omics approach was adopted to longitudinally characterise the unique responses to radiation of the heart base (including ventricular base and right atrium), or the heart apex. Ventricular base irradiation induced a cardiomyopathy phenotype, with pronounced molecular perturbations in metabolism and electrical conduction, while changes related to tissue structure were predominant following apex-directed RT. In the right atrium, irradiation drives fibrotic tissue remodelling, leading to an increased propensity for atrial fibrillation, underpinned by changes in sarcomere organisation. This study represents a comprehensive characterisation of differential spatiotemporal radiation effects in the heart and highlights biological and functional pathways that are potentially clinically actionable for cardiac radioprotection and monitoring.

cell biology↗

bioRxiv2025

Protease-activated receptor 1-mediated matrix metalloprotease signalling in sensory neurons

Visceral pain is a prevalent and debilitating symptom of inflammatory bowel disease (IBD). However, current pain therapies are often ineffective, raising the possibility that novel disease mediators might be contributing to pain during inflammation. Our study provides new insights into how matrix metalloproteinases (MMPs), which are elevated in IBD, stimulate sensory neurons. We demonstrate that MMP3, MMP8, and MMP9 induce intracellular Ca2+ release in dorsal root ganglion (DRG) neurons through activation of protease-activated receptor 1 (PAR1) and subsequent activation of phospholipase C (PLC). Characterisation of the neuronal populations stimulated by these MMPs suggests that a subset is likely nociceptive. In contrast, MMP2 and MMP13, although capable of cleaving PAR1 in other cell types, do not induce Ca2+ mobilisation in DRG neurons. Interestingly, pre-treatment with MMP2 or MMP13 reduces the neuronal response to MMP3 or PAR1 agonist, suggesting that MMP2 and MMP13 act on PAR1 in a manner which prevents further activation. Additionally, MMP10 induces Ca2+ mobilisation in DRG neurons but through a PAR1-independent mechanism. These findings uncover a previously unrecognised role for MMP signalling in sensory neurons, highlighting a potential mechanism by which MMPs could contribute to the pro-nociceptive environment in the inflamed bowel.

neuroscience↗

bioRxiv2025

SCD1 and SCD5 Modulate PARP-Dependent DNA Repair via Fatty Acid Desaturation in Glioblastoma

Glioblastoma (GBM) relies on fatty acid metabolism to sustain its aggressive growth. While the role of stearoyl-CoA desaturase-1 (SCD1) in GBM is established, the function of its brain-enriched isoform, SCD5, remains unexplored. Here, we demonstrate that SCD5 is essential for glioblastoma stem cell (GSC) maintenance and genomic stability, with elevated expression in GSCs that declines upon differentiation, underscoring its role in tumor initiation. Through shotgun lipidomics, 13C metabolic flux analysis, and functional genomics, we reveal that SCD1 and SCD5 play non-redundant roles in fatty acid desaturation, with SCD5 preferentially desaturating C18:0 and uniquely contributing to sphingolipid remodeling. Genetic silencing of either isoform disrupts cell cycle progression, impairs DNA damage repair, and reduces GSC viability, while SCD5 knockdown significantly extends survival in orthotopic GBM models. Mechanistically, loss of SCD activity or saturated fatty acid accumulation triggers PARP1 hyperactivation and subsequent degradation, depleting RAD51 to compromise homologous recombination and induce parthanatos. These findings uncover a lipid-mediated vulnerability in GBM, linking fatty acid desaturation to PARP1-dependent genome integrity. Targeting SCD5 may offer a novel therapeutic strategy to eliminate therapy-resistant GSCs and enhance the efficacy of genotoxic or immunotherapeutic interventions.

cancer biology↗

bioRxiv2025

Dietary lipids, not ketone body metabolites, influence intestinal tumorigenesis in a ketogenic diet

Diet composition shapes tissue function and disease risk by modulating nutrient availability, metabolic state, and cellular dynamics. In the gastrointestinal tract, obesogenic high-fat diets enhance intestinal stem cell activity and tumorigenesis. However, the impact of ketogenic diets (KD), which contain even higher lipid content but induce ketogenesis, remains poorly understood. This is particularly relevant for patients with familial adenomatous polyposis (FAP), who face a high risk of small intestinal tumours. Here, we combine dietary, genetic, and metabolic manipulations in mouse models of spontaneous intestinal adenoma formation to dissect the role of systemic and epithelial ketogenesis in intestinal cancer. We show that KD accelerates tumour burden and shortens survival, independent of ketone body production. Through genetic manipulation of the ketogenic pathway, we modulate local and systemic ketone body production; however, neither inhibition nor augmentation of the ketogenic enzyme HMGCS2 nor disruption of ketolysis altered tumour progression. In contrast, inhibition of fatty acid oxidation did limit adenomatous formation. These findings reveal that dietary lipid content, through FAO rather than ketone body metabolism, influences intestinal tumorigenesis and highlight the need for nuanced consideration of dietary strategies for cancer prevention in genetically susceptible populations.

cancer biology↗

bioRxiv2025

A cold-inducible phospholipid--protein interaction in brown fat mitochondria optimizes thermogenic capacity

Cold stress elicits dynamic remodeling of the mitochondrial lipidome in brown adipose tissue (BAT), marked by an increase in arachidonoyl-phosphatidylethanolamine (AA-PE). However, the function of membrane lipid rewiring in thermoregulatory physiology has been a longstanding mystery. Here, we identify LPCAT3 as a cold-regulated O-acyltransferase driving the highly selective accrual of AA-PE in BAT mitochondria. Lipid-based proteomics, molecular dynamics simulations, and bioenergetic analyses reveal that AA-PE partitions at the COX4I1 interface of the Cytochrome c oxidase complex, enhancing electron transport chain (ETC) efficiency. Accordingly, fat-specific Lpcat3-knockout mice have defects in respiratory-dependent BAT thermogenesis and cold tolerance, despite intact {beta}-adrenergic signaling and UCP1 function. Under cold acclimation, Lpcat3-/-BAT exhibits ETC dysfunction and activation of the integrated stress-response. Thus, our study illuminates a cold-regulated lipid-protein interaction as a gating factor in UCP1-dependent thermogenesis.

physiology↗

bioRxiv2025

Plakophilin-2 Coordinates Energy Metabolism and Contractility in Cardiomyocytes, Revealing Its Roles beyond Desmosomes

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a familial cardiac disease associated with ventricular arrhythmias and an increased risk of sudden cardiac death. Mutations in the desmosome gene Plakophilin-2, PKP2, lead to reduction in PKP2 protein and collapse of desmosomes that is known to compromise contractility and electrical stability of cardiomyocytes. Our previous studies demonstrated the efficacy of adeno-associated virus 9 (AAV9)-mediated restoration of PKP2 expression in a cardiac specific knock-out mouse model of Pkp2 and revealed profound changes in mRNA signatures of metabolic enzymes that were reversed by the gene replacement approach. In this study, we used PKP2-deficient mouse hearts and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to identify changes in steady-state metabolite levels associated with impaired lipid homeostasis, glycolysis, and glucose oxidation. These metabolic phenotypes align with human ARVC metabolic data and reflect an intrinsic impairment of cellular energy metabolism. Here we showed for the first time that these intracellular metabolic defects were associated specifically with poor contractility of cardiomyocytes. AAV9:PKP2 restored contractility, improved electrophysiological properties and Ca2+ transients. In contrast, we observed that treating PKP2-deficient cardiomyocytes pharmacologically with small molecule metabolic enhancers improved contractility but not electrophysiological properties and Ca2+ transients, suggesting differential sensitivity of structure-mediated functions in response to metabolic perturbance. Our study modeled and revealed a direct intracellular connection between compromised PKP2 function and metabolic impairment. We proposed that an increased risk of decoupling energy-responsive contractility from less energy-responsive electrical activities can be a new arrhythmogenic mechanism, potentially responsible for exercise-triggered cardiac adversity in ARVC disease development and progression.

cell biology↗

bioRxiv2024

CCR2-driven monocyte recruitment is protective against radiotherapy-induced intestinal toxicity

Radiotherapy (RT) is essential in treating abdominal and pelvic cancers but often damages the healthy tissues, particularly the intestines, leading to radiation-induced toxicities with limited treatment options. While the immune system is known to help regulate tissue damage, immune mechanisms involved in RT-induced intestinal toxicity are not fully understood. Following CT-guided localised intestinal irradiation, single-cell RNA sequencing (scRNA-seq) and flow cytometry revealed RT-induced chemokine-dependent recruitment of innate immune cells. Deletion of C-C chemokine receptor (Ccr)1, Ccr2, Ccr3 and Ccr5, blocked recruitment and worsened radiation induced toxicities, suggesting an important role for an innate immune cell population in limiting RT-mediated bowel damage. Furthermore, CCR2-deficient mice showed exacerbated weight loss and intestinal permeability, while the transfer of Ly6C+ monocytes alleviated symptoms. Mechanistically, IL-17 cytokine production by group 3 innate lymphoid cells (ILC3s), a critical factor in maintaining intestinal barrier integrity, was found to be reduced in irradiated CCR2-/-, however the transfer of Ly6C+ monocytes resulted in increased IL-17 levels. These findings demonstrate the critical importance of CCR2-mediated monocyte recruitment in mitigating RT-induced toxicities. One Sentence SummaryCCR2-mediated monocyte recruitment protects against RT-induced intestinal toxicity via IL-17, highlighting a therapeutic target.

immunology↗

bioRxiv2024

Dietary control of peripheral adipose storage capacity through membrane lipid remodelling

Complex genetic and dietary cues contribute to the development of obesity, but how these are integrated on a molecular level is incompletely understood. Here, we show that PPAR{gamma} supports hypertrophic expansion of adipose tissue via transcriptional control of LPCAT3, a membrane-bound O-acyltransferase that enriches diet-derived omega-6 (n-6) polyunsaturated fatty acids (PUFAs) in the phospholipidome. In high-fat diet-fed mice, lowering membrane n-6 PUFA levels by adipocyte-specific Lpcat3 knockout (Lpcat3AKO) or by dietary lipid manipulation leads to dysfunctional triglyceride (TG) storage, ectopic fat deposition and insulin resistance. Aberrant lipolysis of stored TGs in Lpcat3AKO adipose tissues instigates a non-canonical adaptive response that engages a futile lipid cycle to increase energy expenditure and limit further body weight gain. Mechanistically, we find that adipocyte LPCAT3 activity promotes TG storage by selectively enriching n-6 arachidonoyl-phosphatidylethanolamine at the ER-lipid droplet interface, which in turn favours the budding of large droplets that exhibit greater resistance to ATGL-dependent hydrolysis. Thus, our study highlights the PPAR{gamma}-LPCAT3 pathway as a molecular link between dietary n-6 PUFA intake, adipose expandability and systemic energy balance.

physiology↗

bioRxiv2024

Flow-Mediated Modulation of the Endothelial Cell Lipidome

The luminal surface of the endothelium is exposed to dynamic blood flow patterns that are known to affect endothelial cell phenotype. While many studies have documented the phenotypic changes by gene or protein expression, less is known about the role of blood flow pattern on the endothelial cell (EC) lipidome. In this study, shotgun lipidomics was conducted on human aortic ECs (HAECs) exposed to unidirectional laminar flow (UF), disturbed flow (DF), or static conditions for 48 hrs. A total of 520 individual lipid species from 17 lipid subclasses were detected. Total lipid abundance was significantly increased for HAECs exposed to DF compared to UF conditions. Despite the increase in the total lipid abundance, HAECs maintained equivalent composition of each lipid subclass (% of total lipid) under both DF and UF. However, by lipid composition (% of total subclass), 28 lipid species were significantly altered between DF and UF. Complimentary RNA sequencing of HAECs exposed to UF or DF revealed changes in transcripts involved in lipid metabolism. Shotgun lipidomics was also performed on HAECs exposed to pro-inflammatory agonists lipopolysaccharide (LPS) or Pam3CSK4 (Pam3) for 48 hrs. Exposure to LPS or Pam3 reshaped the EC lipidome in both unique and overlapping ways. In conclusion, exposure to flow alters the EC lipidome and ECs undergo stimulus-specific lipid reprogramming in response to pro-inflammatory agonist exposure. Ultimately, this work provides a resource to profile the transcriptional and lipidomic changes that occur in response to applied flow that can be accessed by the vascular biology community to further dissect and extend our understanding of endothelial lipid biology.

cell biology↗

bioRxiv2024

IL-4 Licenses B Cell Activation Through Cholesterol Synthesis

Lymphocyte activation involves a transition from quiescence and associated catabolic metabolism to a metabolic state with noted similarities to cancer cells such as heavy reliance on aerobic glycolysis for energy demands and increased nutrient requirements for biomass accumulation and cell division1-3. Following antigen receptor ligation, lymphocytes require spatiotemporally distinct "second signals". These include costimulatory receptor or cytokine signaling, which engage discrete programs that often involve remodeling of organelles and increased nutrient uptake or synthesis to meet changing biochemical demands4-6. One such signaling molecule, IL-4, is a highly pleiotropic cytokine that was first identified as a B cell co-mitogen over 30 years ago7. However, how IL-4 signaling mechanistically supports B cell proliferation is incompletely understood. Here, using single cell RNA sequencing we find that the cholesterol biosynthetic program is transcriptionally upregulated following IL-4 signaling during the early B cell response to influenza virus infection, and is required for B cell activation in vivo. By limiting lipid availability in vitro, we determine cholesterol to be essential for B cells to expand their endoplasmic reticulum, progress through cell cycle, and proliferate. In sum, we demonstrate that the well-known ability of IL-4 to act as a B cell growth factor is through a previously unknown rewiring of specific lipid anabolic programs, relieving sensitivity of cells to environmental nutrient availability.

immunology↗

bioRxiv2024

Targeting SCD triggers lipotoxicity of cancer cells and enhances anti-tumor immunity in breast cancer brain metastasis mouse models

Breast cancer brain metastases (BCBM) are a significant cause of mortality and are incurable. Thus, identifying BCBM targets that reduce morbidity and mortality is critical. BCBM upregulate Stearoyl-CoA Desaturase (SCD), an enzyme that catalyzes the synthesis of monounsaturated fatty acids, suggesting a potential metabolic vulnerability of BCBM. In this study, we tested the effect of a brain-penetrant clinical-stage inhibitor of SCD (SCDi), on breast cancer cells and mouse models of BCBM. Lipidomics, qPCR, and western blot were used to study the in vitro effects of SCDi. Single-cell RNA sequencing was used to explore the effects of SCDi on cancer and immune cells in a BCBM mouse model. Pharmacological inhibition of SCD markedly reshaped the lipidome of breast cancer cells and resulted in endoplasmic reticulum stress, DNA damage, loss of DNA damage repair, and cytotoxicity. Importantly, SCDi alone or combined with a PARP inhibitor prolonged the survival of BCBM-bearing mice. When tested in a syngeneic mouse model of BCBM, scRNAseq revealed that pharmacological inhibition of SCD enhanced antigen presentation by dendritic cells, was associated with a higher interferon signaling, increased the infiltration of cytotoxic T cells, and decreased the proportion of exhausted T cells and regulatory T cells in the tumor microenvironment (TME). Additionally, pharmacological inhibition of SCD decreased engagement of immunosuppressive pathways, including the PD-1:PD-L1/PD-L2 and PVR/TIGIT axes. These findings suggest that SCD inhibition could be an effective strategy to intrinsically reduce tumor growth and reprogram anti-tumor immunity in the brain microenvironment to treat BCBM.

cancer biology↗

bioRxiv2024

Stepwise neofunctionalization of the NF-κB family member c-Rel during vertebrate evolution

Adaptive immunity and the five vertebrate NF-{kappa}B/Rel family members first appeared in cartilaginous fish, suggesting that divergence and specialization within the NF-{kappa}B family helped facilitate the evolution of adaptive immunity. One specialized function of the NF-{kappa}B c-Rel protein in macrophages is the activation of Il12b, which encodes a key regulator of T-cell development. We found that c-Rel is a far more potent regulator of Il12b than of any other inducible genes in macrophages, with c-Rel regulation of Il12b dependent on its heightened intrinsic DNA-binding affinity. c-Rel homodimers regulate Il12b transcription in part via motifs with little resemblance to canonical NF-{kappa}B motifs. ChIP-seq experiments further defined distinct c-Rel DNA-binding preferences genome-wide, and X-ray crystallography of a c-Rel/RelA chimeric protein identified key amino acid changes that support the unique c-Rel properties. Unexpectedly, these changes, along with the c-Rel/RelA binding affinity differences, were largely restricted to mammalian species. Together, our findings reveal how a transcription factor family member can undergo a structural transition at a late stage of vertebrate evolution, resulting in an increased intrinsic DNA binding affinity and with clear functional consequences, presumably to support the increasing complexity of immune regulation.

immunology↗

bioRxiv2023

HER2 overexpression initiates breast tumorigenesis non-cell-autonomously by inducing oxidative stress in the tissue microenvironment

HER2 is considered as a driver oncogene responsible for the HER2+ subtype of breast cancer. However, it is still unclear how HER2 induces the oncogenic transformation of breast cancer stem cells (BCSCs) and initiates tumorigenesis during premalignant stage breast cancer. Here, we used clinical samples and mouse models of HER2+ breast cancer to demonstrate that neither BCSCs nor their cell-of-origin express HER2/Neu in early-stage breast tumors. Instead, our results demonstrate that Neu overexpression results in the transformation of BCSCs in a non-cell-autonomous manner via triggering DNA damage and somatic mutagenesis in their Neu-negative cell-of-origin. This is caused by the increased oxidative stress in the tissue microenvironment generated by altered energy metabolism and increased reactive oxygen species levels in Neu-overexpressing mammary ducts. Therefore, our findings illustrate a previously unrecognized mechanism of HER2-induced breast tumor initiation in vivo with potential impacts on future preventive treatments for HER2+ premalignant breast cancer.

cancer biology↗

bioRxiv2023

Comparative Analysis of Molecular Pathogenic Mechanisms and Antiviral Development Targeting Old and New World Hantaviruses

BackgroundHantaviruses - dichotomized into New World (i.e. Andes virus, ANDV; Sin Nombre virus, SNV) and Old-World viruses (i.e. Hantaan virus, HTNV) - are zoonotic viruses transmitted from rodents to humans. Currently, no FDA-approved vaccines against hantaviruses exist. Given the recent breakthrough to human-human transmission by the ANDV, an essential step is to establish an effective pandemic preparedness infrastructure to rapidly identify cell tropism, infective potential, and effective therapeutic agents through systematic investigation. MethodsWe established human cell model systems in lung (airway and distal lung epithelial cells), heart (pluripotent stem cell-derived (PSC-) cardiomyocytes), and brain (PSC-astrocytes) cell types and subsequently evaluated ANDV, HTNV and SNV tropisms. Transcriptomic, lipidomic and bioinformatic data analyses were performed to identify the molecular pathogenic mechanisms of viruses in different cell types. This cell-based infection system was utilized to establish a drug testing platform and pharmacogenomic comparisons. ResultsANDV showed broad tropism for all cell types assessed. HTNV replication was predominantly observed in heart and brain cells. ANDV efficiently replicated in human and mouse 3D distal lung organoids. Transcriptomic analysis showed that ANDV infection resulted in pronounced inflammatory response and downregulation of cholesterol biosynthesis pathway in lung cells. Lipidomic profiling revealed that ANDV-infected cells showed reduced level of cholesterol esters and triglycerides. Further analysis of pathway-based molecular signatures showed that, compared to SNV and HTNV, ANDV infection caused drastic lung cell injury responses. A selective drug screening identified STING agonists, nucleoside analogues and plant-derived compounds that inhibited ANDV viral infection and rescued cellular metabolism. In line with experimental results, transcriptome data shows that the least number of total and unique differentially expressed genes were identified in urolithin B- and favipiravir-treated cells, confirming the higher efficiency of these two drugs in inhibiting ANDV, resulting in host cell ability to balance gene expression to establish proper cell functioning. ConclusionsOverall, our study describes advanced human PSC-derived model systems and systems-level transcriptomics and lipidomic data to better understand Old and New World hantaviral tropism, as well as drug candidates that can be further assessed for potential rapid deployment in the event of a pandemic.

microbiology↗

bioRxiv2023

Aster-dependent non-vesicular transport facilitates dietary cholesterol uptake

Intestinal cholesterol absorption is an important contributor to systemic cholesterol homeostasis. Niemann-Pick C1 Like 1 (NPC1L1), the target of the drug ezetimibe (EZ), assists in the initial step of dietary cholesterol uptake. However, how cholesterol moves downstream of NPC1L1 is unknown. Here we show that Aster-B and Aster-C are critical for non-vesicular cholesterol movement in enterocytes, bridging NPC1L1 at the plasma membrane (PM) and ACAT2 in the endoplasmic reticulum (ER). Loss of NPC1L1 diminishes accessible PM cholesterol in enterocytes and abolishes Aster recruitment to the intestinal brush border. Enterocytes lacking Asters accumulate cholesterol at the PM and display evidence of ER cholesterol depletion, including decreased cholesterol ester stores and activation of the SREBP-2 transcriptional pathway. Aster-deficient mice have impaired cholesterol absorption and are protected against diet-induced hypercholesterolemia. Finally, we show that the Aster pathway can be targeted with a small molecule inhibitor to manipulate dietary cholesterol uptake. These findings identify the Aster pathway as a physiologically important and pharmacologically tractable node in dietary lipid absorption. One-Sentence SummaryIdentification of a targetable pathway for regulation of dietary cholesterol absorption

physiology↗

bioRxiv2023

Type 4 pili mediated natural competence in Fusobacterium nucleatum

Many bacterial species naturally take up DNA from their surroundings and recombine it into their chromosome through homologous gene transfer (HGT) to aid in survival and gain advantageous functions. Herein we present the first characterization of Type 4 pili mediated natural competence in Fusobacterium nucleatum, which are Gram-negative, anaerobic bacteria that participate in a range of infections and diseases including periodontitis, preterm birth, and cancer. We bioinformatically identified components of the Type 4 conjugal pilus machinery and show this is a conserved system within the Fusobacterium genus. We next validate Type 4 pili in natural competence in F. nucleatum strain 23726 and show that gene deletions in key components of pilus deployment (pilQ) and cytoplasmic DNA import (comEC) abolish DNA uptake and chromosomal incorporation. We next show that natural competence may require native F. nucleatum DNA methylation to bypass restriction modification systems and allow subsequent genomic homologous recombination. In summary, this proof of principle study provides the first characterization of natural competence in Fusobacterium nucleatum and highlights the potential to exploit this DNA import mechanism as a genetic tool to characterize virulence mechanisms of an opportunistic oral pathogen.

microbiology↗