Biology subjects

Sanford, L.

Publications and source records attributed to Sanford, L..

10 recordsLinked to original sources

bioRxiv2026

Metal homeostasis is remodeled in response to different quiescence triggers

Cells can enter a reversible non-proliferative state called quiescence either spontaneously or in response to nutrient deprivations. Metal ions are essential nutrients and play wide-ranging regulatory and signaling roles in biological processes. We previously showed that zinc is an essential nutrient for the mammalian cell cycle as zinc deprivation drives cells into quiescence, and this quiescent state is associated with changes in iron, copper, and manganese, suggesting broad remodeling of metal homeostasis. Here we examine whether metal remodeling is a general feature of quiescence by inducing quiescence via different triggers (zinc deficiency, serum starvation, and growth factor withdrawal) in MCF10A cells. Fluorescence microscopy and elemental analysis reveal significant trigger-dependent changes in the labile and total metal pools of quiescent cells. To gain insight into these differences, we carried out RNA sequencing and differential expression analysis, focusing on metal associated, metal regulatory, and metal homeostasis genes. While core quiescence pathways are shared across triggers, quiescence states remain molecularly distinct. A significant percent of 2458 metal homeostasis annotated genes are differentially expressed including 55% in starvation-induced quiescence, 50% in zinc deficiency-induced quiescence and 21% in growth factor withdrawal-induced quiescence. Our results also showed unique alteration of genes involved in major metal dependent processes including antioxidant activity, oxidative phosphorylation, heme metabolism, and chromatin accessibility in different quiescence states. Overall, this work demonstrates that metal homeostasis is systematically rewired during cellular quiescence with associated effects on genes that regulate critical biological processes. Significance StatementCells constantly evaluate their nutrient and energy status and integrate these signals into proliferation-quiescence decisions. Quiescence prevents cells from passing damage to daughter cells. Metals are essential micronutrients for biological processes. While limitation of zinc can drive cells into quiescence and alter other metals, how metals are remodeled and whether this is a common feature of quiescence was unknown. Here we examined three quiescence triggers by modifying growth media and serum, the primary source of metals. We report significant changes in labile and total Cu, Fe, Zn and Mn pools, and metal associated genes in response to distinct quiescence triggers. These changes converge on mitochondrial function, cellular antioxidant activity, and heme biosynthesis in a trigger specific manner.

cell biology↗

bioRxiv2026

An ancient retrotransposon provides species-specific tuning of IL-18 inflammatory signaling

Species differ markedly in how they regulate immune responses, yet the molecular basis of this variation remains incompletely understood. Here, we characterize an underexplored regulatory mechanism of the pro-inflammatory IL-18 pathway, centered on a truncated IL18R1 isoform with striking species-specific expression differences. This isoform, IL18R1-Short, derives from an ancient LINE2 retrotransposon insertion that provides an intronic polyadenylation signal, producing a receptor that lacks the intracellular signaling domain. Using RNA sequencing across nine mammals, we find that although this cis-regulatory element is broadly conserved, robust IL18R1-Short expression is species- and tissue-restricted: bats and mice express it at high levels, particularly in barrier tissues (lung, skin, intestine), whereas most other species, including humans, show little or no expression. Functionally, IL18R1-Short dampens IL-18-induced NF{kappa}B signaling in human, mouse, and bat systems, and knockdown of the mouse ortholog enhances IL-18-driven immune and inflammatory gene expression in mouse T cells. Together, these results identify IL18R1-Short as a transposable element-derived decoy receptor and highlight alternative transcription as a source of species-specific immune regulation.

evolutionary biology↗

bioRxiv2025

Multi-omics and biochemical reconstitution reveal CDK7-dependent mechanisms controlling RNA polymerase II function at gene 5'- and 3'-ends

CDK7 regulates RNA polymerase II (RNAPII) initiation, elongation, and termination through incompletely understood mechanisms. Because contaminating kinases precluded CDK7 analysis with nuclear extracts, we completed biochemical assays with purified factors. Reconstitution of RNAPII transcription initiation showed CDK7 inhibition slowed and/or paused RNAPII promoter-proximal transcription, which reduced re-initiation. These CDK7-regulatory functions were Mediator- and TFIID-dependent. Similarly in human cells, CDK7 inhibition reduced transcription by suppressing RNAPII activity at promoters, consistent with reduced initiation and/or re-initiation. Moreover, widespread 3-end readthrough transcription was observed in CDK7-inhibited cells; mechanistically, this occurred through rapid nuclear depletion of RNAPII elongation and termination factors, including high-confidence CDK7 targets. Collectively, these results define how CDK7 governs RNAPII function at gene 5-ends and 3-ends, and reveal that nuclear abundance of elongation and termination factors is kinase-dependent. Because 3-readthrough transcription is commonly induced during stress, our results further suggest regulated suppression of CDK7 activity may enable this RNAPII transcriptional response.

biochemistry↗

bioRxiv2024

A transcription factor (TF) inference method that broadly measures TF activity and identifies mechanistically distinct TF networks

TF profiler is a method of inferring transcription factor regulatory activity, i.e. when a TF is present and actively regulating transcription, directly directly from nascent sequencing assays such as PRO-seq and GRO-seq. Transcription factors orchestrate transcription and play a critical role in cellular maintenance, identity and response to external stimuli. While ChIP assays have measured DNA localization, they fall short of identifying when and where transcription factors are actively regulating transcription. Our method, on the other hand, uses RNA polymerase activity to infer TF activity across hundreds of data sets and transcription factors. Based on these classifications we identify three distinct classes of transcription factors: ubiquitous factors that play roles in cellular homeostasis, driving basal gene programs across tissues and cell types, tissue specific factors that act almost exclusively at enhancers and are themselves regulated at transcription, and stimulus responsive TFs which are regulated post-transcriptionally but act predominantly at enhancers. TF profiler is broadly applicable, providing regulatory insights on any PRO-seq sample for any transcription factor with a known binding motif.

genomics↗

bioRxiv2023

Atlas of nascent RNA transcripts reveals enhancer to gene linkages

Gene transcription is controlled and modulated by regulatory regions, including enhancers and promoters. These regions are abundant in unstable, non-coding bidirectional transcription. Using nascent RNA transcription data across hundreds of human samples, we identified over 800,000 regions containing bidirectional transcription. We then identify highly correlated transcription between bidirectional and gene regions. The identified correlated pairs, a bidirectional region and a gene, are enriched for disease associated SNPs and often supported by independent 3D data. We present these resources as an SQL database which serves as a resource for future studies into gene regulation, enhancer associated RNAs, and transcription factors.

genomics↗

bioRxiv2023

Cellular zinc status alters chromatin accessibility and binding of transcription factor TP53 to genomic sites

Zinc (Zn2+) is an essential metal required by approximately 2500 proteins. Nearly half of these proteins act on DNA, including > 850 human transcription factors, polymerases, DNA damage response factors, and proteins involved in chromatin architecture. How these proteins acquire their essential Zn2+ cofactor and whether they are sensitive to changes in the labile Zn2+ pool in cells remain open questions. Here, we examine how changes in the labile Zn2+ pool affect chromatin accessibility and transcription factor binding to DNA. We observed both increases and decreases in accessibility in different chromatin regions via ATAC-seq upon treating MCF10A cells with elevated Zn2+ or the Zn2+-specific chelator tris(2-pyridylmethyl)amine (TPA). Transcription factor enrichment analysis was used to correlate changes in chromatin accessibility with transcription factor motifs, revealing 477 transcription factor motifs that were differentially enriched upon Zn2+ perturbation. 186 of these transcription factor motifs were enriched in Zn2+ and depleted in TPA, and the majority correspond to Zn2+ finger transcription factors. We selected TP53 as a candidate to examine how changes in motif enrichment correlate with changes in transcription factor occupancy by ChIP-qPCR. Using publicly available ChIP-seq and nascent transcription datasets, we narrowed the 50,000+ ATAC-seq peaks to 2164 TP53 targets and subsequently selected 6 high-probability TP53 binding sites for testing. ChIP-qPCR revealed that for 5 of the 6 targets, TP53 binding correlates with the local accessibility determined by ATAC-seq. These results demonstrate that changes in labile zinc directly alter chromatin accessibility and transcription factor binding to DNA.

genomics↗

bioRxiv2023

Mediator kinase inhibition suppresses hyperactive interferon signaling in Down syndrome

Hyperactive interferon (IFN) signaling is a hallmark of Down syndrome (DS), a condition caused by trisomy 21 (T21); strategies that normalize IFN signaling could benefit this population. Mediator-associated kinases CDK8 and CDK19 drive inflammatory responses through incompletely understood mechanisms. Using sibling-matched cell lines with/without T21, we investigated Mediator kinase function in the context of hyperactive IFN in DS over a 75min - 24h timeframe. Activation of IFN-response genes was suppressed in cells treated with the CDK8/CDK19 inhibitor cortistatin A (CA), via rapid suppression of IFN-responsive transcription factor (TF) activity. We also discovered that CDK8/CDK19 affect splicing, a novel means by which Mediator kinases control gene expression. To further probe Mediator kinase function, we completed cytokine screens and metabolomics experiments. Cytokines are master regulators of inflammatory responses; by screening 105 different cytokine proteins, we show that Mediator kinases help drive IFN-dependent cytokine responses at least in part through transcriptional regulation of cytokine genes and receptors. Metabolomics revealed that Mediator kinase inhibition altered core metabolic pathways in cell type-specific ways, and broad up-regulation of anti-inflammatory lipid mediators occurred specifically in kinase-inhibited cells during hyperactive IFN{gamma} signaling. A subset of these lipids (e.g. oleamide, desmosterol) serve as ligands for nuclear receptors PPAR and LXR, and activation of these receptors occurred specifically during hyperactive IFN signaling in CA-treated cells, revealing mechanistic links between Mediator kinases, lipid metabolism, and nuclear receptor function. Collectively, our results establish CDK8/CDK19 as context-specific metabolic regulators, and reveal that these kinases control gene expression not only via TFs, but also through metabolic changes and splicing. Moreover, we establish that Mediator kinase inhibition antagonizes IFN signaling through transcriptional, metabolic, and cytokine responses, with implications for DS and other chronic inflammatory conditions.

biochemistry↗

bioRxiv2021

Nascent RNA sequencing defines dynamic aryl hydrocarbon receptor signaling responses to wood smoke particles

Heterogeneity of respirable particulates and compounds complicates understanding transcriptional responses to air pollution. Here, we address this problem through applying precision nuclear run-on sequencing (PRO-seq) to measure nascent transcription and the assay for transposase-accessible chromatin using sequencing (ATAC-seq) to airway epithelial cells after wood smoke particle (WSP) exposure. We used transcription factor enrichment analysis to agnostically identify temporally distinct roles for the TCF/SRF family, the aryl hydrocarbon receptor (AHR), and NFkB in regulating transcriptional changes induced by WSP. Transcription of canonical targets of the AHR, such as CYP1A1 and AHRR, was robustly increased after just 30 minutes of WSP exposure, and we discovered novel AHR-regulated pathways and targets including the DNA methyltransferase, DNMT3L. Transcription of these genes and associated enhancers rapidly returned to near baseline by 120 minutes. The kinetics of AHR- and NFkB-regulated responses to WSP were distinguishable based on the timing of both transcriptional responses and chromatin remodeling, with induction of several cytokines implicated in maintaining NFkB responses through 120 minutes of exposure. In aggregate, our data establish a direct and primary role for AHR in mediating airway epithelial responses to WSP and identify crosstalk between AHR and NFkB signaling in controlling pro-inflammatory gene expression. This work also defines an integrated genomics-based strategy for deconvoluting multiplexed transcriptional responses to heterogeneous environmental exposures.

genomics↗

bioRxiv2020

Dissociated hippocampal neurons exhibit distinct Zn2+ dynamics in a stimulation method-dependent manner

Ionic Zn2+ has increasingly been recognized as an important neurotransmitter and signaling ion in glutamatergic neuron pathways. Intracellular Zn2+ transiently increases as a result of neuronal excitation, and this Zn2+ signal is essential for neuron plasticity, but the source and regulation of the signal is still unclear. In this study we rigorously quantified Zn2+, Ca2+ and pH dynamics in dissociated mouse hippocampal neurons stimulated with bath application of high KCl or glutamate. While both stimulation methods yielded Zn2+ signals, Ca2+ influx, and acidification, glutamate stimulation induced more sustained high intracellular Ca2+ and a larger increase in intracellular Zn2+. However, the stimulation-induced pH change was similar between conditions, indicating that a different cellular change is responsible for the stimulation-dependent difference in Zn2+ signal. This work provides the first robust quantification of Zn2+ dynamics in neurons using different methods of stimulation.

neuroscience↗

bioRxiv2018

Gramicidin increases lipid flip-flop in symmetric and asymmetric lipid vesicles

Unlike most transmembrane proteins, phospholipids can migrate from one leaflet of the membrane to the other. Because this spontaneous lipid translocation (flip-flop) tends to be very slow, cells facilitate the process with enzymes that catalyze the transmembrane movement and thereby regulate the transbilayer lipid distribution. Non-enzymatic membrane-spanning proteins with unrelated primary functions have also been found to accelerate lipid flip-flop in a nonspecific manner and by various hypothesized mechanisms. Using deuterated phospholipids, we examined the acceleration of flip-flop by gramicidin channels which have well-defined structures and known function, features that make them ideal candidates for probing the protein-membrane interactions underlying lipid flip-flop. To study compositionally and isotopically asymmetric proteoliposomes containing gramicidin, we expanded a recently developed protocol for the preparation and characterization of lipid-only asymmetric vesicles. Channel incorporation, conformation, and function were examined with small-angle X-ray scattering, circular dichroism and a stopped-flow spectrofluorometric assay, respectively. As a measure of lipid scrambling we used differential scanning calorimetry to monitor the effect of gramicidin on the melting transition temperatures of the two bilayer leaflets. The two calorimetric peaks of the individual leaflets merged into a single peak over time suggestive of scrambling activity, and the effect of the channel on the transbilayer lipid distribution in both symmetric POPC and asymmetric POPC/DMPC vesicles was quantified from proton NMR measurements. Our results show that gramicidin increases lipid flip-flop in a complex, concentration-dependent manner. To determine the molecular mechanism of the process we used molecular dynamics simulations and further computational analysis of the trajectories to estimate the amount of membrane deformation in the samples. Together, the experimental and computational approaches were found to constitute an effective means for studying the effects of transmembrane proteins on lipid distribution in both symmetric and asymmetric model membranes.

biophysics↗