Biology subjects

Clark, S.

Publications and source records attributed to Clark, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Epigenetic priming of embryonic enhancer elements coordinates developmental gene networks

Embryonic development requires the accurate spatiotemporal execution of cell lineage-specific gene expression programs, which are controlled by transcriptional enhancers. Developmental enhancers adopt a primed chromatin state prior to their activation; however how this primed enhancer state is established, maintained, and how it affects the regulation of developmental gene networks remains poorly understood. Here, we use comparative multi-omic analyses of human and mouse early embryonic development to identify subsets of post-gastrulation lineage-specific enhancers which are epigenetically primed ahead of their activation, marked by the histone modification H3K4me1 within the epiblast. We show that epigenetic priming occurs at lineage-specific enhancers for all three germ layers, and that epigenetic priming of enhancers confers lineage-specific regulation of key developmental gene networks. Surprisingly in some cases, lineage-specific enhancers are epigenetically marked already in the zygote, weeks before their activation during lineage specification. Moreover, we outline a generalisable strategy to use naturally occurring human genetic variation to delineate important sequence determinants of primed enhancer function. Our findings identify an evolutionarily conserved program of enhancer priming and begin to dissect the temporal dynamics and mechanisms of its establishment and maintenance during early mammalian development.

developmental biology↗

bioRxiv2024

Structural basis of bulk lipid transfer by bridge-like lipid transfer protein LPD-3

Bridge-like lipid transport proteins (BLTPs) are an evolutionarily conserved family of proteins that localize to membrane contact sites and are thought to mediate the bulk transfer of lipids from a donor membrane, typically the endoplasmic reticulum (ER), to an acceptor membrane, such as a that of the cell or an organelle 1. Despite the fundamental importance of BLTPs for cellular function, the architecture, composition, and lipid transfer mechanisms remain poorly characterized. Here, we present the subunit composition and the cryo-electron microscopy structure of the native LPD-3 BLTP complex isolated from transgenic C. elegans. LPD-3 folds into an elongated, rod-shaped tunnel whose interior is filled with ordered lipid molecules that are coordinated by a track of ionizable residues that line one side of the tunnel. LPD-3 forms a complex with two previously uncharacterized proteins, here named "Intake" and "Spigot", both of which interact with the N-terminal end of LPD-3 where lipids enter the tunnel. Intake has three transmembrane helices, one of which borders the entrance to the tunnel; Spigot has one transmembrane helix and extends 80 [A] along the cytosolic surface of LPD-3. Experiments in multiple model systems indicate that Spigot plays a conserved role in ER-PM contact site formation. Our LPD-3 complex structural data, together with molecular dynamics simulations of the transmembrane region in a lipid bilayer, reveal protein-lipid interactions that suggest a model for how the native LPD-3-complex mediates bulk lipid transport and provide a foundation for mechanistic studies of BLTPs.

biochemistry↗

bioRxiv2024

Streptococcus pneumoniae infection of lung epithelial cells induces internalization of surface GPI-anchored proteins through pneumolysin-mediated activation of host Rho GTPases

A return to homeostasis after infection-associated cellular injury can be accelerated by a rapid damage response. S. pneumoniae, a typically asymptomatic colonizer of the host upper respiratory tract, can cause serious and life-threatening infections when it gains access to the lungs and other organs. The cholesterol binding S. pneumoniae pore-forming toxin, pneumolysin (PLY), is central to the induction of host cell damage. Here, we first found that mouse lung infection by S. pneumoniae diminished pulmonary expression of CD73, a glycosylphosphatidylinositol anchored protein (GPI-AP) that modulates inflammation. Infection of the human pulmonary epithelial cell line H292 resulted in a PLY-dependent reduction of not only cell surface CD73, but also the population of surface expressed GPI-APs. The decrease in cell surface GPI-APs was rapid, required pore-forming activity, and could be recapitulated by purified PLY and other cholesterol binding cytolysins. In response to PLY-mediated insult, GPI-APs were not released from the surface of epithelial cells in extracellular vesicles but rather internalized by a mechanism dependent on the Rho GTPases RhoA and Cdc42. Internalization of GPI-APs was associated with lower levels of PLY-induced apoptosis and membrane permeabilization. These findings suggest that internalization of GPI-APs from epithelial cell membranes may constitute a rapid innate repair response to cell damage induced by PLY and other pore forming toxins that could help bacteria evade host defenses as many GPI-APs have roles in immunity. Author summaryStreptococcus pneumoniae causes serious infections that can result in mortality. The pore- forming toxin, pneumolysin (PLY) produced by these bacteria is important for their ability to cause disease. Understanding how the host responds to damage by this toxin can result in better treatment against infection. In this study, we found that PLY-mediated injury results in decreased expression of glycosylphosphatidylinositol anchored proteins (GPI-AP) from the cell surface by internalization. GPI-AP co-localize in cholesterol-rich areas of the membrane where PLY inserts to form pores and cells with decreased surface GPI-APs were associated with less of PLY-induced cell death and membrane permeabilization. These results suggest that GPI-AP are internalized as part of repair mechanisms activated in response to infection-induced cell injury. As many GPI-APs have important roles in the immune response, their removal from the cell may inadvertently help the bacteria establish better infection.

microbiology↗

bioRxiv2024

UHRF1 ubiquitin ligase activity supports the maintenance of low-density CpG methylation

The RING E3 ubiquitin ligase UHRF1 is an established cofactor for DNA methylation inheritance. Nucleosomal engagement through histone and DNA interactions directs UHRF1 ubiquitin ligase activity toward lysines on histone H3 tails, creating binding sites for DNMT1 through ubiquitin interacting motifs (UIM1 and UIM2). Here, we profile contributions of UHRF1 and DNMT1 to genome-wide DNA methylation inheritance and dissect specific roles for ubiquitin signaling in this process. We reveal DNA methylation maintenance at low-density CpGs is vulnerable to disruption of UHRF1 ubiquitin ligase activity and DNMT1 ubiquitin reading activity through UIM1. Hypomethylation of low-density CpGs in this manner induces formation of partially methylated domains (PMD), a methylation signature observed across human cancers. Furthermore, disrupting DNMT1 UIM2 function abolishes DNA methylation maintenance. Collectively, we show DNMT1-dependent DNA methylation inheritance is a ubiquitin-regulated process and suggest a disrupted UHRF1-DNMT1 ubiquitin signaling axis contributes to the development of PMDs in human cancers.

genomics↗

bioRxiv2023

Single molecule studies of the native hair cell mechanosensory transduction complex

Hearing and balance rely on the conversion of a mechanical stimulus into an electrical signal, a process known as mechanosensory transduction (MT). In vertebrates, this process is accomplished by an MT complex that is located in hair cells of the inner ear. While the past three decades of research have identified many subunits that are important for MT and revealed interactions between these subunits, the composition and organization of a functional complex remains unknown. The major challenge associated with studying the MT complex is its extremely low abundance in hair cells; current estimates of MT complex quantity range from 3-60 attomoles per cochlea or utricle, well below the detection limit of most biochemical assays that are used to characterize macromolecular complexes. Here we describe the optimization of two single molecule assays, single molecule pull-down (SiMPull) and single molecule array (SiMoA), to study the composition and quantity of native mouse MT complexes. We demonstrate that these assays are capable of detecting and quantifying low attomoles of the native MT subunits protocadherin-15 (PCDH15) and lipoma HMGIC fusion partner-like protein 5 (LHFPL5). Our results illuminate the stoichiometry of PCDH15- and LHFPL5-containing complexes and establish SiMPull and SiMoA as productive methods for probing the abundance, composition, and arrangement of subunits in the native MT complex. Impact StatementIn the present work, the authors develop and employ single molecule methods to detect, characterize, and quantitate attomole quantities of the hair cell mechanosensory transduction complex.

biochemistry↗

bioRxiv2023

Structure of C. elegans TMC-2 complex suggests roles of lipid-mediated subunit contacts in mechanosensory transduction

Mechanotransduction is the process by which a mechanical force, such as touch, is converted into an electrical signal. Transmembrane channel-like (TMC) proteins are an evolutionarily-conserved family of ion channels whose function has been linked to a variety of mechanosensory processes, including hearing and balance sensation in vertebrates and locomotion in Drosophila. The molecular features that tune homologous TMC ion channel complexes to diverse mechanical stimuli are unknown. Caenorhabditis elegans express two TMC homologs, TMC-1 and TMC-2, both of which are the likely pore-forming subunits of mechanosensitive ion channels but differ in their expression pattern and functional role in the worm. Here we present the single particle cryo-electron microscopy structure of the native TMC-2 complex isolated from C. elegans. The complex is composed of two copies each of the pore-forming TMC-2 subunit, the calcium and integrin binding protein CALM-1 and the transmembrane inner ear protein TMIE. Comparison of the TMC-2 complex to the recently published cryo-EM structure of the C. elegans TMC-1 complex reveals differences in subunit composition and highlights conserved protein-lipid interactions, as well as other structural features, that together suggest a mechanism for TMC-mediated mechanosensory transduction. Significance StatementOne mechanism by which organisms sense their environment is through the perception of mechanical stimuli such as sound, touch, and vibration. Transmembrane channel-like (TMC) proteins are ion channels whose function has been linked to a variety of mechanosensitive processes, including hearing and balance in vertebrates and touch sensation in worms. The molecular mechanisms by which TMCs respond to mechanical stimuli are unknown. Here we present the structure of the TMC-2 complex isolated from worms. Comparison of the TMC-2 complex to the recently solved structure of the worm TMC-1 complex highlights common structural features that are likely important for sensing mechanical stimuli yet also illuminates key differences that may explain the distinct functional roles of TMC-1 and TMC-2 in the worm.

biochemistry↗

bioRxiv2023

Primordial germ cell DNA demethylation and development require DNA translesion synthesis.

Mutations in DNA damage response (DDR) factors are associated with human infertility, which affects up to 15% of the population. It remains unclear if the role of DDR is solely in meiosis. One pathway implicated in human fertility is DNA translesion synthesis (TLS), which allows replication impediments to be bypassed. We find that TLS is essential for pre-meiotic germ cell development in the embryo. Loss of the central TLS component, REV1, significantly inhibits the induction of human PGC-like cells (hPGCLCs). This is recapitulated in mice, where deficiencies in TLS initiation (Rev1-/- or PcnaK164R/K164R) or extension (Rev7-/-) result in a >150-fold reduction in the number of primordial germ cells (PGCs) and complete sterility. In contrast, the absence of TLS does not impact the growth, function, or homeostasis of somatic tissues. Surprisingly, we find a complete failure in both activation of the germ cell transcriptional program and in DNA demethylation, a critical step in germline epigenetic reprogramming. Our findings show that for normal fertility, DNA repair is required not only for meiotic recombination but for progression through the earliest stages of germ cell development in mammals.

developmental biology↗

bioRxiv2023

Single cell DNA methylation ageing in mouse blood

Ageing is the accumulation of changes and overall decline of the function of cells, organs and organisms over time. At the molecular and cellular level, the concept of biological age has been established and biomarkers of biological age have been identified, notably epigenetic DNA-methylation based clocks. With the emergence of single-cell DNA methylation profiling methods, the possibility to study biological age of individual cells has been proposed, and a first proof-of-concept study, based on limited single cell datasets mostly from early developmental origin, indicated the feasibility and relevance of this approach to better understand organismal changes and cellular ageing heterogeneity. Here we generated a large single-cell DNA methylation and matched transcriptome dataset from mouse peripheral blood samples, spanning a broad range of ages (10-101 weeks of age). We observed that the number of genes expressed increased at older ages, but gene specific changes were small. We next developed a robust single cell DNA methylation age predictor (scEpiAge), which can accurately predict age in a broad range of publicly available datasets, including very sparse data and it also predicts age in single cells. Interestingly, the DNA methylation age distribution is wider than technically expected in 19% of single cells, suggesting that epigenetic age heterogeneity is present in vivo and may relate to functional differences between cells. In addition, we observe differences in epigenetic ageing between the major blood cell types. Our work provides a foundation for better single-cell and sparse data epigenetic age predictors and highlights the significance of cellular heterogeneity during ageing. Highlights- Model to estimate DNA methylation age in single cells - Large multi-omics dataset of single cells from murine blood - Epigenetic age deviations from chronological age are greater than technical expected from technical variability - Number of genes expressed increases with chronological and epigenetic age

cell biology↗

bioRxiv2022

Wnt signalling, cell fate determination and anteroposterior polarity of the skate gill arch skeleton

The gill skeleton of cartilaginous fishes (sharks, skates, rays and holocephalans) exhibits a striking anterior-posterior polarity, with a series of fine appendages called branchial rays projecting from the posterior margin of the gill arch cartilages. We previously demonstrated in the skate (Leucoraja erinacea) that branchial rays derive from a posterior domain of pharyngeal arch mesenchyme that is responsive to Shh signalling from a distal gill arch epithelial ridge (GAER) signalling centre. However, how branchial ray progenitors are specified exclusively within posterior gill arch mesenchyme is not known. Here we show that genes encoding several Wnt ligands are expressed in the ectoderm immediately adjacent to the skate GAER, and that these Wnt signals are transduced largely in the anterior arch environment. Using pharmacological manipulation, we show that inhibition of Wnt signalling results in an anterior expansion of Shh signal transduction in developing skate gill arches, and in the formation of ectopic anterior branchial ray cartilages. Our findings demonstrate that ectodermal Wnt signalling contributes to gill arch skeletal polarity in skate by restricting Shh signal transduction and chondrogenesis to the posterior arch environment and highlights the importance of signalling interactions at embryonic tissue boundaries for cell fate determination in vertebrate pharyngeal arches.

developmental biology↗

bioRxiv2022

Structure of C. elegans TMC-1 complex illuminates auditory mechanosensory transduction

The initial step in the sensory transduction pathway underpinning hearing and balance in mammals involves the conversion of force into the gating of a mechanosensory transduction (MT) channel. Despite the profound socioeconomic impacts of hearing disorders and the fundamental biological significance of understanding MT, the composition, structure and mechanism of the MT complex has remained elusive. Here we report the single particle cryo-EM structure of the native MT TMC-1 complex isolated from C. elegans. The 2-fold symmetric complex is composed of 2 copies each of the pore-forming TMC-1 subunit, the calcium-binding protein CALM-1 and the transmembrane inner ear protein TMIE. CALM-1 makes extensive contacts with the cytoplasmic face of the TMC-1 subunits while the single-pass TMIE subunits reside on the periphery of the complex, poised like the handles of an accordion. A subset of particles in addition harbors a single arrestin-like protein, ARRD-6, bound to a CALM-1 domain. Single- particle reconstructions and molecular dynamics simulations show how the MT complex deforms the membrane bilayer and suggest crucial roles for lipid-protein interactions in the mechanism by which mechanical force is transduced to ion channel gating.

biochemistry↗

bioRxiv2022

Hypoxia shapes the immune landscape in lung injury promoting inflammation persistence

Acute Respiratory Distress Syndrome (ARDS), an often-fatal complication of pulmonary or systemic inflammation, has no cure. Hypoxemia is a defining feature, yet its impact on inflammation is often neglected. Patients with ARDS are monocytopenic early in the onset of the disease. Endotoxin or Streptococcus pneumoniae acute lung injury (ALI) in the context of hypoxia replicates this finding, through hypoxia-driven suppression of type I interferon signalling. This results in failed lung monocyte-derived interstitial macrophages (IM) niche expansion and unchecked neutrophilic inflammation. Administration of colony stimulating factor 1 (CSF1) rescues the monocytopenia, alters the circulating classical monocyte phenotype in hypoxic endotoxin-driven ALI and enables lung IM population expansion, thus limiting lung injury in endotoxin- and virally-induced hypoxic ALI. Hypoxia directly alters immune dynamics to the detriment of the host and manipulation of this aberrant response offers new therapeutic strategies for ARDS.

cell biology↗

bioRxiv2022

Effects of Experimental Design, Genetic Architecture and Threshold on Power and False Positive Rate of Genome-Wide Association Studies

Genome-Wide Association Studies are an important tool for identifying genetic markers associated with a trait, but it has been plagued by the multiple testing problem, which necessitates a multiple testing correction method. While many multiple testing methods have been suggested, e.g. Bonferroni and Benjamini-Hochbergs False Discovery Rate, the quality of the adjusted threshold based on these methods is not as well investigated. The aim of this study was to evaluate the balance between power and false positive rate of a Genome-Wide Association Studies experiment with Bonferroni and Benjamini-Hochbergs False Discovery Rate multiple testing correction methods and to test the effects of various experimental design and genetic architecture parameters on this balance. Our results suggest that when the markers are independent the threshold from Benjamini-Hochbergs False Discovery Rate provides a better balance between power and false positive rate in an experiment. However, with correlations between markers the threshold of Benjamini-Hochbergs False Discovery Rate becomes too lenient with an excessive number of false positives. Experimental design parameters such as sample size and number of markers used, as well as genetic architecture of a trait affect the balance between power and false positive rate. This experiment provided guidance in selecting an appropriate experimental design and multiple testing correction method when conducting an experiment.

genetics↗

bioRxiv2021

Single-Cell Atlas of Common Variable Immunodeficiency reveals germinal center-associated epigenetic dysregulation in B cell responses

Common variable immunodeficiency (CVID), the most prevalent symptomatic primary immunodeficiency, is characterized by impaired terminal B-cell differentiation and defective antibody responses. Incomplete genetic penetrance and a wide range of phenotypic expressivity in CVID suggest the participation of additional pathogenic mechanisms. Monozygotic (MZ) twins discordant for CVID are uniquely valuable for studying the contribution of epigenetics to the disease. We used single-cell epigenomics and transcriptomics to create a cell census of naive-to-memory B cell differentiation in a pair of CVID-discordant MZ twins. Our analysis identifies DNA methylation, chromatin accessibility and transcriptional defects in memory B cells that mirror defective cell-cell communication defects following activation. These findings were validated in a cohort of CVID patients and healthy donors. Our findings provide a comprehensive multi-omics map of alterations in naive-to-memory B-cell transition in CVID and reveal links between the epigenome and immune cell cross-talk. Our resource, publicly available at the Human Cell Atlas, paves the way for future diagnosis and treatments of CVID patients.

immunology↗

bioRxiv2021

mTOR inhibition via Rapamycin treatment partially reverts the deficit in energy metabolism caused by FH loss in RPE cells

Age-related macular degeneration (AMD) is a complex degenerative disease of the retina with multiple risk-modifying factors, including ageing, genetics and lifestyle choices. The combination of these factors leads to oxidative stress, inflammation and metabolic failure in the retinal pigment epithelium (RPE) with subsequent degeneration of photoreceptors in the retina. The alternative complement pathway is tightly linked to AMD. In particular, the genetic variant in the complement factor H gene (CFH), that leads to the Y402H polymorphism in the factor H protein (FH), confers the second highest risk for the development and progression of AMD. While the association between the FH Y402H variant and increased complement system activation is known, recent studies have uncovered novel FH functions not tied to this activity and highlighted functional relevance for intracellular FH. In our previous studies, we show that loss of CFH expression in RPE cells causes profound disturbances in cellular metabolism, increases the vulnerability towards oxidative stress and modulates the activation of pro-inflammatory signaling pathways, most importantly the NF-kB pathway. Here, we silenced CFH in hTERT-RPE1 cells to investigate the mechanism by which intracellular FH regulates RPE cell homeostasis. We found that silencing of CFH results in hyperactivation of mTOR signaling along with decreased mitochondrial respiration and that mTOR inhibition via rapamycin can partially rescue these metabolic defects. To obtain mechanistic insight into the function of intracellular FH in hTERT-RPE1 cells, we analyzed the interactome of FH via immunoprecipitation followed by Mass spectrometry-based analysis. We found that FH interacts with essential components of the ubiquitin-proteasomal pathway (UPS) as well as with factors associated with RB1/E2F signalling in a complement-pathway independent manner. Moreover, we found, that FH silencing affects mRNA levels of the E3 Ubiquitin-Protein Ligase Parkin and PTEN induced putative kinase (Pink1), both of them are associated with UPS. As inhibition of mTORC1 has been previoulsy shown to result in increased overall protein degradation via UPS and as FH mRNA and protein levels were shown to be affected by inhibition of UPS, our data stress a potential regulatory link between endogenous FH activity and the UPS.

molecular biology↗

bioRxiv2021

Molecular structure and conformation of stereocilia tip-links elucidated by cryo-electron tomography

Mechanosensory transduction (MT), the conversion of mechanical stimuli into electrical signals, underpins hearing and balance and is carried out within hair cells in the inner ear. Hair cells harbor actin-filled stereocilia, arranged in rows of descending heights, where the tips of stereocilia are connected to their taller neighbors by a filament composed of protocadherin 15 (PCDH15) and cadherin 23 (CDH23), deemed the tip-link. Tension exerted on the tip-link opens an ion channel at the tip of the shorter stereocilia, thus converting mechanical force into an electrical signal. While biochemical and structural studies have provided insights into the molecular composition and structure of isolated portions of the tip-link, the architecture, location and conformational states of intact tip-links, on stereocilia, remains unknown. Here we report in situ cryo-electron microscopy imaging of the tip-link in mouse stereocilia. We observe individual PCDH15 molecules at the tip and shaft of stereocilia and determine their stoichiometry, conformational heterogeneity, and their complexes with CDH23. The PCDH15/CDH23 complexes occur in clusters, frequently with more than one copy of PCDH15 at the tip of stereocilia, suggesting that tip-links might consist of more than one copy of the PCDH15/CDH23 heterotetramer and by extension, might include multiple MT complexes.

neuroscience↗

bioRxiv2021

In vitro Functional Analysis of pgRNA Sites Regulating Assembly of Hepatitis B Virus.

The roles of RNA sequence/structure motifs, Packaging Signals (PSs), for regulating assembly of an HBV genome transcript have been investigated in an efficient in vitro assay containing only core protein (Cp) and RNA. Variants of three conserved PSs, within the genome of a strain not used previously, preventing correct presentation of a Cp-recognition loop motif are differentially deleterious for assembly of nucleocapsid-like particles (NCPs). Cryo-electron microscopy reconstruction of the T=4 NCPs formed with the wild-type gRNA transcript, reveal that the interior of the Cp shell is in contact with lower resolution density, potentially encompassing the arginine-rich protein domains and gRNA. Symmetry-relaxation of this reconstruction reveals that such contacts are made at every symmetry axis. We infer from their regulation of assembly that some of these contacts would involve gRNA PSs, and confirmed this by X-ray RNA footprinting. Mutation of the {varepsilon} stem-loop in the gRNA, where polymerase binds in vivo, produces a poor RNA assembly substrate with Cp alone, largely due to alterations in its conformation. The results show that RNA PSs regulate assembly of HBV genomic transcripts in vitro, and therefore may play similar roles in vivo, in concert with other molecular factors.

microbiology↗

bioRxiv2021

RNA X-ray footprinting reveals the consequences of an in vivo acquired determinant of viral infectivity

The secondary structures of the bacteriophage MS2 ssRNA genome, frozen in defined states, were determined with minimal perturbation using constraints from X-ray synchrotron footprinting (XRF). The footprints of the gRNA in the virion and as transcript are consistent with single, dominant but distinct conformations, and reveal the presence of multiple Packaging Signals potentially involved in assembly regulation that have not been detected by other techniques. XRF also reveals the dramatic effect of the unique Maturation Protein (MP) on both the capsid lattice, and the gRNA conformation inside the phage compared with a virus-like-particle composed only of coat protein subunits. Aspects of genome organisation in the phage, their impacts on the capsid shell, and the distortion of lattice geometry by MP, are hallmarks of molecular frustration. Phage assembly therefore appears to prepare the particle for the next step of the infectious cycle.

molecular biology↗

bioRxiv2021

Single-cell bisulfite sequencing of spermatozoa from lean and obese humans reveals potential for the transmission of epimutations

Epigenetic marks in gametes modulate developmental programming after fertilization. Spermatozoa from obese men exhibit distinct epigenetic signatures compared to lean men, however, whether epigenetic differences are concentrated in a sub-population of spermatozoa or spread across the ejaculate population is unknown. Here, by using whole-genome single-cell bisulfite sequencing on 87 motile spermatozoa from 8 individuals (4 lean and 4 obese), we found that spermatozoa within single ejaculates are highly heterogeneous and contain subsets of spermatozoa with marked imprinting defects. Comparing lean and obese subjects, we discovered methylation differences across two large CpG dense regions located near PPM1D and LINC01237. These findings confirm that sperm DNA methylation is altered in human obesity and indicate that single ejaculates contain subpopulations of spermatozoa carrying distinct DNA methylation patterns. Distinct epigenetic patterns of spermatozoa within an ejaculate may result in different intergenerational effects and therefore influence strategies aiming to prevent epigenetic-related disorders in the offspring.

genomics↗