Biology subjects

Buckley, M.

Publications and source records attributed to Buckley, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Physiological load via voluntary wheel running maintains Achilles tendon homeostasis by region-specific cellular responses

Physiological load is vital for maintaining tendon homeostasis, preserving the organized extracellular matrix that enables tendons to withstand extreme forces. Although tenocytes are regarded as the primary regulators of extracellular matrix production, precisely how cells facilitate the maintenance of homeostasis in response to physiological load is poorly understood. Here, we used Voluntary Wheel Running (VWR) as a model of physiological load to delineate the specific cellular contributions to mouse Achilles tendon homeostasis. Eight weeks of VWR led to a smaller cross-sectional area, increased mechanical and material properties at the midsubstance, which corresponded to a decreased proportion of small (0-60 nm) collagen fibrils and an increased proportion of larger (100-60 nm) collagen fibrils compared to sedentary controls. Using Visium HD spatial transcriptomics, we identified region-specific cell clusters (insertion vs. midsubstance). In response to physiological load, cells in the insertion and midsubstance upregulate distinct genes that reinforce the fibrocartilage interface and collagen-rich tendon matrix, respectively. Notably, Clu, Myoc, and Ccdc80 were upregulated with VWR in the midsubstance, with previously uncharacterized roles in tendon homeostasis. Together, our findings suggest that in response to physiological load, tendon cells maintain homeostasis by region-specific responses. Given that insertional and midsubstance tendinopathy is function-limiting and painful, defining the region-specific cellular responses will be key to advancing therapeutic prospects for tendon health. New and NoteworthyThis study is the first spatially rigorous characterization of the tendon response to physiological load using a Voluntary Wheel Running (VWR) model. VWR led to smaller, stronger, but not stiffer tendons at the midsubstance compared to sedentary controls. This corresponded with significant decreased proportion of small collagen fibrils and a shift toward an increased proportion of large collagen fibrils. Using Visium HD spatial transcriptomics, we identified region-specific transcriptional responses to physiological load that maintain homeostasis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=67 SRC="FIGDIR/small/722285v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@13aa717org.highwire.dtl.DTLVardef@1afe3f1org.highwire.dtl.DTLVardef@9e1137org.highwire.dtl.DTLVardef@17c47bc_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2026

Combinatorial pioneer transcription factor binding reinforces bivalent epigenetic states to preserve lineage fidelity

Cooperative and collaborative modes of transcription factor (TF) binding are central to eukaryotic gene activation, providing regulatory specificity and robustness in cell fate control. However, gene activation is only half the story of transcriptional regulation, and cells also require mechanisms to repress inappropriate gene programs. It remains largely unknown how broadly expressed repressive TFs and epigenetic regulators achieve context-specific repression required for proper cell differentiation. Here, we show that the repressive TF PRDM1 cooperates with lineage- and stage-specific pioneer TFs during human endoderm differentiation. Notably, the number of diverse pioneer TFs bound at enhancers determines the strength of PRDM1-mediated enhancer repression. Mechanistically, collaborative binding by multiple pioneer TFs synergistically promotes local nucleosome remodeling and stabilizes PRDM1 occupancy, culminating in the formation of hyper-bivalent enhancers that are characterized by strongly elevated levels of H3K4me1 and Polycomb-associated modifications. These enhancers reinforce the repression of alternative-lineage, and past and future developmental programs. Together, our findings redefine the roles of TF cooperation and chromatin accessibility beyond the prevailing activation-centric view. We propose a collaborative repression model in which diverse pioneer TFs establish focal chromatin accessibility that reinforces the context-specific assembly of repressive regulators, thereby safeguarding lineage and developmental fidelity.

genomics↗

bioRxiv2026

Cognitive control networks in human and macaque

A much-replicated finding in human brain imaging is a distributed 'multiple-demand' or MD system, increasing in activity for many kinds of cognitive demand, and centrally involved in cognitive control. MD regions are proposed to encode a distributed mental model of critical task events, bound together in the roles and relationships needed to direct action selection. Though previous data hint at a corresponding network in the macaque, there has been no direct comparison to human data. Here we used functional magnetic resonance imaging to measure whole brain activation in a multi-step saccadic maze task, compared to a control requiring similar moves but without goal-based decisions. Human data were a close match to the canonical MD network, extended to include adjacent regions and in particular much of the canonical dorsal attention network. Data from two monkeys suggested correspondences in dorsomedial frontal, insula/orbitofrontal and posterior temporal cortex. In lateral frontal cortex there was just a single, largely dorsal activation patch, in contrast to multiple distinct human patches. In one animal, as in human, there was accompanying activation of dorsal and medial parietal cortex. In macaque as in human, together with previous data, our findings suggest an extended and strongly interconnected brain network recruited by increased cognitive challenge.

neuroscience↗

bioRxiv2026

3D Light Sheet Microscopy Reveals Aging Osteoarthritis-associated Joint-innervated Nerve Remodeling in Mouse Knee Joints

ObjectivesOsteoarthritis (OA) is a prevalent age related joint disease-causing chronic pain. This study investigated how nociceptive and sympathetic nerve innervation in the mouse knee joint changes with age and OA progression, and how these changes relate to pain and disease severity. MethodsThirty-eight mice were assigned to four groups: young male (My), aged male (Ma), young female (Fy), and aged female (Fa). Pain sensitivity was evaluated via Pressure Application Measurement (PAM), and joint damage was graded using OARSI scoring. CGRP and PIEZO2 expressions in dorsal root ganglia (DRG) were also assessed. We employed iDISCO tissue clearing and 3D light sheet fluorescence microscopy to visualize total (PGP9.5), nociceptive (CGRP), and sympathetic (TH) nerve fibers in anterior regions of mouse knee joints. A MATLAB-based tool quantified nerve architecture. ResultsThe Ma group displayed the highest OA severity and markedly lower mechanical withdrawal thresholds compared with My (H = 11.59, {varepsilon}{superscript 2} = 0.64), suggesting an age effect on pain-related behavior. This phenotype was accompanied by a higher total PGP9.5 nerve fiber density in the knee joint (mean difference -0.3127, 95% CI -0.4551 to -0.1704) and increased CGRP nociceptive innervation. In contrast, female mice showed no age-dependent change in PAM withdrawal thresholds, consistent with preserved cartilage integrity and stable OARSI scores, and no detectable age-related differences in PGP9.5 or CGRP innervation. The TH sympathetic fiber distribution was comparable across sexes and ages. Consistent with joint-level findings, DRG analyses demonstrated increased CGRP and PIEZO2 expression in the Ma group, whereas females exhibited no significant change. ConclusionEnhanced nociceptive but not sympathetic nerve remodeling in knee cavities is associated with increased OA severity and knee pain in the Ma group. These findings emphasize the role of peripheral sensory plasticity in OA pain and demonstrate the value of 3D imaging for visualizing neuroanatomical changes in joint disorders.

neuroscience↗

bioRxiv2025

Neural dynamics in ventrolateral prefrontal cortex underlie learning from feedback

Learning often depends on feedback, yet how positive and negative outcomes reorganize target representations to support later memory retrieval remains poorly understood. Accumulating evidence suggests that the ventrolateral prefrontal cortex (vlPFC) acts as a central hub linking learning and retrieval, raising the possibility that it plays a critical role in this process. Here we analysed spiking activity and local field potentials (LFPs) recorded from vlPFC while monkeys performed a multi-cycle object-learning task. During the initial learning cycle, correct and incorrect feedback elicited distinct vlPFC neural responses in both spiking and LFPs. In particular, positive feedback produced elevated theta power and enhanced phase-amplitude coupling (PAC) between theta phase and high-frequency amplitude, associated with sustained suppression of neural spiking. Incorrect feedback induced stronger beta power. Despite comparable levels of object information under both feedback conditions, decoders trained and tested within the same feedback state outperformed those tested across states, revealing feedback-dependent coding formats. State-space and cross-period generalisation analyses further showed that object representations following positive feedback were geometrically closer to and shared a common coding format with those reinstated during later retrieval, indicating that feedback reshapes neural geometry toward retrieval-compatible states. Moreover, these geometric and generalisation effects were selectively expressed on electrodes showing stronger PAC or beta power, suggesting that oscillatory coordination may regulate how feedback signals are transformed into stable target codes. Together, our results reveal how vlPFC serves as a critical bridge between learning and memory retrieval, with feedback-driven dynamics reorganizing population geometry through rhythmic coordination and bringing successful outcome states closer to future retrieval representations.

neuroscience↗

bioRxiv2024

Vaccines combining slow delivery and follicle targeting of antigens increase germinal center B cell clonal diversity and clonal expansion

Vaccines incorporating slow delivery, multivalent antigen display, or immunomodulation through adjuvants have an important role to play in shaping the humoral immune response. Here we analyzed mechanisms of action of a clinically relevant combination adjuvant strategy, where phosphoserine (pSer)-tagged immunogens bound to aluminum hydroxide (alum) adjuvant (promoting prolonged antigen delivery to draining lymph nodes) are combined with a potent saponin nanoparticle adjuvant termed SMNP (which alters lymph flow and antigen entry into lymph nodes). When employed with a stabilized HIV Env trimer antigen in mice, this combined adjuvant approach promoted substantial enhancements in germinal center (GC) and antibody responses relative to either adjuvant alone. Using scRNA-seq and scBCR-seq, we found that the alum-pSer/SMNP combination both increased the diversity of GC B cell clones and increased GC B cell clonal expansion, coincident with increases in the expression of Myc and the proportion of S-phase GC B cells. To gain insight into the source of these changes in the GC response, we analyzed antigen biodistribution and structural integrity in draining lymph nodes and found that the combination adjuvant approach, but not alum-pSer delivery or SMNP alone, promoted accumulation of highly intact antigen on follicular dendritic cells, reflecting an integration of the slow antigen delivery and altered lymph node uptake effects of these two adjuvants. These results demonstrate how adjuvants with complementary mechanisms of action impacting vaccine biodistribution and kinetics can synergize to enhance humoral immunity.

immunology↗

bioRxiv2024

Mechano-adaptation: Exercise-driven Piezo1 & Piezo2 augmentation and chondroprotection in articular cartilage

Chondrocytes in adult joints are mechanosensitive post-mitotic quiescent cells with robustly expressed both Piezo1 and Piezo2 ion channels. Here, we examined the mechano-adaptation and Piezo modulations in articular chondrocytes using a mouse exercise model. We first found differential expression patterns of PIEZO1 and PIEZO2 in articular chondrocytes of healthy knee joints; chondrocytes in tibial cartilage (T) exhibit significantly higher PIEZO1 and PIEZO2 than femoral chondrocytes (F). Interestingly, a few weeks of exercise caused both PIEZO1 and PIEZO2 augmentation in F and T compared to the sedentary control group. Despite the increased expression levels of these mechanosensors, chondrocytes in exercised cartilage exhibit significantly reduced mechanical susceptibility against 1mJ impact. PIEZO1 modulation was relatively more rapid than PIEZO2 channels post-exercise. We tested the exercise-induced effect using Piezo1-conditional knockout (Pz1-cKO; Agc1CreERT2;Piezo1fl/fl). Pz1-cKO mice exhibit diminished exercise-driven chondroprotection against 1mJ impact, suggesting essential roles of Piezo1-mediated mechanotransduction for physiologic-induced cartilage matrix homeostasis. In addition, using a mouse OA model, we further found the modulated PIEZO1 in chondrocytes, consistent with reports in Ren et al., but without PIEZO2 modulations over OA progression. In summary, our data reveal the distinctly tuned Piezo1 and Piezo2 channels in chondrocytes post-exercise and post-injury, in turn modulating the mechanical susceptibility of chondrocytes. We postulate that Piezo1 is a tightly-regulated biphasic biomarker; Piezo1 antagonism may increase cellular survival post-injury and Piezo1 (with Piezo2) agonism to promote cartilage ECM restoration.

bioengineering↗

bioRxiv2024

Visualizing lipid nanoparticle trafficking for mRNA vaccine delivery in non-human primates

mRNA delivered using lipid nanoparticles (LNPs) has become an important subunit vaccine modality, but mechanisms of action for mRNA vaccines remain incompletely understood. Here, we synthesized a metal chelator-lipid conjugate enabling positron emission tomography (PET) tracer labeling of LNP/mRNA vaccines for quantitative visualization of vaccine trafficking in live non-human primates (NHPs). Following i.m. injection, we observed LNPs distributing through injected muscle tissue, simultaneous with rapid trafficking to draining lymph nodes (dLNs). Deltoid injection of LNPs mimicking human vaccine administration led to stochastic LNP delivery to 3 different sets of dLNs. LNP uptake in dLNs was confirmed by histology, and cellular analysis of tissues via flow cytometry identified antigen-presenting cells as the primary cell type responsible for early LNP uptake and mRNA translation. These results provide insights into the biodistribution of mRNA vaccines administered at clinically relevant doses, injection volumes, and injection sites in an important large animal model for vaccine development.

bioengineering↗

bioRxiv2024

High-throughput screening of human genetic variants by pooled prime editing

Understanding the effects of rare genetic variants remains challenging, both in coding and non-coding regions. While multiplexed assays of variant effect (MAVEs) have enabled scalable functional assessment of variants, established MAVEs are limited by either exogenous expression of variants or constraints of genome editing. Here, we introduce a pooled prime editing (PE) platform in haploid human cells to scalably assay variants in their endogenous context. We first optimized delivery of variants to HAP1 cells, defining optimal pegRNA designs and establishing a co-selection strategy for improved efficiency. We characterize our platform in the context of negative selection by testing over 7,500 pegRNAs targeting SMARCB1 for editing activity and observing depletion of highly active pegRNAs installing loss-of-function variants. We next assess variants in MLH1 via 6-thioguanine selection, assaying 65.3% of all possible SNVs in a 200-bp region spanning exon 10 and distinguishing LoF variants with high accuracy. Lastly, we assay 362 non-coding MLH1 variants across a 60 kb region in a single experiment, identifying pathogenic variants acting via multiple mechanisms with high specificity. Our analyses detail how filtering for highly active pegRNAs can facilitate both positive and negative selection screens. Accordingly, our platform promises to enable highly scalable functional assessment of human variants.

genomics↗

bioRxiv2024

Long Amplicon Nanopore Sequencing for Dual-Typing RdRp and VP1 Genes of Norovirus Genogroups I and II in Wastewater

Noroviruses (NoV) are the leading cause of non-bacterial gastroenteritis across the globe with societal costs of US$60.3 billion per annum. Development of a long amplicon nanopore-based method for dual-typing the RNA-dependent RNA polymerase (RdRp) and major structural protein (VP1) regions from a single RNA fragment could improve existing norovirus typing methods. Its application to wastewater-based epidemiology (WBE) and environmental testing could enable the discovery of novel types and improve tracking throughout the population and into aquaculture and recreational water settings. Here, we develop and optimise such a method for wastewater as the sample matrix. Reverse transcription (RT), PCR and library pooling were optimised and a consensus-based bioinformatics pipeline was developed. Inhibitor removal and LunaScript(R) RT gave robust amplification of the {approx}1000 bp RdRP+VP1 amplicon. Platinum Taq polymerase showed good sensitivity and reduced levels non-specific amplification (NSA) when compared to other polymerases. Optimised PCR annealing temperatures significantly reduced NSA (51.3% and 42.4% for GI and GII), increased yield (86.5% for GII) and increased taxa richness (57.7%) for GII. Analysis of three NoV positive faecal samples showed 100% nucleotide similarity with Sanger sequencing. Eight GI genotypes, 11 polymerase types (p-types) and 13 combinations were detected in wastewater along with 4 GII genotypes, 4 p-types and 8 combinations; highlighting the diversity of norovirus taxa present in wastewater in England. The most common genotypes detected in clinical samples were all detected in wastewater while we also commonly detected several GI genotypes not reported in the clinical data. Application of this method into a WBE scheme, therefore, may allow for more accurate measurement of norovirus diversity within the population.

molecular biology↗

bioRxiv2024

Cell non-autonomous signaling through the conserved C. elegans glycopeptide hormone receptor FSHR-1 regulates cholinergic neurotransmission

Modulation of neurotransmission is key for organismal responses to varying physiological contexts such as during infection, injury, or other stresses, as well as in learning and memory and for sensory adaptation. Roles for cell autonomous neuromodulatory mechanisms in these processes have been well described. The importance of cell non-autonomous pathways for inter-tissue signaling, such as gut-to-brain or glia-to-neuron, has emerged more recently, but the cellular mechanisms mediating such regulation remain comparatively unexplored. Glycoproteins and their G protein-coupled receptors (GPCRs) are well-established orchestrators of multi-tissue signaling events that govern diverse physiological processes through both cell-autonomous and cell non-autonomous regulation. Here, we show that follicle stimulating hormone receptor, FSHR-1, the sole Caenorhabditis elegans ortholog of mammalian glycoprotein hormone GPCRs, is important for cell non-autonomous modulation of synaptic transmission. Inhibition of fshr-1 expression reduces muscle contraction and leads to synaptic vesicle accumulation in cholinergic motor neurons. The neuromuscular and locomotor defects in fshr-1 loss-of-function mutants are associated with an underlying accumulation of synaptic vesicles, build-up of the synaptic vesicle priming factor UNC-10/RIM, and decreased synaptic vesicle release from cholinergic motor neurons. Restoration of FSHR-1 to the intestine is sufficient to restore neuromuscular activity and synaptic vesicle localization to fshr-1-deficient animals. Intestine-specific knockdown of FSHR-1 reduces neuromuscular function, indicating FSHR-1 is both necessary and sufficient in the intestine for its neuromuscular effects. Re-expression of FSHR-1 in other sites of endogenous expression, including glial cells and neurons, also restored some neuromuscular deficits, indicating potential cross-tissue regulation from these tissues as well. Genetic interaction studies provide evidence that downstream effectors gsa-1/GS, acy-1/adenylyl cyclase and sphk-1/sphingosine kinase and glycoprotein hormone subunit orthologs, GPLA-1/GPA2 and GPLB-1/GPB5, are important for FSHR-1 modulation of the NMJ. Together, our results demonstrate that FSHR-1 modulation directs inter-tissue signaling systems, which promote synaptic vesicle release at neuromuscular synapses.

neuroscience↗

bioRxiv2023

Saturation Genome Editing Resolves the Functional Spectrum of Pathogenic VHL Alleles

To maximize the impact of precision medicine approaches, it is critical to accurately identify genetic variants in cancer-associated genes with functional consequences. Yet, our knowledge of rare variants conferring clinically relevant phenotypes and the mechanisms through which they act remains highly limited. A tumor suppressor gene exemplifying the challenge of variant interpretation is VHL. VHL encodes an E3 ubiquitin ligase that regulates the cellular response to hypoxia. Germline pathogenic variants in VHL predispose patients to tumors including clear cell renal cell carcinoma (ccRCC) and pheochromocytoma, and somatic VHL mutations are frequently observed in sporadic renal cancer. Here, we optimize and apply Saturation Genome Editing (SGE) to assay nearly all possible single nucleotide variants (SNVs) across VHLs coding sequence. To delineate mechanisms, we quantify mRNA dosage effects over time and compare effects in isogenic cell lines. Function scores for 2,268 VHL SNVs identify a core set of pathogenic alleles driving ccRCC with perfect accuracy, inform differential risk across tumor types, and reveal novel mechanisms by which variants impact function. These results have immediate utility for classifying VHL variants encountered in both germline testing and tumor profiling and illustrate how precise functional measurements can resolve pleiotropic and dosage-dependent genotype-phenotype relationships across complete genes.

genomics↗

bioRxiv2023

A late Neanderthal reveals genetic isolation in their populations before extinction

Neanderthal genomes have been recovered from sites across Eurasia, painting an increasingly complex picture of their populations structure, mostly indicating that late European Neanderthals belonged to a single metapopulation with no significant evidence of deep population structure. Here we report the discovery of a late Neanderthal individual, nicknamed "Thorin", from Grotte Mandrin in Mediterranean France, and his genome. These dentognathic fossils, including a rare example of distomolars, are associated with a rich archeological record of their final technological traditions in this region [~]50-42 thousand years ago. Thorins genome reveals a deep divergence with other late Neanderthals. Thorin belonged to a population with small group size that showed no genetic introgression with other known late European Neanderthals, revealing genetic isolation of his lineage despite them living in neighboring regions. These results have important implications for resolving competing hypotheses about causes of the Neanderthals disappearance. One Sentence SummaryA new French Neanderthal fossil and its genome reveal complex population dynamics during the past 100,000 years.

genomics↗

bioRxiv2023

The ENCODE Uniform Analysis Pipelines

The Encyclopedia of DNA elements (ENCODE) project is a collaborative effort to create a comprehensive catalog of functional elements in the human genome. The current database comprises more than 19000 functional genomics experiments across more than 1000 cell lines and tissues using a wide array of experimental techniques to study the chromatin structure, regulatory and transcriptional landscape of the Homo sapiens and Mus musculus genomes. All experimental data, metadata, and associated computational analyses created by the ENCODE consortium are submitted to the Data Coordination Center (DCC) for validation, tracking, storage, and distribution to community resources and the scientific community. The ENCODE project has engineered and distributed uniform processing pipelines in order to promote data provenance and reproducibility as well as allow interoperability between genomic resources and other consortia. All data files, reference genome versions, software versions, and parameters used by the pipelines are captured and available via the ENCODE Portal. The pipeline code, developed using Docker and Workflow Description Language (WDL; https://openwdl.org/) is publicly available in GitHub, with images available on Dockerhub (https://hub.docker.com), enabling access to a diverse range of biomedical researchers. ENCODE pipelines maintained and used by the DCC can be installed to run on personal computers, local HPC clusters, or in cloud computing environments via Cromwell. Access to the pipelines and data via the cloud allows small labs the ability to use the data or software without access to institutional compute clusters. Standardization of the computational methodologies for analysis and quality control leads to comparable results from different ENCODE collections - a prerequisite for successful integrative analyses. Database URL: https://www.encodeproject.org/

bioinformatics↗

bioRxiv2022

Pioneer transcription factors coordinate active and repressive gene expression states to regulate cell fate

Pioneer transcription factors (TFs) regulate cell fate by establishing transcriptionally primed and active states. However, cell fate control requires the coordination of both lineage-specific gene activation and repression of alternative lineage programs, a process that is poorly understood. Here, we demonstrate that the pioneer TF Forkhead box A (FOXA), required for endoderm lineage commitment, coordinates with the PR domain zinc finger 1 (PRDM1) TF to recruit Polycomb repressive complexes, which establish bivalent enhancers and repress alternative lineage programs. Similarly, the pioneer TF OCT4 coordinates with PRDM14 to repress cell differentiation programs in pluripotent stem cells, suggesting this is a common feature of pioneer TFs. We propose that pioneer and PRDM TFs coordinate recruitment of Polycomb complexes to safeguard cell fate. One-Sentence SummaryPioneer and PRDM transcription factors repress alternative lineage programs.

cell biology↗

bioRxiv2022

Inducible depletion of Scleraxis-lineage cells during tendon healing impairs multi-scale restoration of tendon structure

Tendons are composed of a heterogeneous cell environment, with Scleraxis-lineage (ScxLin) cells being the predominant population. Although ScxLin cells are required for maintenance of tendon homeostasis, their functions during tendon healing are unknown. To this end, we first characterized the spatiotemporal dynamics of ScxLin cells during tendon healing, and identified that the overall ScxLin pool continuously expands up to early remodeling healing phase. To better define the function of ScxLin cells during the late proliferative phase of healing, we inducibly depleted ScxLin cells from day 14-18 post-surgery using the Scx-Cre; Rosa-DTR mouse model, with local administration of diphtheria toxin inducing apoptosis of ScxLin cells in the healing tendon. At D28 post-surgery, ScxLin cell depleted tendons (DTR) had substantial impairments in structure and function, relative to WT, demonstrating the importance of ScxLin cells during tendon healing. Next, bulk RNAseq was utilized to identify the underlying mechanisms that were impaired with depletion and revealed that ScxLin depletion induced molecular and morphological stagnation of the healing process at D28. However, this stagnation was transient, such that by D56 tendon mechanics in DTR were not significantly different than wildtype repairs. Collectively, these data offer fundamental knowledge on the dynamics and roles of ScxLin cells during tendon healing.

cell biology↗

bioRxiv2021

Altered TGFB1 regulated pathways promote accelerated tendon healing in the superhealer MRL/MpJ mouse

To better understand the molecular mechanisms of tendon healing, we investigated the Murphy Roths Large (MRL) mouse, which is considered a model of mammalian tissue regeneration. We show that compared to C57Bl/6J (C57) mice, injured MRL tendons have reduced fibrotic adhesions and cellular proliferation, with accelerated improvements in biomechanical properties. Transcriptional analysis of biological drivers showed positive enrichment of TGFB1 in both C57 and MRL healing tendons. However, only MRL tendons exhibited downstream transcriptional effects of cell cycle regulatory genes, with negative enrichment of the cell senescence-related regulators, compared to the positively-enriched inflammatory and ECM organization pathways in the C57 tendons. Serum cytokine analysis revealed decreased levels of circulating senescence-associated circulatory proteins (SASP) in response to injury in the MRL mice compared to the C57 mice. These data collectively demonstrate altered TGFB1 regulated inflammatory, fibrosis, and cell cycle pathways in flexor tendon repair.

cell biology↗

bioRxiv2020

DNA methylation is required to maintain DNA replication timing precision and 3D genome integrity

DNA replication timing and three-dimensional (3D) genome organisation occur across large domains associated with distinct epigenome patterns to functionally compartmentalise genome regulation. However, it is still unclear if alternations in the epigenome, in particular cancer-related DNA hypomethylation, can directly result in alterations to cancer higher order genome architecture. Here, we use Hi-C and single cell Repli-Seq, in the colorectal cancer DNMT1 and DNMT3B DNA methyltransferases double knockout model, to determine the impact of DNA hypomethylation on replication timing and 3D genome organisation. First, we find that the hypomethylated cells show a striking loss of replication timing precision with gain of cell-to-cell replication timing heterogeneity and loss of 3D genome compartmentalisation. Second, hypomethylated regions that undergo a large change in replication timing also show loss of allelic replication timing, including at cancer-related genes. Finally, we observe the formation of broad ectopic H3K4me3-H3K9me3 domains across hypomethylated regions where late replication is maintained, that potentially prevent aberrant transcription and loss of genome organisation after DNA demethylation. Together, our results highlight a previously underappreciated role for DNA methylation in maintenance of 3D genome architecture.

molecular biology↗