Biology subjects

Ford, E.

Publications and source records attributed to Ford, E..

7 recordsLinked to original sources

bioRxiv2026

Coordinated IL-2 and TGF-β Signaling via a Novel Fusion Protein Selectively Expands and Activates Regulatory T Cells

Establishing immune tolerance requires the integration of cytokine signals that favor regulatory T cell (Treg) function over effector differentiation, yet how such coordination can be achieved selectively in vivo remains incompletely defined. Regulatory T cells are essential for maintaining immune homeostasis and peripheral tolerance, and both interleukin-2 (IL-2) and transforming growth factor-{beta} (TGF-{beta}) contribute to Treg activation, differentiation, and stability in a highly context-dependent manner. Here, we examine the consequences of coordinated IL-2 and TGF-{beta}3 signaling using an engineered cytokine construct combining attenuated IL-2 with receptor-masked TGF-{beta}3. In vitro, attenuated IL-2/TGF-{beta}3 signaling promoted conversion of naive CD4 T cells into Foxp3 induced Tregs, expanded endogenous Tregs, and suppressed inflammatory cytokine production by memory and effector CD4 T cells. In vivo, a single administration selectively increased the frequency of CD4Foxp3 regulatory T cells exhibiting an activated and stable phenotype, while minimally activating Foxp3- conventional CD4 T cells relative to IL-2 alone. In a Treg-deficiency-driven adoptive transfer model of autoimmunity, early exposure to coordinated IL-2/TGF-{beta}3 signaling enhanced Treg activation, reduced tissue infiltration by autoreactive T cells, and conferred sustained protection from autoimmune gastritis in the absence of continued treatment. Together, these findings identify a mode of coordinated IL-2 and TGF-{beta}3 signaling sufficient to stabilize regulatory T cell responses and promote immune tolerance while limiting activation of conventional CD4 T cells, highlighting signal integration as a determinant of tolerogenic immune regulation in vivo. One Sentence SummaryCUE-401 coordinates IL-2 and TGF-{beta} signaling to expand and activate regulatory T cells and restrain autoimmune responses.

immunology↗

bioRxiv2021

Coordinated activation of both TGFβ and BMP canonical pathways regulates autophagy and tissue regeneration in acetaminophen induced liver injury

Transforming Growth Factor-{beta}s (TGF{beta}s)/Activins and Bone Morphogenetic Proteins (BMPs) have been implicated in numerous aspects of hepatic pathophysiology. However, the way by which hepatocytes integrate and decode the interplay between the TGF{beta}/Activin and BMP branches in health and disease is still not fully understood. To address this, TGF{beta}/BMP Smad- responsive double transgenic reporter mice were generated and utilized to map patterns of TGF{beta}- and/or BMP-pathway activation during acetaminophen- induced liver injury. TGF{beta} signaling was blocked either pharmacologically or by Smad7 over-expression and the transcriptomes of canonical TGF{beta}- and/or BMP4-treated hepatospheres and Smad7-treated livers were analyzed to highlight TGF{beta}-superfamily-regulated pathways and processes. Acetaminophen administration led to dynamically evolving, stage- and context-specific, patterns of hepatic TGF{beta}/Activin and BMP-reporter expression. TGF{beta}-superfamily signaling was activated in an autophagy prone zone at the borders between healthy and injured tissue. Inhibition of TGF{beta}-superfamily signaling attenuated autophagy, exacerbated liver histopathology, and finally led to accelerated tissue-recovery. Hallmarks of this process were the paraptosis-like cell death and the attenuation of immune and reparatory cell responses. Transcriptomic analysis highlighted autophagy as a prominent TGF{beta}1- and BMP4-regulated process and recognized Trp53inp2 as the top TGF{beta}-superfamily-regulated autophagy-related gene. Collectively, these findings implicate the coordinated activation of both canonical TGF{beta}-superfamily signalling branches in balancing autophagic response and tissue-reparatory and -regenerative processes upon acetaminophen-induced hepatotoxicity, highlighting opportunities and putative risks associated with their targeting for treatment of hepatic diseases.

physiology↗

bioRxiv2020

The First Mitotic Division of the Human Embryo is Highly Error-prone

Aneuploidy in human embryos is surprisingly prevalent and increases drastically with maternal age, resulting in miscarriages, infertility and birth defects. Frequent errors during the meiotic divisions cause this aneuploidy, while age-independent errors during the first cleavage divisions of the embryo also contribute. However, the underlying mechanisms are poorly understood, largely because these events have never been visualised in living human embryos. Here, using cell-permeable DNA dyes, we film chromosome segregation during the first and second mitotic cleavage divisions in human embryos from women undergoing assisted reproduction following ovarian stimulation. We show that the first mitotic division takes several hours to complete and is highly variable. Timings of key mitotic events were, however, largely consistent with clinical videos of embryos that gave rise to live births. Multipolar divisions and lagging chromosomes during anaphase were frequent with no maternal age association. In contrast, the second mitosis was shorter and underwent mostly bipolar divisions with no detectable lagging chromosomes. We propose that the first mitotic division in humans is a unique and highly error-prone event, which contributes to fetal aneuploidies.

cell biology↗

bioRxiv2020

Dopamine-gated memory selection during slow wave sleep

The human brain selectively stores knowledge of the world to optimise future behaviour, automatically rehearsing, contextualising or discarding information to create a robust record of experiences. Storage or forgetting evolves over time, particularly during sleep. We have previously shown that dopamine given in the form of L-DOPA tablets improves long-term memory in Parkinsons disease, but only when given overnight. L-DOPA is already prescribed widely with a good safety profile and could potentially be rapidly repurposed to improve cognitive performance and improve quality of life in, for example, early Alzheimers Disease, if we understood the best time of day to prescribe. Therefore, we sought to test how dopamine shaped long-term memory formation before and during sleep in a double-blind randomised placebo-controlled cross-over trial of healthy older adults (n = 35). We administered L-DOPA after word-list learning to be active during repeat exposure to a proportion of the words and during subsequent nocturnal sleep. Nocturnal dopamine accelerated forgetting for words presented once but it did not affect memory for words presented twice. During slow wave sleep, L-DOPA also increased spindle amplitude around slow oscillation peaks. Larger dopamine-induced difference in word memory was associated with a larger increase in spindle amplitude. Dopamine-dependent memory processing may therefore modulate spindles dependent on slow-oscillation phase. Further, overnight dopamine increased total slow wave sleep duration by approximately 11%. This pharmaceutical modification of slow wave sleep may have potential health-enhancing benefits in old age that could include cognitive enhancement and Alzheimers prevention. One Sentence SummaryDopamine before sleep promotes forgetting of weak memory traces associated with increased spindle amplitude around the peak of a slow oscillations.

neuroscience↗

bioRxiv2020

TINC - a method to dissect transcriptional complexes at single locus resolution - reveals novel Nanog regulators in mouse embryonic stem cells

Cellular identity is ultimately controlled by transcription factors (TFs), which bind to specific regulatory elements (REs) within the genome to regulate gene expression and cell fate changes. While recent advances in genome-wide epigenetic profiling techniques have significantly increased our understanding of which REs are utilized in which cell type, it remains largely unknown which TFs and cofactors interact with these REs to modulate gene expression. A major hurdle in dissecting the whole composition of a multi-protein complex formed at a specific RE is the shortage of appropriate techniques. We have developed a novel method termed TALE-mediated Isolation of Nuclear Chromatin (TINC). TINC utilizes epitope-tagged TALEs to isolate a specific genomic region from the mammalian genome and includes a nuclei isolation and chromatin enrichment step for increased specificity. Upon cross-linking of the cells and isolation of the chromatin, the target region is purified based on affinity purification of the TALE and associated nucleic acid and protein molecules can be subjected to further analyses. A key TF in the pluripotency network and therefore in embryonic stem cells (ESCs) is NANOG. It is currently not fully understood how Nanog expression is regulated and consequently it remains unclear how the ESC state is maintained. Using TINC we dissected the protein complex formed at the Nanog promoter in mouse ESCs and identified many known and numerous novel factors.

genomics↗

bioRxiv2019

Dorsal raphe oxytocin receptors regulate the neurobehavioral consequences of social touch

Prosocial interactions are essential for group-living animals and are regulated by tactile cues shared among the group members. Neurobiological mechanisms through which social touch influences prosociality and related affective behaviors are relatively unknown. Using the evolutionarily ancient mother-young dyad as a model, we hypothesized that neurobehavioral consequences of social touch involves an interaction between central oxytocin (released during social touch) and serotonin (regulating affect and neuroplasticity). New mother rats showed upregulation of numerous aspects of the oxytocin system in the midbrain dorsal raphe (DR; source of forebrain serotonin) compared to non-maternal females. Preventing this upregulation by OTR knockdown in the maternal DR elicited infanticide, reduced nursing, increased aggression, and decreased active coping behavior. OTR knockdown also decreased serotonin-immunoreactive fibers, and increased neuroplasticity-restricting perineuronal nets, in the primary somatosensory cortex. Thus, oxytocin signaling in the DR regulates mechanisms involved in serotonin-induced cortical plasticity, which refines the tactile processing underlying prosocial behaviors.

neuroscience↗

bioRxiv2018

A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9-DNMT3A constructs

AbstractDNA methylation is a covalent modification of the genome that plays important roles in genome regulation and vertebrate development. Although detection of this modification in the genome has been possible for several decades, the ability to deliberately and specifically manipulate local DNA methylation states in the genome has been extremely limited. Consequently, this has impeded the direct determination of the consequence of DNA methylation on transcriptional regulation and transcription factor binding in the native chromatin context. Thus, highly specific targeted epigenome editing tools are needed to address this outstanding question. Recent adaptations of genome editing technologies, such as the fusion of the DNMT3A methyltransferase catalytic domain to catalytically inactive Cas9 (dC9-D3A), have aimed to provide new tools for altering DNA methylation at desired loci. Here, we performed a deeper analysis of the performance of these tools, revealing consistent off-target binding events and DNA methylation deposition in the genome, limiting the capacity of these tools to unambiguously assess the functional consequences of DNA methylation. To address this, we developed a modular dCas9-SunTag (dC9Sun-D3A) system that can recruit multiple DNMT3A catalytic domains to a target site for editing DNA-methylation. dC9Sun-D3A is tunable, specific and exhibits much higher induction of DNA methylation at target sites than the dC9-D3A direct fusion protein. Importantly, genome-wide characterization of dC9Sun-D3A binding sites and DNA methylation revealed minimal off-target protein binding and induction of DNA methylation with dC9Sun-D3A, compared to pervasive off-target binding and methylation by the dC9-D3A direct fusion construct. Furthermore, we used dC9Sun-D3A to test the impact of DNA methylation upon the DNA binding of CTCF and NRF1 upon targeted methylation of their core binding sites, demonstrating the binding sensitivity of these proteins to DNA methylation in situ. Overall, this modular dC9Sun-D3A system enables precise DNA methylation deposition with the lowest amount of off-target DNA methylation reported to date, allowing accurate functional determination of the role of DNA methylation at single loci.

molecular biology↗