Biology subjects

Day, R. C.

Publications and source records attributed to Day, R. C..

6 recordsLinked to original sources

bioRxiv2025

Commensal skin bacteria interact with the innate immunesystem to promote tail regeneration in Xenopus laevis tadpoles.

Xenopus laevis tadpoles regenerate their tails following partial amputation, however, for a brief developmental window, some undertake wound healing rather than regeneration. Inspired by links between microbiomes and human inflammatory disease, we asked how tadpole skin microbiomes and innate immunity influence the ability to regenerate tails. Having previously shown that lipopolysaccharide-Tlr4 signalling promotes regeneration, here, we demonstrate that the peptidoglycan-Tlr2 pathway is also involved. While tadpoles acquire their commensal skin bacteria from their mothers, analysis of mitochondrial haplotypes did not explain familial regenerative bias. Levels of endogenous lipopolysaccharides on tail tips were also not predictive of regenerative success, and shotgun sequencing indicated no difference in bacterial loads. To see if microbiome composition correlated with regenerative success, we sequenced 16S rRNA amplicons from 503 tadpole tail tips and mapped these to regenerative outcomes. While no one taxon appeared critical for regenerative success, higher proportions of Gram-positives correlated with successful regeneration. Our results suggest a previously undocumented role for Tlr2, peptidoglycan and Gram-positive commensal skin bacteria in tipping the balance from wound repair to regenerative programmes in Xenopus laevis refractory stage tadpoles.

developmental biology↗

bioRxiv2022

Gene expression analysis of the Xenopus laevis early limb bud proximodistal axis.

BackgroundLimb buds develop as bilateral outgrowths of the lateral plate mesoderm and are patterned along three axes. Current models of proximal to distal patterning of early amniote limb buds suggest that two signals, a distal organising signal from the apical epithelial ridge (AER, Fgfs) and an opposing proximal (retinoic acid) act early on pattern this axis. ResultsTranscriptional analysis of stage 51 Xenopus laevis hindlimb buds sectioned along the proximal-distal axis, showed that the distal region is distinct from the rest of the limb. Expression of capn8.3, a novel calpain, was located in cells immediately flanking the AER. The Wnt antagonist Dkk1 was AER-specific in Xenopus limbs. Two transcription factors, sall1 and zic5, were expressed in distal mesenchyme. Zic5 has no described association with limb development. We also describe expression of two proximal genes, gata5 and tnn, not previously associated with limb development. Differentially expressed genes were associated with Fgf, Wnt and retinoic acid (RA) signalling as well as differential cell adhesion and proliferation. ConclusionsWe identify new candidate genes for early proximodistal limb patterning. Our analysis of RA-regulated genes supports a role for transient RA gradients in early limb bud in proximal-to-distal patterning in this anamniote model organism.

developmental biology↗

bioRxiv2022

Nanobodies identify an activated state of the TRIB2 pseudokinase

Tribbles proteins (TRIB1-3) are a pseudokinase-only branch of the human kinome, which recruit substrates to the COP1 ubiquitin-ligase for ubiquitination. TRIB2 was the first Tribbles ortholog to be implicated as a myeloid leukaemia oncogene, by way of recruiting the C/EBPa transcription factor for degradation by COP1. Here we report selection and characterisation of nanobodies against the TRIB2 pseudokinase domain from a synthetic yeast surface-display library. We identified nanobodies that bind the TRIB2 pseudokinase domain with low nanomolar affinity. A crystal structure of Nb4.103 in complex with TRIB2 identified a mode of binding to the N-terminal lobe of the pseudokinase, in a manner that enables specific recognition of TRIB2 over TRIB1 and TRIB3. In the nanobody-stabilised state, TRIB2 adopts an activated conformation that is remarkably similar to the C/EBPa-bound state of TRIB1. Characterization in solution revealed that Nb4.103 can stabilise a TRIB2 pseudokinase domain dimer in a face-to-face manner. Conversely, a distinct nanobody (Nb4.101) binds through a similar epitope but does not readily promote dimerization. In combination, this study identifies features of TRIB2 that could be exploited for the development of inhibitors, and nanobody tools for future investigation of TRIB2 function.

biochemistry↗

bioRxiv2021

Locus-Specific DNA Methylation Editing in Mammalian Cells using a CRISPR-Based System

DNA methylation is a key epigenetic modification implicated in the pathogenesis of numerous human diseases, including cancer development and metastasis. Gene promoter methylation changes are widely associated with transcriptional deregulation and disease progression. The advent of CRISPR-based technologies has provided a powerful toolkit for locus-specific manipulation of the epigenome. Here, we describe a comprehensive global workflow for the design and application of a dCas9-SunTag-based tool for editing a DNA methylation locus in human melanoma cells, alongside protocols for downstream techniques used to evaluate subsequent methylation and gene expression changes in methylation-edited cells. Using transient system delivery, we demonstrate both highly efficacious methylation and demethylation of the EBF3 promoter, a putative epigenetic driver of melanoma metastasis, achieving up to 304.00% gain of methylation and 99.99% relative demethylation, respectively. Further, we employ a novel, targeted screening approach to confirm minimal off-target activity and high on-target specificity of our editing sys-tem within our target locus.

molecular biology↗

bioRxiv2019

BET inhibition prevents aberrant RUNX1 and ERG transcription in STAG2 mutant leukaemia cells.

Mutations in the subunits of the cohesin complex, particularly in the STAG2 subunit, have been identified in a range of myeloid malignancies, but it is unclear how these mutations progress leukaemia. Here, we created isogenic K562 erythromyeloid leukaemia cells with and without the known leukemic STAG2 null mutation, R614*. STAG2 null cells acquired stem cell and extracellular matrix gene expression signatures that accompanied an adherent phenotype. Chromatin accessibility was dramatically altered in STAG2 null K562 cells, consistent with gene expression changes. Enhanced chromatin accessibility was observed at genes encoding hematopoietic transcription factors, ERG and RUNX1. Upon phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic differentiation, STAG2-null cells showed precocious spike in RUNX1 transcription from its P2 promoter. A similar precocious spike was observed in transcription of ERG. Interestingly, spikes in RUNX1-P2 and ERG only occurred as immediate early response to differentiation induction. Treatment of STAG2 null cells with enhancer-blocking BET inhibitor, JQ1, dampened precocious RUNX1 P2 expression and led to a complete loss of RUNX1 P1 and ERG transcription during PMA stimulation in both parental and STAG2 null K562 cells. These results suggest that precocious RUNX1 and ERG expression in STAG2 null cells is enhancer-driven. Furthermore, JQ1 treatment reduced stem cell-associated KIT expression in STAG2 null cells. We conclude that STAG2 depletion in leukemic cells amplifies an enhancer-driven transcriptional response to differentiation signals, and this characteristic is dampened by BET inhibition. The results have relevance to the development of therapeutic strategies for myeloid leukaemia

cancer biology↗

bioRxiv2018

Integrated Cell Expression Toolkit for low input transcript profiling

Several methods exist for constructing sequencing libraries from single-cell amounts of RNA. However, different library preparation methods are required to enable full length or strand specific 3 end profiling. The Integrated Cell Expression Toolkit (ICE-T) maximizes functionality by integrating these approaches with novel strategies that provide a range of profiling options.\n\nTranscriptome profiling at high cellular resolution has many applications in biology but is hampered by a need to boost the representation of RNA to guard against losses during processing and enable efficient use of high throughput technologies. RNA amplification techniques developed for profiling limiting amounts of RNA by microarray have recently been adapted to NGS1-5. These various, but distinct, techniques enable both full length profiling3 and strand specific 3 end profiling2, 4 ...

genomics↗