Biology subjects

Horsfield, J. A.

Publications and source records attributed to Horsfield, J. A..

10 recordsLinked to original sources

bioRxiv2026

Loss of cohesin subunit Stag1 in zebrafish limits cell cycle progression and is compensated by altered BMP signalling and metabolic pathways

Cohesin is a large multisubunit protein complex that plays essential roles in cell proliferation, genome organisation, and gene regulation in metazoans. Germline mutations in cohesin subunits or regulators cause a group of human developmental disorders collectively known as cohesinopathies. Increasing evidence indicates that individual cohesin subunits can confer distinct molecular functions to the complex; for example, STAG1 and STAG2 have both overlapping and non-overlapping roles in genome organisation. The zebrafish tailbud provides an excellent developmental model for investigating the coordination of cell proliferation and differentiation, processes in which cohesin has crucial functions. We previously demonstrated that loss of Stag2 disrupts Wnt signalling and mesoderm patterning in the zebrafish tailbud. Here, we show that, unlike mammals, zebrafish can tolerate complete loss of Stag1 from embryogenesis through to adulthood. In contrast to Stag2 deficiency, loss of Stag1 impairs cell cycle progression, activates p53 signalling, and induces a metabolic shift towards catabolism. BMP signalling is reduced in Stag1-deficient embryos and is accompanied by expansion of BMP antagonist chordin expression. Stag1 loss also alters chromatin accessibility at the chordin locus and affects accessibility at chromatin domain boundaries. We propose that modulation of growth and signalling pathways compensates for the absence of Stag1, allowing embryonic development to proceed correctly. Together, these findings reveal distinct contributions of Stag1 and Stag2 to cell-cycle regulation, chromatin architecture, and developmental signalling during vertebrate embryogenesis.

developmental biology↗

bioRxiv2025

The ratio of Wnt signaling activity to Sox2 transcription factor levels predicts neuromesodermal fate potential

Neuromesodermal progenitors (NMPs) are a vertebrate cell type that contribute descendants to both the spinal cord and the mesoderm. The undifferentiated bipotential NMP state is maintained when both Wnt signaling is active and Sox2 is present. We used transgenic reporter lines to live-image both Wnt activity and Sox2 levels in NMPs and observed a unique cellular ratio in NMPs compared to NMP-derived mesoderm or neural tissue. We used this unique signature to identify the previously unknown anatomical position of a progenitor population that gives rise to the midline tissues of the floor plate of the spinal cord and the mesodermal notochord. Thus, quantification of the active Wnt signaling to Sox2 ratio can be used to predict and identify cells with neuromesodermal potential. We also developed the auxin inducible degron 2 system for use in zebrafish to test the temporal role that Sox2 plays during midline formation. We found ectopic Sox2 in the presence of Wnt activity holds cells in the undifferentiated floor plate/notochord progenitor state, and that degradation of the ectopic Sox2 is required for cells to adopt a notochord fate.

developmental biology↗

bioRxiv2023

Cohesin composition and dosage independently affect early development in zebrafish

Cohesin, a chromatin-associated protein complex with four core subunits (Smc1a, Smc3, Rad21 and either Stag1 or 2), has a central role in cell proliferation and gene expression in metazoans. Human developmental disorders termed "cohesinopathies" are characterised by germline mutations in cohesin or its regulators that do not entirely eliminate cohesin function. However, it is not clear if mutations in individual cohesin subunits have independent developmental consequences. Here we show that zebrafish rad21 or stag2b mutants independently influence embryonic tailbud development. Both mutants have altered mesoderm induction, but only homozygous or heterozygous rad21 mutation affects cell cycle gene expression. stag2b mutants have narrower notochords and reduced Wnt signaling in neuromesodermal progenitors as revealed by single cell RNA-sequencing. Stimulation of Wnt signaling rescues transcription and morphology in stag2b, but not rad21 mutants. Our results suggest that mutations altering the quantity versus composition of cohesin have independent developmental consequences, with implications for the understanding and management of cohesinopathies. Summary StatementViable zebrafish mutants show that cohesin complex quantity versus composition lead to different transcriptional and developmental outcomes in the early embryo.

developmental biology↗

bioRxiv2021

Genetic Variation as a Long-Distance Modulator of RAD21 Expression in Humans

Mutations and changes in expression in RAD21 are common across cancers types and outside of cancer can result in cohesinopathy. As such, exploration of variants that modify RAD21 enhancer activity, across the genome, may also provide insights into mechanisms by which distinct variants impact healthy human development and disease. We searched 42,953,834 genomic variants for a spatial-eQTL association with the transcription of RAD21. We identified 123 significant associations (FDR < 0.05), which are local (cis) or long-distance (trans) regulators of RAD21 expression. The 123 variants co-regulate a further seven genes, enriched for having Sp2 transcription factor binding sites in their promoter regions. The Sp2 transcription factor and six of the seven genes had previously been associated with cancer onset, progression, and metastasis. Our results suggest that genome-wide variation in non-coding regions impacts on RAD21 transcript levels in addition to other genes, which then could impact on oncogenesis and the process of ubiquitination. This identification of distant co-regulation of oncogenes represents a strategy for discovery of novel genetic regions which impact cancer onset and a potential for diagnostics.

bioinformatics↗

bioRxiv2021

Leptin regulates glucose homeostasis via the canonical WNT pathway.

Leptin is a body weight regulatory hormone, but it is arguably even more potent at regulating blood glucose levels. To further our understanding of the molecular mechanisms by which leptin controls glucose homeostasis, we have used transgenic zebrafish models and conditional deletion of beta catenin in the mediobasal hypothalamus of adult mice to show that Wnt signalling in the brain mediates glucoregulatory effects of leptin. In zebrafish, under normal feeding conditions, leptin regulates glucose homeostasis but not adipostasis. In times of nutrient excess, we found that leptin also regulates body weight and size in this species. Using a Wnt signalling reporter fish, we show that leptin directly activates the canonical Wnt pathway in vivo. Pharmacological inhibition of this pathway prevented the leptin-induced improvement in glucose tolerance. In adult mice, conditional deletion of the key Wnt effector molecule, {beta}-catenin, in the mediobasal hypothalamus of male mice confirmed the essential role of the Wnt pathway in mediating leptin action and the neuroendocrine regulation of glucose homeostasis. Adult-onset {beta}-catenin deletion in the mediobasal hypothalamus led to glucose intolerance, exacerbation of caloric intake and body weight gain under high fat diet, as well as resistance to exogenous leptin.

physiology↗

bioRxiv2020

Cohesin components Stag1 and Stag2 differentially influence haematopoietic mesoderm development in zebrafish embryos

Cohesin is a multiprotein complex made up of core subunits Smc1, Smc3 and Rad21, and either Stag1 or Stag2. Normal haematopoietic development relies on crucial functions of cohesin in cell division and regulation of gene expression via three-dimensional chromatin organisation. Cohesin subunit STAG2 is frequently mutated in myeloid malignancies, but the individual contributions of Stag variants to haematopoiesis or malignancy are not fully understood. Zebrafish have four Stag paralogues (Stag1a, Stag1b, Stag2a and Stag2b), allowing detailed genetic dissection of the contribution of Stag1-cohesin and Stag2-cohesin to development. Here we characterize for the first time the expression patterns and functions of zebrafish stag genes during embryogenesis. Using loss-of-function CRISPR-Cas9 zebrafish mutants, we show that stag1a and stag2b contribute to primitive embryonic haematopoiesis. Both stag1a and stag2b mutants present with erythropenia by 24 hours post-fertilisation. Homozygous loss of either paralog alters the number of haematopoietic/vascular progenitors in the lateral plate mesoderm. The lateral plate mesoderm zone of scl-positive cells is expanded in stag1a mutants with concomitant loss of kidney progenitors, and the number of spi1-positive cells are increased, consistent with skewing toward primitive myelopoiesis. In contrast, stag2b mutants have reduced haematopoietic/vascular mesoderm and downregulation of primitive erythropoiesis. Our results suggest that Stag1 and Stag2 proteins cooperate to balance the production of primitive haematopoietic/vascular progenitors from mesoderm.

developmental biology↗

bioRxiv2020

Cohesin mutations are synthetic lethal with stimulation of WNT signaling

Mutations in genes encoding subunits of the cohesin complex are common in several cancers, but may also expose druggable vulnerabilities. We generated isogenic MCF10A cell lines with deletion mutations of genes encoding cohesin subunits SMC3, RAD21 and STAG2 and screened for synthetic lethality with 3,009 FDA-approved compounds. The screen identified several compounds that interfere with transcription, DNA damage repair and the cell cycle. Unexpectedly, one of the top hits was a GSK3 inhibitor, an agonist of Wnt signaling. We show that sensitivity to GSK3 inhibition is likely due to stabilization of {beta}-catenin in cohesin mutant cells, and that Wnt-responsive gene expression is highly sensitized in STAG2-mutant CMK leukemia cells. Moreover, Wnt activity is enhanced in zebrafish mutant for cohesin subunit rad21. Our results suggest that cohesin mutations could progress oncogenesis by enhancing Wnt signaling, and that targeting the Wnt pathway may represent a novel therapeutic strategy for cohesin mutant cancers.

cancer biology↗

bioRxiv2017

Cohesin facilitates zygotic genome activation in zebrafish

At zygotic genome activation (ZGA), changes in chromatin structure are associated with new transcription immediately following the maternal-to-zygotic transition (MZT). The nuclear architectural proteins, cohesin and CCCTC-binding factor (CTCF), contribute to chromatin structure and gene regulation. We show here that normal cohesin function is important for ZGA in zebrafish. Depletion of cohesin subunit Rad21 delays ZGA without affecting cell cycle progression. In contrast, CTCF depletion has little effect on ZGA whereas complete abrogation is lethal. Genome wide analysis of Rad21 binding reveals a change in distribution from pericentromeric satellite DNA, and few locations including the miR-430 locus (whose products are responsible for maternal transcript degradation), to genes, as embryos progress through the MZT. After MZT, a subset of Rad21 binding occurs at genes dysregulated upon Rad21 depletion and overlaps pioneer factor Pou5f3, which activates early expressed genes. Rad21 depletion disrupts the formation of nucleoli and RNA polymerase II foci, suggestive of global defects in chromosome architecture. We propose that Rad21/cohesin redistribution to active areas of the genome is key to the establishment of chromosome organization and the embryonic developmental program.\n\nAuthor SummaryDuring the first few hours of existence, early zygotic cellular events are regulated by maternally inherited molecules. From a defined timepoint, the zygotic genome gradually becomes active and is transcribed. How the zygotic genome is first held inactive before becoming rapidly activated is poorly understood. Both gene repression and activation mechanisms are involved, but one aspect that has not yet been investigated is how 3-dimensional chromosome structure influences genome activation. In this study, we used zebrafish embryos to model zygotic genome activation.\n\nThe multi-subunit protein complex, cohesin, and the DNA-binding protein CCCTC-binding factor (CTCF) both have well known and overlapping roles in 3-dimensional genome organization. We depleted cohesin subunit Rad21, or CTCF, to determine their effects on zygotic genome activation. Moderate Rad21 depletion delayed transition to zygotic gene expression, without disrupting the cell cycle. By contrast, moderate CTCF depletion had very little effect; however, strong depletion of CTCF was lethal. We surveyed genome-wide binding of Rad21 before and after the zygotic genome is activated, and determined what other chromatin factors and transcription factors coincide with Rad21 binding. Before genome activation, Rad21 was located at satellite DNA and a few noncoding genes, one of which (miR-430) is responsible for degrading maternal transcripts. Following genome activation, there was a mass relocation of Rad21 to genes, particularly active genes and those that are targets of transcriptional activators when the zygotic genome is switched on. Depletion of Rad21 also affected global chromosome structure.\n\nOur study shows that cohesin binding redistributes to active RNA Polymerase II genes at the onset of zygotic gene transcription. Furthermore, we suggest that cohesin contributes to dynamic changes in chromosome architecture that occur upon zygotic genome activation.

developmental biology↗

bioRxiv2017

Variants at the ADAMTS13, BGALT5, SSBP2 and TKT Loci are associated with Post-term birth.

Gestation is a crucial timepoint in human development. Deviation from a term gestational age correlates with both acute and long-term adverse health effects for the child. Both being born pre and post-term, i.e. having short and long gestational ages, are heritable and influenced by the pre- and perinatal environment. Despite the obvious heritable component, specific genetic influences underlying differences in gestational age are poorly understood. Here we identify one globally significant intronic genetic variant within the ADAMTS13 gene that is associated with prolonged gestation in 9,141 white European individuals from the 1966 and 1986 Northern Finland birth cohorts. Additional variants that reached suggestive levels of significance were identified within introns at the TKT, and ARGHAP42 genes, and in the upstream (5) intergenic regions of the B3GALT5 and SSBP2 genes. The variants near the ADAMTS13, B3GALT5, SSBP2 and TKT loci are linked to alterations in gene expression levels (cis-eQTLs). Luciferase assays confirmed the allele specific enhancer activity for the BGALT5 and TKT loci. Our findings provide the first evidence of a specific genetic influence associated with prolonged gestation.

genetics↗

bioRxiv2017

A DNA Contact Map for the Mouse Runx1 Gene Identifies Novel Hematopoietic Enhancers

The transcription factor Runx1 is essential for definitive hematopoiesis, and the RUNX1 gene is frequently translocated or mutated in leukemia. Runx1 is transcribed from two promoters, P1 and P2, to give rise to different protein isoforms. Although the expression of Runx1 must be tightly regulated for normal blood development, the mechanisms that regulate Runx1 isoform expression during hematopoiesis remain poorly understood. Gene regulatory elements located in non-coding DNA are likely to be important for Runx1 transcription. Here we use circular chromosome conformation capture sequencing to identify DNA interactions with the P1 and P2 promoters of Runx1, and the previously identified +24 enhancer, in the mouse multipotent hematopoietic progenitor cell line HPC-7. The active promoter, P1, interacts with nine non-coding regions that are occupied by transcription factors within a 1 Mb topologically associated domain. Eight of nine regions function as blood-specific enhancers in zebrafish. Interestingly, the +24 enhancer interacted with multiple distant regions on chromosome 16, indicating it may regulate the expression of additional genes. The Runx1 DNA contact map identifies connections with multiple novel hematopoietic enhancers that are likely to be involved in regulating Runx1 expression in hematopoietic progenitor cells.

molecular biology↗