Biology subjects

Ratcliffe, P. J.

Publications and source records attributed to Ratcliffe, P. J..

6 recordsLinked to original sources

bioRxiv2026

Discrete genetic effects of VHL and PBRM1 inactivation co-operate to disrupt epithelial homeostasis and promote ccRCC

Inactivation of VHL is a truncal alteration in clear cell renal cell carcinoma, but additional events are required for oncogenesis, most commonly PBRM1 inactivation. To better understand this co-operation, we used an oncogenic cell-tagging strategy to analyze the earliest transcriptional and cellular consequences of Vhl and/or Pbrm1 inactivation in the renal tubular epithelium, in vivo, at single-cell resolution. Pbrm1 inactivation did not globally alter HIF-dependent transcription or increase early tubular proliferation induced by Vhl inactivation. Instead, it had independent effects on epithelial organization. Combined genetic and morphological analyses suggested that Pbrm1 inactivation allows cells to sustain Vhl/HIF-dependent proliferation by disrupting tubular architectures that ordinarily restrain this proliferation, resulting in extra-tubular cell accumulation, multilayered epithelia, and tumor formation. Our findings frame a new model for the VHL-PBRM1 interaction that explains loss of epithelial homeostasis through an interaction between discrete effects that drive proliferation and remove structural tissue restraints on that proliferation. Statement of SignificanceVHL and PBRM1 are frequently co-inactivated in ccRCC. Combining transcriptomic and histological analyses of Vhl and/or Pbrm1-inactivated renal cells in vivo, this study highlights independent effects on transcription and epithelial organization that converge to promote sustained proliferation and dysplasia. The work illuminates how tissue homeostasis is disrupted by oncogenic co-operation.

cancer biology↗

bioRxiv2025

Developmental changes in the carotid body transcriptome accompanying the maturation of chemosensitivity

The carotid body (CB) chemoreceptors mediate rapid cardiorespiratory reflexes to hypoxia, which mature peri-natally and are vital for fetal hypoxia tolerance and post-natal ventilatory control. This maturation is associated with an increase in the sensitivity of the CB electrophysiological response to hypoxia (chemosensitivity): a process that is incompletely understood but critical to systemic oxygen homeostasis. Hypothesizing that perinatal CB gene expression changes would reveal candidate mechanisms for oxygen chemosensitivity, we studied the CB transcriptome in sheep, where peri-natal CB physiology is well-characterised. CB-mediated cardiovascular reflexes are detectable at fetal day 120, and robust by term (day 145), while hypoxic ventilatory responses are established by post-natal day 15. We performed RNA sequencing on sheep CBs at each of these stages, and adults, along with the superior cervical ganglion (SCG) as an oxygen-insensitive control. This allowed us to define tissue-specific changes in the CB transcriptome correlating with chemosensitivity maturation. Striking, progressive CB enrichment is observed in genes implicated in murine CB chemosensitivity, including potassium channels (KCNK9), mitochondrial complex IV regulators (NDUFA4L2, HIGD1C), and HIF-2 (EPAS1). Genes with this expression pattern are also enriched for regulators of diacylglycerol (DAG), particularly the DAG kinase DGKH: one of the most abundant CB transcripts increasing in parallel with chemosensitivity. Across developmental stages, the CB also exhibits marked down-regulation of metabolic pathways and ATP/GTP consuming processes, potentially providing a state permissive to metabolic signal detection. Together, this builds a detailed picture of the CB transcriptional signature, with core features established in fetal life and conserved across species. Key pointsO_LIThe carotid body (CB) chemoreceptors mediate rapid cardiorespiratory responses to hypoxia, which maintain systemic oxygen homeostasis, but CB dysfunction is also implicated in pathologies including hypertension, heart failure and sudden infant death. C_LIO_LICB-mediated chemoreflexes mature during the peri-natal period, with increasing sensitivity of the oxygen chemosensory response. C_LIO_LIWe performed RNA-seq of CBs from sheep across 3 peri-natal stages and adults, enabling us to identify gene expression changes that correlate with functional state. C_LIO_LIWe describe a CB transcriptomic signature that is conserved across species, established in fetal life, and correlates with maturation. This includes features of a unique metabolic phenotype, and up-regulation of genes encoding the extracellular matrix and diacylglycerol signalling. The top transcription factor correlating with functional maturation is EPAS1/ HIF-2. C_LIO_LIWe anticipate that this data set will be a valuable resource in generating novel hypotheses on mechanisms of oxygen chemosensory function, development and CB-associated pathologies. C_LI

physiology↗

bioRxiv2024

HIFalpha isoform specific activities drive cell-type specificity of VHL-associated oncogenesis

Cancers arising from dysregulation of generally operative signaling pathways are often tissue specific, but the mechanisms underlying this paradox are poorly understood. Based on striking cell-type specificity, we postulated that these mechanisms must operate early in cancer development and set out to study them in a model of von Hippel Lindau (VHL) disease. Biallelic mutation of the VHL ubiquitin ligase leads to constitutive activation of hypoxia inducible factors HIF1A and HIF2A and is generally a truncal event in clear cell renal carcinoma. We used an oncogenic tagging strategy in which VHL-mutant cells are marked by tdTomato, enabling their observation, retrieval, and analysis early after VHL-inactivation. Our findings reveal markedly different consequences of HIF1A and HIF2A activation, but that both contribute to renal cell-type specific consequences of VHL-inactivation in the kidney. Early involvement of HIF2A in promoting proliferation within proximal tubular epithelium supports therapeutic targeting of HIF2A early in VHL disease.

cancer biology↗

bioRxiv2023

Comparative analysis of N-terminal cysteine dioxygenation and prolyl-hydroxylation as oxygen sensing pathways in mammalian cells

In animals, adaptation to changes in cellular oxygen levels is coordinated largely by the 2-oxoglutarate dependent prolyl-hydroxylase domain (PHD) dioxygenase family, which regulate the stability of their hypoxia-inducible factor (HIF) substrates to promote expression of genes that adapt cells to hypoxia. Recently, 2-aminoethanethiol dioxygenase (ADO) was identified as a novel O2-sensing enzyme in animals. Through N-terminal cysteine dioxygenation and the N-degron pathway, ADO regulates the stability of a set of non-transcription factor substrates; the regulators of G-protein signalling 4, 5 and 16, and interleukin-32. Here, we set out to compare and contrast the in cellulo characteristics of ADO and PHD enzymes in an attempt to better understand their co-evolution in animals. We find that ADO operates to regulate the stability of its substrates rapidly and with similar O2-sensitivity to the PHD/HIF pathway. ADO appeared less sensitive to iron chelating agents or transition metal exposure than the PHD enzymes, possibly due to tighter catalytic-site Fe2+ coordination. Unlike the PHD/HIF pathway, the ADO/N-degron pathway was not subject to feedback by hypoxic induction of ADO and induction of ADO substrates was well sustained in response to prolonged hypoxia. The data also reveal strong interactions between proteolytic regulation of targets by ADO and transcriptional induction of those targets, that shape integrated cellular responses to hypoxia.

cell biology↗

bioRxiv2021

Isoform resolved measurements of absolute translational efficiency define interplay of HIF and mTOR dysregulation in kidney cancer

Hypoxia inducible factor (HIF) and mammalian target of rapamycin (mTOR) pathways orchestrate responses to oxygen and nutrient availability. These pathways are frequently dysregulated in cancer, but their interplay is poorly understood, in part because of difficulties in simultaneous measurement of global and mRNA-specific translation. Here we describe a workflow for measurement of absolute translational efficiency of mRNAs resolved by their transcription start sites (TSSs). Its application to kidney cancer cells revealed a remarkable extent of translational reprogramming by mTOR, strongly affecting many metabolic enzymes and pathways. By contrast, global effects of HIF on translation were limited, and we did not observe reported translational activation by HIF2A. In contrast, HIF-dependent alterations in TSS usage were associated with robust changes in translational efficiency in a subset of genes. Analyses of the interplay of HIF and mTOR revealed that specific classes of HIF1A and HIF2A transcriptional target gene manifest markedly different sensitivity to mTOR, in a manner that supports combined use of HIF2A and mTOR inhibitors in treatment of kidney cancer.

molecular biology↗

bioRxiv2017

The SIN3A histone deacetylase complex is required for a complete transcriptional response to hypoxia

Cells adapt to environmental changes, including fluctuations in oxygen levels, through the induction of specific gene expression programs. To identify genes regulated by hypoxia at the transcriptional level, we pulse-labeled HUVEC cells with 4-thiouridine and sequenced nascent transcripts. Then, we searched genome-wide binding profiles from the ENCODE project for factors that correlated with changes in transcription and identified binding of several components of the Sin3A co-repressor complex, including SIN3A, SAP30 and HDAC1/2, proximal to genes repressed by hypoxia. SIN3A interference revealed that it participates in the downregulation of 75% of the hypoxia-repressed genes in endothelial cells. Unexpectedly, it also blunted the induction of 47% of the upregulated genes, suggesting a role for this corepressor in gene induction. In agreement, ChIP-seq experiments showed that SIN3A preferentially localizes to the promoter region of actively transcribed genes and that SIN3A signal was enriched in hypoxia-repressed genes, prior exposure to the stimulus. Importantly, SINA3 occupancy was not altered by hypoxia in spite of changes in H3K27ac signal. In summary, our results reveal a prominent role for SIN3A in the transcriptional response to hypoxia and suggest a model where modulation of the associated histone deacetylase activity, rather than its recruitment, determines the transcriptional output.

genomics↗