From transcriptome to biological function: environmental stress in an ectothermic vertebrate, the coral reef fish Pomacentrus moluccensis

BackgroundOur understanding of the importance of transcriptional regulation for biological function is continuously improving. We still know, however, comparatively little about how environmentally induced stress affects gene expression in vertebrates, and the consistency of transcriptional stress responses to different types of environmental stress. In this study, we used a multi-stressor approach to identify components of a common stress response as well as components unique to different types of environmental stress. We exposed individuals of the coral reef fish Pomacentrus moluccensis to hypoxic, hyposmotic, cold and heat shock and measured the responses of approximately 16,000 genes in liver. We also compared winter and summer responses to heat shock to examine the capacity for such responses to vary with acclimation to different ambient temperatures.ResultsWe identified a series of gene functions that were involved in all stress responses examined here, suggesting some common effects of stress on biological function. These common responses were achieved by the regulation of largely independent sets of genes; the responses of individual genes varied greatly across different stress types. In response to heat exposure over five days, a total of 324 gene loci were differentially expressed. Many heat-responsive genes had functions associated with protein turnover, metabolism, and the response to oxidative stress. We were also able to identify groups of co-regulated genes, the genes within which shared similar functions.ConclusionThis is the first environmental genomic study to measure gene regulation in response to different environmental stressors in a natural population of a warm-adapted ectothermic vertebrate. We have shown that different types of environmental stress induce expression changes in genes with similar gene functions, but that the responses of individual genes vary between stress types. The functions of heat-responsive genes suggest that prolonged heat exposure leads to oxidative stress and protein damage, a challenge of the immune system, and the re-allocation of energy sources. This study hence offers insight into the effects of environmental stress on biological function and sheds light on the expected sensitivity of coral reef fishes to elevated temperatures in the future

PINA v2.0: Mining interactome modules

Peer reviewe

Targeting DNA Damage Response and Replication Stress in Pancreatic Cancer

Background and aims: Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. The study aim was to investigate the association between DNA damage response (DDR), replication stress and novel therapeutic response in PC to develop a biomarker driven therapeutic strategy targeting DDR and replication stress in PC. Methods: We interrogated the transcriptome, genome, proteome and functional characteristics of 61 novel PC patient-derived cell lines to define novel therapeutic strategies targeting DDR and replication stress. Validation was done in patient derived xenografts and human PC organoids. Results: Patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, co-segregates with response to platinum (P < 0.001) and PARP inhibitor therapy (P < 0.001) in vitro and in vivo. We generated a novel signature of replication stress with which predicts response to ATR (P < 0.018) and WEE1 inhibitor (P < 0.029) treatment in both cell lines and human PC organoids. Replication stress was enriched in the squamous subtype of PC (P < 0.001) but not associated with DDR deficiency. Conclusions: Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR proficient PC, and post-platinum therapy

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

A microarray approach to understanding stress in a coral reef fish

Coral reef fishes are expected to experience a rise in sea surface temperatures due to climate change. How well tropical reef fishes will respond to these increased temperatures and which genes are important in the response to elevated temperatures is not known. Microarray technology provides a powerful tool for gene discovery studies, but the development of microarrays for individual species can be expensive and time-consuming. There often are, however, microarrays available for related species. I show that inter-species genomic hybridisation experiments can be used to assess which genes are conserved enough for microarray analysis across species, and thus introduce a novel application of microarray technology. I performed a series of tests to determine whether a microarray developed for the zebrafish Danio rerio is useful for measuring gene regulation in the coral reef fish Pomacentrus moluccensis.\ud I hybridised genomic DNA from both taxa onto the D. rerio microarray, and based on significant cross-hybridisation, inferred that most genes share significant sequence similarity between the two taxa. I also sequenced eight nuclear genes.\ud \ud These genes showed an average sequence similarity of 81%. Finally, I used quantitative real-time PCR to validate the microarray data for differential expression.\ud The results of the genomic hybridisation experiments, direct sequence comparisons, and quantitative real-time PCR indicate that the D. rerio microarray is useful for measuring gene regulation in P. moluccensis. I then used the D. rerio microarray to characterise the transcriptional responses of P. moluccensis to elevated temperatures over five days. Heat stress elicited differential expression of 324 genes. The functions of heat-responsive genes indicated that prolonged heat exposure leads to oxidative stress and protein damage, challenges the immune system, and causes reallocation of energy sources. I have shown that a temperature increase of three degrees above normal can lead to significant gene regulation in a coral reef fish suggesting that climate change will have measurable impacts upon coral reef fish physiology. In order to identify upstream regulators of the transcriptional responses observed and test for the presence of a general stress response, I measured the early gene responses of P. moluccensis to hypoxic, hyposmotic, cold and heat shock. Early stress responses three hours after exposure were generally associated with a suppression of transcription, but the responses of individual genes varied depending on the type of stressor applied. Only a few genes showed consistent regulation across stress treatments. However, a series of gene functions showed consistent responses across stress treatments, suggesting that there are common effects of stress on biological function. I present a conceptual model of the interactions between stress responses at different levels of biological organisation. I propose that stress commonly leads to a reduction in cellular oxygen levels and oxidative stress.\ud \ud Reduced cellular oxygen levels initiate endocrine, cardio-respiratory, and cellular responses many of which are aimed at restoring cellular oxygen balance. Oxidative stress in turn activates certain signal transduction pathways, immediate early genes, and transcription factors. The transcriptional stress profiles measured in environmental genomic studies are likely the result of the activation of these redoxsensitive signalling pathways and transcription factors. Further, I tested whether genes with stress-related functions evolve at accelerated rates. To do this, I competitively hybridised genomic DNA from D. rerio and P. moluccensis to a D.\ud rerio microarray. 985 genes showed evidence of accelerated rates of sequence evolution between D. rerio and P. moluccensis. Rapidly diverging genes were over-represented for receptor, transcription co-activator, and cell signalling functions, but not for stress-related gene functions. I obtained orthologous sequences to D. rerio for the teleosts Takifugu rubripes and Gasterosteus aculeatus. A selection of rapidly diverging candidate genes showed accelerated rates of sequence evolution across multiple teleost lineages. I have shown that genomic hybridisation experiments on microarrays can be successfully used to identify rapidly diverging genes, in particular in species that currently lack genome sequence data and for which bioinformatic approaches are thus not applicable.\u

Sequencing transcriptomes in toto

The development of next-generation sequencing technologies has enabled the transcriptome to be measured and characterized at a level which was previously unattainable. Shot gun sequencing of RNAs, or RNA-Seq as it is known, is providing the means to simultaneously survey locus activity, transcript-specific expression, sequence content of transcripts and transcriptome discovery. This article discusses the current state of RNA-Seq, its potential for redefining transcriptomics and some of the challenges associated with this revolutionary technology

From transcriptome to biological function: environmental stress in an ectothermic vertebrate, the coral reef fish Pomacentrus moluccensis

Background Our understanding of the importance of transcriptional regulation for biological function is continuously improving. We still know, however, comparatively little about how environmentally induced stress affects gene expression in vertebrates, and the consistency of transcriptional stress responses to different types of environmental stress. In this study, we used a multi-stressor approach to identify components of a common stress response as well as components unique to different types of environmental stress. We exposed individuals of the coral reef fish Pomacentrus moluccensis to hypoxic, hyposmotic, cold and heat shock and measured the responses of approximately 16,000 genes in liver. We also compared winter and summer responses to heat shock to examine the capacity for such responses to vary with acclimation to different ambient temperatures. Results We identified a series of gene functions that were involved in all stress responses examined here, suggesting some common effects of stress on biological function. These common responses were achieved by the regulation of largely independent sets of genes; the responses of individual genes varied greatly across different stress types. In response to heat exposure over five days, a total of 324 gene loci were differentially expressed. Many heat-responsive genes had functions associated with protein turnover, metabolism, and the response to oxidative stress. We were also able to identify groups of co-regulated genes, the genes within which shared similar functions. Conclusion This is the first environmental genomic study to measure gene regulation in response to different environmental stressors in a natural population of a warm-adapted ectothermic vertebrate. We have shown that different types of environmental stress induce expression changes in genes with similar gene functions, but that the responses of individual genes vary between stress types. The functions of heat-responsive genes suggest that prolonged heat exposure leads to oxidative stress and protein damage, a challenge of the immune system, and the re-allocation of energy sources. This study hence offers insight into the effects of environmental stress on biological function and sheds light on the expected sensitivity of coral reef fishes to elevated temperatures in the future

Cerebellar output in zebrafish: an analysis of spatial patterns and topography in eurydendroid cell projections

The cerebellum is a brain region responsible for motor coordination and for refining motor programs. While a great deal is known about the structure and connectivity of the mammalian cerebellum, fundamental questions regarding its function in behaviour remain unanswered. Recently, the zebrafish has emerged as a useful model organism for cerebellar studies, owing in part to the similarity in cerebellar circuits between zebrafish and mammals. While the cell types composing their cerebellar cortical circuits are generally conserved with mammals, zebrafish lack deep cerebellar nuclei, and instead a majority of cerebellar output comes from a single type of neuron: the eurydendroid cell. To describe spatial patterns of cerebellar output in zebrafish, we have used genetic techniques to label and trace eurydendroid cells individually and en masse. We have found that cerebellar output targets the thalamus and optic tectum, and have confirmed the presence of presynaptic terminals from eurydendroid cells in these structures using a synaptically targeted GFP. By observing individual eurydendroid cells, we have shown that different medial-lateral regions of the cerebellum have eurydendroid cells projecting to different targets. Finally, we found topographic organisation in the connectivity between the cerebellum and the optic tectum, where more medial eurydendroid cells project to the rostral tectum while lateral cells project to the caudal tectum. These findings indicate that there is spatial logic underpinning cerebellar output in zebrafish with likely implications for cerebellar function

Evolution of gene function and regulatory control after whole-genome duplication: Comparative analyses in vertebrates

The significance of whole-genome duplications (WGD) for vertebrate evolution remains controversial, in part because the mechanisms by which WGD contributed to functional evolution or speciation are still incompletely characterized. Fish genomes provide an ideal context in which to examine the consequences of WGD, because the teleost lineage experienced an additional WGD soon after divergence from tetrapods and because five teleost genomes are available for comparative analysis. Here we present an integrated approach to characterize these post-duplication genomes based on genome-scale synteny, phylogenetic, temporal, and spatial gene expression and on protein sequence data. A minimum of 3%–4% of protein-coding loci have been retained in two copies in each of the five fish genomes, and many of these duplicates are key developmental genes that function as transcription factors or signaling molecules. Almost all duplicate gene pairs we examined have diverged in spatial and/or temporal expression during embryogenesis. A quarter of duplicate pairs have diverged in function via the acquisition of novel protein domains or via changes in the subcellular localization of their encoded proteins. We compared the spatial expression and protein domain architecture of zebrafish WGD-duplicates to those of their single mouse ortholog and found many examples supporting a model of neofunctionalization. WGD-duplicates have acquired novel protein domains more often than have single-copy genes. Post-WGD changes at the gene regulatory level were more common than changes at the protein level. We conclude that the most significant consequence of WGD for vertebrate evolution has been to enable more-specialized regulatory control of development via the acquisition of novel spatiotemporal expression domains. We find limited evidence that reciprocal gene loss led to reproductive isolation and speciation in this lineage