Germline and Somatic Genetic Variants in the p53 Pathway Interact to Affect Cancer Risk, Progression, and Drug Response.

Insights into oncogenesis derived from cancer susceptibility loci (SNP) hold the potential to facilitate better cancer management and treatment through precision oncology. However, therapeutic insights have thus far been limited by our current lack of understanding regarding both interactions of these loci with somatic cancer driver mutations and their influence on tumorigenesis. For example, although both germline and somatic genetic variation to the p53 tumor suppressor pathway are known to promote tumorigenesis, little is known about the extent to which such variants cooperate to alter pathway activity. Here we hypothesize that cancer risk-associated germline variants interact with somatic TP53 mutational status to modify cancer risk, progression, and response to therapy. Focusing on a cancer risk SNP (rs78378222) with a well-documented ability to directly influence p53 activity as well as integration of germline datasets relating to cancer susceptibility with tumor data capturing somatically-acquired genetic variation provided supportive evidence for this hypothesis. Integration of germline and somatic genetic data enabled identification of a novel entry point for therapeutic manipulation of p53 activities. A cluster of cancer risk SNPs resulted in increased expression of prosurvival p53 target gene KITLG and attenuation of p53-mediated responses to genotoxic therapies, which were reversed by pharmacologic inhibition of the prosurvival c-KIT signal. Together, our results offer evidence of how cancer susceptibility SNPs can interact with cancer driver genes to affect cancer progression and identify novel combinatorial therapies. SIGNIFICANCE: These results offer evidence of how cancer susceptibility SNPs can interact with cancer driver genes to affect cancer progression and present novel therapeutic targets.

ETS factors are required but not sufficient for specific patterns of enhancer activity in different endothelial subtypes.

Correct vascular differentiation requires distinct patterns of gene expression in different subtypes of endothelial cells. Members of the ETS transcription factor family are essential for the transcriptional activation of arterial and angiogenesis-specific gene regulatory elements, leading to the hypothesis that they play lineage-defining roles in arterial and angiogenic differentiation directly downstream of VEGFA signalling. However, an alternative explanation is that ETS binding at enhancers and promoters is a general requirement for activation of many endothelial genes regardless of expression pattern, with subtype-specificity provided by additional factors. Here we use analysis of Ephb4 and Coup-TFII (Nr2f2) vein-specific enhancers to demonstrate that ETS factors are equally essential for vein, arterial and angiogenic-specific enhancer activity patterns. Further, we show that ETS factor binding at these vein-specific enhancers is enriched by VEGFA signalling, similar to that seen at arterial and angiogenic enhancers. However, while arterial and angiogenic enhancers can be activated by VEGFA in vivo, the Ephb4 and Coup-TFII venous enhancers are not, suggesting that the specificity of VEGFA-induced arterial and angiogenic enhancer activity occurs via non-ETS transcription factors. These results support a model in which ETS factors are not the primary regulators of specific patterns of gene expression in different endothelial subtypes.

Venous identity requires BMP signalling through ALK3.

Venous endothelial cells are molecularly and functionally distinct from their arterial counterparts. Although veins are often considered the default endothelial state, genetic manipulations can modulate both acquisition and loss of venous fate, suggesting that venous identity is the result of active transcriptional regulation. However, little is known about this process. Here we show that BMP signalling controls venous identity via the ALK3/BMPR1A receptor and SMAD1/SMAD5. Perturbations to TGF-ß and BMP signalling in mice and zebrafish result in aberrant vein formation and loss of expression of the venous-specific gene Ephb4, with no effect on arterial identity. Analysis of a venous endothelium-specific enhancer for Ephb4 shows enriched binding of SMAD1/5 and a requirement for SMAD binding motifs. Further, our results demonstrate that BMP/SMAD-mediated Ephb4 expression requires the venous-enriched BMP type I receptor ALK3/BMPR1A. Together, our analysis demonstrates a requirement for BMP signalling in the establishment of Ephb4 expression and the venous vasculature.

MEF2 transcription factors are key regulators of sprouting angiogenesis.

Angiogenesis, the fundamental process by which new blood vessels form from existing ones, depends on precise spatial and temporal gene expression within specific compartments of the endothelium. However, the molecular links between proangiogenic signals and downstream gene expression remain unclear. During sprouting angiogenesis, the specification of endothelial cells into the tip cells that lead new blood vessel sprouts is coordinated by vascular endothelial growth factor A (VEGFA) and Delta-like ligand 4 (Dll4)/Notch signaling and requires high levels of Notch ligand DLL4. Here, we identify MEF2 transcription factors as crucial regulators of sprouting angiogenesis directly downstream from VEGFA. Through the characterization of a Dll4 enhancer directing expression to endothelial cells at the angiogenic front, we found that MEF2 factors directly transcriptionally activate the expression of Dll4 and many other key genes up-regulated during sprouting angiogenesis in both physiological and tumor vascularization. Unlike ETS-mediated regulation, MEF2-binding motifs are not ubiquitous to all endothelial gene enhancers and promoters but are instead overrepresented around genes associated with sprouting angiogenesis. MEF2 target gene activation is directly linked to VEGFA-induced release of repressive histone deacetylases and concurrent recruitment of the histone acetyltransferase EP300 to MEF2 target gene regulatory elements, thus establishing MEF2 factors as the transcriptional effectors of VEGFA signaling during angiogenesis.

An Intronic Flk1 Enhancer Directs Arterial-Specific Expression via RBPJ-Mediated Venous Repression.

OBJECTIVE: The vascular endothelial growth factor (VEGF) receptor Flk1 is essential for vascular development, but the signaling and transcriptional pathways by which its expression is regulated in endothelial cells remain unclear. Although previous studies have identified 2 Flk1 regulatory enhancers, these are dispensable for Flk1 expression, indicating that additional enhancers contribute to Flk1 regulation in endothelial cells. In the present study, we sought to identify Flk1 enhancers contributing to expression in endothelial cells. APPROACH AND RESULTS: A region of the 10th intron of the Flk1 gene (Flk1in10) was identified as a putative enhancer and tested in mouse and zebrafish transgenic models. This region robustly directed reporter gene expression in arterial endothelial cells. Using a combination of targeted mutagenesis of transcription factor-binding sites and gene silencing of transcription factors, we found that Gata and Ets factors are required for Flk1in10 enhancer activity in all endothelial cells. Furthermore, we showed that activity of the Flk1in10 enhancer is restricted to arteries through repression of gene expression in venous endothelial cells by the Notch pathway transcriptional regulator Rbpj. CONCLUSIONS: This study demonstrates a novel mechanism of arterial-venous identity acquisition, indicates a direct link between the Notch and VEGF signaling pathways, and illustrates how cis-regulatory diversity permits differential expression outcomes from a limited repertoire of transcriptional regulators.

Robust homeostasis of Presenilin1 protein levels by transcript regulation.

The Presenilin proteins are essential facilitators of numerous developmental and cell signaling pathways. Point mutations in the human PRESENILIN genes (including mutations affecting splicing) have been linked to familial Alzheimer's disease. Zebrafish possess orthologues of the human PRESENILIN1 and PRESENILIN2 genes. We previously investigated forced aberrant splicing of zebrafish presenilin1 and discovered that high levels of incorporation into spliced transcripts of the intron cognate with human PRESENILIN1 intron 8 resulted in little or no change in Presenilin1 protein level and no identifiable embryonic phenotype. We now demonstrate that zebrafish embryos maintain relatively stable levels of normal Presenilin1 transcript and protein despite accumulating large amounts of aberrantly spliced presenilin1 transcript. We also show that increasing the levels of Presenilin1 protein decreases normal presenilin1 transcription. These two independent lines of evidence and the fact that blockage of Presenilin1 translation increases presenilin1 transcription support that regulation of presenilin1 transcript levels plays a major role in the homeostasis of Presenilin1 protein levels, presumably via a feedback mechanism that monitors the levels of Presenilin1 protein.

Transgenic zebrafish recapitulating tbx16 gene early developmental expression.

We describe the creation of a transgenic zebrafish expressing GFP driven by a 7.5 kb promoter region of the tbx16 gene. This promoter segment is sufficient to recapitulate early embryonic expression of endogenous tbx16 in the presomitic mesoderm, the polster and, subsequently, in the hatching gland. Expression of GFP in the transgenic lines later in development diverges to some extent from endogenous tbx16 expression with the serendipitous result that one line expresses GFP specifically in commissural primary ascending (CoPA) interneurons of the developing spinal cord. Using this line we demonstrate that the gene mafba (valentino) is expressed in CoPA interneurons.

Zebrafish aplnra functions in epiboly.

BACKGROUND: The zebrafish, Danio rerio, possesses the paralogous genes aplnra and aplnrb that are duplicates of an ancestral orthologue of the human APLNR gene encoding a G-protein coupled receptor that binds the peptide ligand APELIN and is required for normal cardiovascular function. aplnrb is required for migration of cells contributing to heart development in zebrafish embryos. aplnra is transcribed in a complex pattern during early development but its function in embryogenesis is largely unknown. FINDINGS: Blockage of translation of aplnra mRNA in zebrafish embryos results in retarded or failed epiboly with the blastoderm apparently disconnected from the nuclei of the yolk syncytial layer. Gastrulation is also defective. Failure of correct tail extension is observed with ectopic structures resembling somites positioned dorsal to the spinal cord. CONCLUSION: aplnra, unlike its duplicate aplnrb, is essential for normal epiboly, although this function appears to be independent of signalling activated by zebrafish Apelin. The defects in epiboly caused by loss of aplnra activity appear, at least partially, to be due to a requirement for aplnra activity in the yolk syncytial layer.

Selective neuronal requirement for huntingtin in the developing zebrafish.

Huntington's disease shares a common molecular basis with eight other neurodegenerative diseases, expansion of an existing polyglutamine tract. In each case, this repeat tract occurs within otherwise unrelated proteins. These proteins show widespread and overlapping patterns of expression in the brain and yet the diseases are distinguished by neurodegeneration in a specific subset of neurons that are most sensitive to the mutation. It has therefore been proposed that expansion of the polyglutamine region in these genes may result in perturbation of the normal function of the respective proteins, and that this perturbation in some way contributes to the neuronal specificity of these diseases. The normal functions of these proteins have therefore become a focus for investigation as potential pathogenic pathways. We have used synthetic antisense morpholinos to inhibit the translation of huntingtin mRNA during early zebrafish development and have previously reported the effects of huntingtin reduction on iron transport and homeostasis. Here we report an analysis of the effects of huntingtin loss-of-function on the developing nervous system, observing distinct defects in morphology of neuromasts, olfactory placode and branchial arches. The potential common origins of these defects were explored, revealing impaired formation of the anterior-most region of the neural plate as indicated by reduced pre-placodal and telencephalic gene expression with no effect on mid- or hindbrain formation. These investigations demonstrate a specific 'rate-limiting' role for huntingtin in formation of the telencephalon and the pre-placodal region, and differing levels of requirement for huntingtin function in specific nerve cell types.

Independent and cooperative action of Psen2 with Psen1 in zebrafish embryos.

Presenilin1 (PSEN1) and presenilin2 (PSEN2) are involved in the processing of type-1 transmembrane proteins including the amyloid precursor protein (APP), Notch and several others. PSEN1 has been shown to be crucial for proteolytic cleavage of Notch in developing animal embryos. Mouse embryos lacking Psen1 function show disturbed neurogenesis and somite formation, resembling Notch pathway mutants. However, loss of Psen2 activity reveals only a minor phenotype. Zebrafish embryos are a valuable tool for analysis of the molecular genetic control of cell differentiation since endogenous gene expression can be modulated in subtle and complex ways to give a phenotypic readout. Using injection of morpholino antisense oligonucleotides to inhibit protein translation in zebrafish embryos, we show that reduced Psen2 activity decreases the number of melanocytes in the trunk but not in the cranial area at 2 days post fertilisation (dpf). Reduced Psen2 activity apparently reduces Notch signalling resulting in perturbed spinal neurogenin1 (neurog1) expression, neurogenesis and trunk and tail neural crest development. Similar effects are seen for reduced Psen1 activity. These results suggest that Psen2 plays a more prominent role in Notch signalling and embryo development in zebrafish than in mammals. Intriguingly, decreased Psen2 activity increases the number of Dorsal Longitudinal Ascending (DoLA) interneurons in the spinal cord while decreased Psen1 activity has no effect. However, the effect on DoLAs of reduced Psen2 can be ameliorated by Psen1 loss. The effects of changes in Psen2 activity on DoLA interneurons and other cells in zebrafish embryos provide bioassays for more detailed dissection of Psen2 function.

Altering presenilin gene activity in zebrafish embryos causes changes in expression of genes with potential involvement in Alzheimer's disease pathogenesis.

Aberrant splicing and point mutations in the human presenilin genes, PSEN1 and PSEN2, have been linked to familial forms of Alzheimer's disease. We have previously described that low-level aberrant splicing of exon 8 in zebrafish psen1 transcripts in zebrafish embryos produces potent dominant negative effects that increase psen1 transcription, cause a dramatic hydrocephalus phenotype, decreased pigmentation and other developmental defects. Similar effects are also observed after low-level interference with splicing of exon 8 of psen2. To determine the molecular etiology of these effects, we performed microarray analyses of global gene expression changes. Of the 100 genes that showed greatest dysregulation after either psen1 or psen2 manipulation, 12 genes were common to both treatments. Five of these have known function and showed increased expression: cyclin G1 (ccng1), prosaposin (psap), cathepsin Lb (ctslb), heat shock protein 70kDa (hsp70) and hatching enzyme 1 (he1). We used phylogenetic and conserved synteny analysis to confirm the orthology of zebrafish ccng1 with human CCNG1. We analyzed the expression of zebrafish ccng1 in developing embryos to 24 hours post fertilization (hpf). Decreased ccng1 expression does not rescue the hydrocephalus or pigmentation phenotypes of embryos with aberrant splicing of psen1 exon 8.

Interference with splicing of Presenilin transcripts has potent dominant negative effects on Presenilin activity.

Missense mutations in the PRESENILIN1 (PSEN1) gene frequently underlie familial Alzheimer's disease (FAD). Nonsense and most splicing mutations result in the synthesis of truncated peptides, and it has been assumed that truncated PSEN1 protein is functionless so that heterozygotes for these mutations are unaffected. Some FAD mutations affecting PSEN1 mRNA splicing cause loss of exon 8 or 9 sequences while maintaining the reading frame. We attempted to model these exon-loss mutations in zebrafish embryos by injecting morpholino antisense oligonucleotides (morpholinos) directed against splice acceptor sites in zebrafish psen1 transcripts. However, this produced cryptic changes in splicing potentially forming mRNAs encoding truncated presenilin proteins. Aberrant splicing in the region between exons 6 and 8 produces potent dominant negative effects on Psen1 protein activity, including Notch signalling, and causes a hydrocephalus phenotype. Reductions in Psen1 activity feedback positively to increase psen1 transcription through a mechanism apparently independent of gamma-secretase. We present evidence that the dominant negative effects are mediated through production of truncated Psen1 peptides that interfere with the normal activity of both Psen1 and Psen2. Mutations causing such truncations would be dominant lethal in embryo development. Somatic cellular changes in ageing cells that interfere with PSEN1 splicing, or otherwise cause protein truncation, might contribute to sporadic Alzheimer's disease, cancer and other diseases.

Developmental control of Presenilin1 expression, endoproteolysis, and interaction in zebrafish embryos.

Dominant mutations in presenilin1 (PS1) and presenilin2 (PS2) are a major cause of early-onset Alzheimer's disease. In this report we analyze the expression of the zebrafish presenilin1 (Psen1) and presenilin2 (Psen2) proteins during embryogenesis. We demonstrate that Psen1 and Psen2 holoproteins are relatively abundant in zebrafish embryos and are proteolytically processed. Psen1 is maternally expressed, whereas Psen2 is expressed at later stages during development. The Psen1 C-terminal proteolytic fragment (CTF) is present at varying levels during embryogenesis, indicating the existence of developmental control mechanisms regulating its production. We examine the codependency of Psen1 and Psen2 expression during early embryogenesis. Forced overexpression of psen2 increases expression of Psen2 holoprotein, but not the N-terminal fragment (NTF), indicating that levels of Psen2 NTF are strictly controlled. Overexpression of psen2 did not alter levels of Psen1 holoprotein, CTF, or higher molecular weight complexes. Reduction of Psen1 activity in zebrafish embryos produces similar developmental defects to those seen for loss of PS1 activity in knockout mice. The relevance of these results to previous work on presenilin protein regulation and function are discussed. Our work shows that zebrafish embryos are a valid and valuable system in which to study presenilin interactions, regulation, and function.

Identification of a second presenilin gene in zebrafish with similarity to the human Alzheimer's disease gene presenilin2.

Presenilins play prominent roles in the molecular pathogenesis of Alzheimer's disease and during embryo development. We have isolated a zebrafish presenilin orthologue (pre2), which shows a high degree of sequence identity to the human PS2 protein. Zebrafish pre2 is maternally and ubiquitously expressed during early embryo development, whereas Pre2 protein expression is initiated between 6 and 12 hours post fertilisation (hpf), suggesting strict regulation of pre2 translation. pre2 expression is especially high in neural-crest-derived melanocytes.