Comprehensive characterisation of compartment-specific long non-coding RNAs associated with pancreatic ductal adenocarcinoma.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDA) is a highly metastatic disease with limited therapeutic options. Genome and transcriptome analyses have identified signalling pathways and cancer driver genes with implications in patient stratification and targeted therapy. However, these analyses were performed in bulk samples and focused on coding genes, which represent a small fraction of the genome. DESIGN: We developed a computational framework to reconstruct the non-coding transcriptome from cross-sectional RNA-Seq, integrating somatic copy number alterations (SCNA), common germline variants associated to PDA risk and clinical outcome. We validated the results in an independent cohort of paired epithelial and stromal RNA-Seq derived from laser capture microdissected human pancreatic tumours, allowing us to annotate the compartment specificity of their expression. We employed systems and experimental biology approaches to interrogate the function of epithelial long non-coding RNAs (lncRNAs) associated with genetic traits and clinical outcome in PDA. RESULTS: We generated a catalogue of PDA-associated lncRNAs. We showed that lncRNAs define molecular subtypes with biological and clinical significance. We identified lncRNAs in genomic regions with SCNA and single nucleotide polymorphisms associated with lifetime risk of PDA and associated with clinical outcome using genomic and clinical data in PDA. Systems biology and experimental functional analysis of two epithelial lncRNAs (LINC00673 and FAM83H-AS1) suggest they regulate the transcriptional profile of pancreatic tumour samples and PDA cell lines. CONCLUSIONS: Our findings indicate that lncRNAs are associated with genetic marks of pancreatic cancer risk, contribute to the transcriptional regulation of neoplastic cells and provide an important resource to design functional studies of lncRNAs in PDA.

The novel enterochromaffin marker Lmx1a regulates serotonin biosynthesis in enteroendocrine cell lineages downstream of Nkx2.2.

Intestinal hormone-producing cells represent the largest endocrine system in the body, but remarkably little is known about enteroendocrine cell type specification in the embryo and adult. We analyzed stage- and cell type-specific deletions of Nkx2.2 and its functional domains in order to characterize its role in the development and maintenance of enteroendocrine cell lineages in the mouse duodenum and colon. Although Nkx2.2 regulates enteroendocrine cell specification in the duodenum at all stages examined, it controls the differentiation of progressively fewer enteroendocrine cell populations when deleted from Ngn3(+) progenitor cells or in the adult duodenum. During embryonic development Nkx2.2 regulates all enteroendocrine cell types, except gastrin and preproglucagon. In developing Ngn3(+) enteroendocrine progenitor cells, Nkx2.2 is not required for the specification of neuropeptide Y and vasoactive intestinal polypeptide, indicating that a subset of these cell populations derive from an Nkx2.2-independent lineage. In adult duodenum, Nkx2.2 becomes dispensable for cholecystokinin and secretin production. In all stages and Nkx2.2 mutant conditions, serotonin-producing enterochromaffin cells were the most severely reduced enteroendocrine lineage in the duodenum and colon. We determined that the transcription factor Lmx1a is expressed in enterochromaffin cells and functions downstream of Nkx2.2. Lmx1a-deficient mice have reduced expression of Tph1, the rate-limiting enzyme for serotonin biosynthesis. These data clarify the function of Nkx2.2 in the specification and homeostatic maintenance of enteroendocrine populations, and identify Lmx1a as a novel enterochromaffin cell marker that is also essential for the production of the serotonin biosynthetic enzyme Tph1.

ßlinc1 encodes a long noncoding RNA that regulates islet ß-cell formation and function.

Pancreatic ß cells are responsible for maintaining glucose homeostasis; their absence or malfunction results in diabetes mellitus. Although there is evidence that long noncoding RNAs (lncRNAs) play important roles in development and disease, none have been investigated in vivo in the context of pancreas development. In this study, we demonstrate that ßlinc1 (ß-cell long intergenic noncoding RNA 1), a conserved lncRNA, is necessary for the specification and function of insulin-producing ß cells through the coordinated regulation of a number of islet-specific transcription factors located in the genomic vicinity of ßlinc1. Furthermore, deletion of ßlinc1 results in defective islet development and disruption of glucose homeostasis in adult mice.

Nkx2.2 is expressed in a subset of enteroendocrine cells with expanded lineage potential.

There are two major stem cell populations in the intestinal crypt region that express either Bmi1 or Lgr5; however, it has been shown that other populations in the crypt can regain stemness. In this study, we demonstrate that the transcription factor NK2 homeobox 2 (Nkx2.2) is expressed in enteroendocrine cells located in the villus and crypt of the intestinal epithelium and is coexpressed with the stem cell markers Bmi1 and Lgr5 in a subset of crypt cells. To determine whether Nkx2.2-expressing enteroendocrine cells display cellular plasticity and stem cell potential, we performed genetic lineage tracing of the Nkx2.2-expressing population using Nkx2.2(Cre/+);R26RTomato mice. These studies demonstrated that Nkx2.2+ cells are able to give rise to all intestinal epithelial cell types in basal conditions. The proliferative capacity of Nkx2.2-expressing cells was also demonstrated in vitro using crypt organoid cultures. Injuring the intestine with irradiation, systemic inflammation, and colitis did not enhance the lineage potential of Nkx2.2-expressing cells. These findings demonstrate that a rare mature enteroendocrine cell subpopulation that is demarcated by Nkx2.2 expression display stem cell properties during normal intestinal epithelial homeostasis, but is not easily activated upon injury.

ATP-binding cassette transporters and sterol O-acyltransferases interact at membrane microdomains to modulate sterol uptake and esterification.

A key component of eukaryotic lipid homeostasis is the esterification of sterols with fatty acids by sterol O-acyltransferases (SOATs). The esterification reactions are allosterically activated by their sterol substrates, the majority of which accumulate at the plasma membrane. We demonstrate that in yeast, sterol transport from the plasma membrane to the site of esterification is associated with the physical interaction of the major SOAT, acyl-coenzyme A:cholesterol acyltransferase (ACAT)-related enzyme (Are)2p, with 2 plasma membrane ATP-binding cassette (ABC) transporters: Aus1p and Pdr11p. Are2p, Aus1p, and Pdr11p, unlike the minor acyltransferase, Are1p, colocalize to sterol and sphingolipid-enriched, detergent-resistant microdomains (DRMs). Deletion of either ABC transporter results in Are2p relocalization to detergent-soluble membrane domains and a significant decrease (53-36%) in esterification of exogenous sterol. Similarly, in murine tissues, the SOAT1/Acat1 enzyme and activity localize to DRMs. This subcellular localization is diminished upon deletion of murine ABC transporters, such as Abcg1, which itself is DRM associated. We propose that the close proximity of sterol esterification and transport proteins to each other combined with their residence in lipid-enriched membrane microdomains facilitates rapid, high-capacity sterol transport and esterification, obviating any requirement for soluble intermediary proteins.

Nkx2.2:Cre knock-in mouse line: a novel tool for pancreas- and CNS-specific gene deletion.

Nkx2.2 is a homeodomain-containing transcriptional regulator necessary for the appropriate differentiation of ventral neuronal populations in the spinal cord and hindbrain, and endocrine cell populations in the pancreas and intestine. In each tissue, Nkx2.2 inactivation leads to reciprocal cell fate alterations. To confirm the cell fate changes are due to respecification of Nkx2.2-expressing progenitors and to provide a novel tool for lineage tracing in the pancreas and CNS, we generated an Nkx2.2:Cre mouse line by knocking in a Cre-EGFP cassette into the Nkx2.2 genomic locus and inactivating endogenous Nkx2.2. The R26R-CAG-LSL-tdTomato reporter was used to monitor the specificity and efficiency of Nkx2.2:Cre activity; the tomato reporter faithfully recapitulated endogenous Nkx2.2 expression and could be detected as early as embryonic day (e) 9.25 in the developing CNS and was initiated shortly thereafter at e9.5 in the pancreas. Lineage analyses in the CNS confirmed the cell populations thought to be derived from Nkx2.2-expressing progenitor domains. Furthermore, lineage studies verified Nkx2.2 expression in the earliest pancreatic progenitors that give rise to all cell types of the pancreas; however they also revealed more robust Cre activity in the dorsal versus ventral pancreas. Thus, the Nkx2.2:Cre line provides a novel tool for gene manipulations in the CNS and pancreas.

Mapping the functional yeast ABC transporter interactome.

ATP-binding cassette (ABC) transporters are a ubiquitous class of integral membrane proteins of immense clinical interest because of their strong association with human disease and pharmacology. To improve our understanding of these proteins, we used membrane yeast two-hybrid technology to map the protein interactome of all of the nonmitochondrial ABC transporters in the model organism Saccharomyces cerevisiae and combined this data with previously reported yeast ABC transporter interactions in the BioGRID database to generate a comprehensive, integrated 'interactome'. We show that ABC transporters physically associate with proteins involved in an unexpectedly diverse range of functions. We specifically examine the importance of the physical interactions of ABC transporters in both the regulation of one another and in the modulation of proteins involved in zinc homeostasis. The interaction network presented here will be a powerful resource for increasing our fundamental understanding of the cellular role and regulation of ABC transporters.

The L6 domain tetraspanin Tm4sf4 regulates endocrine pancreas differentiation and directed cell migration.

The homeodomain transcription factor Nkx2.2 is essential for pancreatic development and islet cell type differentiation. We have identified Tm4sf4, an L6 domain tetraspanin family member, as a transcriptional target of Nkx2.2 that is greatly upregulated during pancreas development in Nkx2.2(-/-) mice. Tetraspanins and L6 domain proteins recruit other membrane receptors to form active signaling centers that coordinate processes such as cell adhesion, migration and differentiation. In this study, we determined that Tm4sf4 is localized to the ductal epithelial compartment and is prominent in the Ngn3(+) islet progenitor cells. We also established that pancreatic tm4sf4 expression and regulation by Nkx2.2 is conserved during zebrafish development. Loss-of-function studies in zebrafish revealed that tm4sf4 inhibits α and ß cell specification, but is necessary for ε cell fates. Thus, Tm4sf4 functional output opposes that of Nkx2.2. Further investigation of how Tm4sf4 functions at the cellular level in vitro showed that Tm4sf4 inhibits Rho-activated cell migration and actin organization in a ROCK-independent fashion. We propose that the primary role of Nkx2.2 is to inhibit Tm4sf4 in endocrine progenitor cells, allowing for delamination, migration and/or appropriate cell fate decisions. Identification of a role for Tm4sf4 during endocrine differentiation provides insight into islet progenitor cell behaviors and potential targetable regenerative mechanisms.

Cumulative mutations affecting sterol biosynthesis in the yeast Saccharomyces cerevisiae result in synthetic lethality that is suppressed by alterations in sphingolipid profiles.

UPC2 and ECM22 belong to a Zn(2)-Cys(6) family of fungal transcription factors and have been implicated in the regulation of sterol synthesis in Saccharomyces cerevisiae and Candida albicans. Previous reports suggest that double deletion of these genes in S. cerevisiae is lethal depending on the genetic background of the strain. In this investigation we demonstrate that lethality of upc2Delta ecm22Delta in the S288c genetic background is attributable to a mutation in the HAP1 transcription factor. In addition we demonstrate that strains containing upc2Delta ecm22Delta are also inviable when carrying deletions of ERG6 and ERG28 but not when carrying deletions of ERG3, ERG4, or ERG5. It has previously been demonstrated that UPC2 and ECM22 regulate S. cerevisiae ERG2 and ERG3 and that the erg2Delta upc2Delta ecm22Delta triple mutant is also synthetically lethal. We used transposon mutagenesis to isolate viable suppressors of hap1Delta, erg2Delta, erg6Delta, and erg28Delta in the upc2Delta ecm22Delta genetic background. Mutations in two genes (YND1 and GDA1) encoding apyrases were found to suppress the synthetic lethality of three of these triple mutants but not erg2Delta upc2Delta ecm22Delta. We show that deletion of YND1, like deletion of GDA1, alters the sphingolipid profiles, suggesting that changes in sphingolipids compensate for lethality produced by changes in sterol composition and abundance.

Mutagenesis of the putative sterol-sensing domain of yeast Niemann Pick C-related protein reveals a primordial role in subcellular sphingolipid distribution.

Lipid movement between organelles is a critical component of eukaryotic membrane homeostasis. Niemann Pick type C (NP-C) disease is a fatal neurodegenerative disorder typified by lysosomal accumulation of cholesterol and sphingolipids. Expression of yeast NP-C-related gene 1 (NCR1), the orthologue of the human NP-C gene 1 (NPC1) defective in the disease, in Chinese hamster ovary NPC1 mutant cells suppressed lipid accumulation. Deletion of NCR1, encoding a transmembrane glycoprotein predominantly residing in the vacuole of normal yeast, gave no phenotype. However, a dominant mutation in the putative sterol-sensing domain of Ncr1p conferred temperature and polyene antibiotic sensitivity without changes in sterol metabolism. Instead, the mutant cells were resistant to inhibitors of sphingolipid biosynthesis and super sensitive to sphingosine and C2-ceramide. Moreover, plasma membrane sphingolipids accumulated and redistributed to the vacuole and other subcellular membranes of the mutant cells. We propose that the primordial function of these proteins is to recycle sphingolipids and that defects in this process in higher eukaryotes secondarily result in cholesterol accumulation.

Transcriptional profiling identifies two members of the ATP-binding cassette transporter superfamily required for sterol uptake in yeast.

In contrast to lipoprotein-mediated sterol uptake, free sterol influx by eukaryotic cells is poorly understood. To identify components of non-lipoprotein-mediated sterol uptake, we utilized strains of Saccharomyces cerevisiae that accumulate exogenous sterol due to a neomorphic mutation in the transcription factor, UPC2. Two congenic upc2-1 strains, differing quantitatively in aerobic sterol uptake due to a modifying mutation in the HAP1 transcription factor, were compared using DNA microarrays. We identified 9 genes as responsive to UPC2 that were also induced under anaerobiosis, when sterol uptake is essential. Deletion mutants in these genes were assessed for sterol influx in the upc2-1 background. UPC2 itself was up-regulated under these conditions and was required for aerobic sterol influx. Deletion of the ATP-binding cassette transporters YOR011w (AUS1) or PDR11, or a putative cell wall protein encoded by DAN1, significantly reduced sterol influx. Sodium azide and vanadate inhibited sterol uptake, consistent with the participation of ATP-binding cassette transporters. We hypothesized that the physiological role of Aus1p and Pdr11p is to mediate sterol uptake when sterol biosynthesis is compromised. Accordingly, expression of AUS1 or PDR11 was required for anaerobic growth and sterol uptake. We proposed similar molecules may be important components of sterol uptake in all eukaryotes.