SUMMARY UHRF1 is an essential regulator of DNA methylation that is highly expressed in many cancers. Here, we use transgenic zebrafish, cultured cells and human tumors to demonstrate that UHRF1 is an oncogene. UHRF1 overexpression in zebrafish hepatocytes destabilizes and delocalizes DNMT1, causes DNA hypomethylation and Tp53-mediated senescence. Hepatocellular carcinoma (HCC) emerges when senescence is bypassed. tp53 mutation both alleviates senescence and accelerates tumor onset. Human HCCs recapitulate this paradigm, as UHRF1 overexpression defines a subclass of aggressive HCCs characterized by genomic instability, TP53 mutation and abrogation of the TP53-mediated senescence program. We propose that UHRF1 overexpression is a mechanism underlying DNA hypomethylation in cancer cells and that senescence is a primary means of restricting tumorigenesis due to epigenetic disruption.

UHRF1 (ubiquitin-like, containing PHD and RING finger domains, 1) recruits DNMT1 to hemimethylated DNA during replication and is essential for maintaining DNA methylation. uhrf1 mutant zebrafish have global DNA hypomethylation and display embryonic defects, including a small liver, and they die as larvae. We make the surprising finding that, despite their reduced organ size, uhrf1 mutants express high levels of genes controlling S-phase and have many more cells undergoing DNA replication, as measured by BrdU incorporation. In contrast to wild-type hepatocytes, which are continually dividing during hepatic outgrowth and thus dilute the BrdU label, uhrf1 mutant hepatocytes retain BrdU throughout outgrowth, reflecting cell cycle arrest. Pulse-chase-pulse experiments with BrdU and EdU, and DNA content analysis indicate that uhrf1 mutant cells undergo DNA re-replication and that apoptosis is the fate of many of the rereplicating and arrested hepatocytes. Importantly, the DNA rereplication phenotype and hepatic outgrowth failure are preceded by global loss of DNA methylation. Moreover, uhrf1 mutants are phenocopied by mutation of dnmt1, and Dnmt1 knockdown in uhrf1 mutants enhances their small liver phenotype. Together, these data indicate that unscheduled DNA replication and failed cell cycle progression leading to apoptosis are the mechanisms by which DNA hypomethylation prevents organ expansion in uhrf1 mutants. We propose that cell cycle arrest leading to apoptosis is a strategy that restricts propagation of epigenetically damaged cells during embryogenesis.

Edited by Qi-Qun Tang Nucleotide synthesis is essential to proliferating cells, but the preferred precursors for de novo biosynthesis are not defined in human cancer tissues. We have employed multiplexed stable isotope-resolved metabolomics to track the metabolism of [ 13 C 6 ]glucose, D 2-glycine, [ 13 C 2 ]glycine, and D 3-serine into purine nucleotides in freshly resected cancerous and matched noncancerous lung tissues from nonsmall cell lung cancer (NSCLC) patients, and we compared the metabolism with established NSCLC PC9 and A549 cell lines in vitro. Surprisingly, [ 13 C 6 ]glucose was the best carbon source for purine synthesis in human NSCLC tissues, in contrast to the noncancerous lung tissues from the same patient, which showed lower mitotic indices and MYC expression. We also observed that D 3-Ser was preferentially incorporated into purine rings over D 2-glycine in both tissues and cell lines. MYC suppression attenuated [ 13 C 6 ]glucose, D 3-serine, and [ 13 C 2 ]glycine incorporation into purines and reduced proliferation in PC9 but not in A549 cells. Using detailed kinetic modeling, we showed that the preferred use of glucose as a carbon source for purine ring synthesis in NSCLC tissues involves cytoplasmic activation/compartmentation of the glucose-to-serine pathway and enhanced reversed one-carbon fluxes that attenuate exogenous serine incorporation into purines. Our findings also indicate that the substrate for de novo nucleotide synthesis differs profoundly between cancer cell lines and fresh human lung cancer tissues; the latter preferred glucose to exogenous serine or glycine but not the former. This distinction in substrate utilization in purine synthesis in human cancer tissues should be considered when targeting one-carbon metabolism for cancer therapy. De novo nucleotide biosynthesis is required to meet the demand for maintaining energy, nucleotide levels, and new nucleic acids in dividing cells (1, 2). Synthesis of pyrimidine nucleotides in cancer cells utilizes glucose and glutamine for the carbon of the uracil and cytosine rings, which then condense with the ribose subunit primarily derived from glucose via the pentose-phosphate pathway (1, 3). The purine nucleotides are synthesized by building the purine ring directly onto phosphoribosyl pyrophosphate (PRPP) 6 using glycine (direct route), CO 2 , and glycine-derived N 10-formyl tetrahydrofolate (CHO-THF, indirect route) as the carbon sources for C4,5, C6, and C2,8, respectively (cf. Fig. 1). Cellular glycine may derive from glucose via de novo synthesis from serine (1, 4), via protein degradation, and/or via uptake from external sources, i.e. common cell culture media (1, 2) or the circulating blood (5). Serine derived from glucose or external sources is converted (through one-carbon metabolism) into Gly and 5,10-methylene THF (CH 2-THF) via the serine hydroxymethyltransferase (SHMT) activity (Fig. 1). CH 2-THF is oxidized to CHO-THF via the methylene tetrahydrofolate cyclohydrolase activity, prior to incorporation into purine rings.

Complex cytoplasmic nucleotide-sensing mechanisms can recognize foreign DNA based on a lack of methylation and initiate an immune response to clear the infection. Zebrafish embryos with global DNA hypomethylation caused by mutations in the () or () genes exhibit a robust interferon induction characteristic of the first line of defense against viral infection. We found that this interferon induction occurred in non-immune cells and examined whether intracellular viral sensing pathways in these cells were the trigger. RNA-seq analysis of and mutants revealed widespread induction of Class I retrotransposons and activation of cytoplasmic DNA viral sensors. Attenuating Sting, phosphorylated Tbk1 and, importantly, blocking reverse transcriptase activity suppressed the expression of interferon genes in mutants. Thus, activation of transposons in cells with global DNA hypomethylation mimics a viral infection by activating cytoplasmic DNA sensors. This suggests that antiviral pathways serve as surveillance of cells that have derepressed intragenomic parasites due to DNA hypomethylation.

BackgroundPrevious studies suggested that the metabolism is differently reprogrammed in the major subtypes of non-small cell lung cancer (NSCLC), squamous cell carcinomas (SCC) and adenocarcinomas (AdC). However, a comprehensive analysis of this differential metabolic reprogramming is lacking.MethodsPublicly available gene expression data from human lung cancer samples and cell lines were analysed. Stable isotope resolved metabolomics were performed on SCC and ADC tumours in human patients and in freshly resected tumour slices.ResultsAnalysis of multiple transcriptomics data from human samples identified a SCC-distinguishing enzyme gene signature. SCC tumours from patients infused with [U-13C]-glucose and SCC tissue slices incubated with stable isotope tracers demonstrated differential glucose and glutamine catabolism compared to AdCs or non-cancerous lung, confirming increased activity through pathways defined by the SCC metabolic gene signature. Furthermore, the upregulation of Notch target genes was a distinguishing feature of SCCs, which correlated with the metabolic signature. Notch and MYC-driven murine lung tumours recapitulated the SCC-distinguishing metabolic reprogramming. However, the differences between SCCs and AdCs disappear in established cell lines in 2D culture.ConclusionsOur data emphasise the importance of studying lung cancer metabolism in vivo. They also highlight potential targets for therapeutic intervention in SCC patients including differentially expressed enzymes that catalyse reactions in glycolysis, glutamine catabolism, serine, nucleotide and glutathione biosynthesis.

Background: L-type calcium channels (LTCC) regulate calcium entry into cardiomyocytes. CACNB2 (b2) LTCC auxiliary subunits traffic the pore-forming CACNA subunit to the membrane and modulate channel kinetics. b2 is a membrane associated guanylate kinase (MAGUK) protein. A major role of MAGUK proteins is to scaffold cellular junctions and multiprotein complexes. Results: To investigate developmental functions for b2.1, we depleted it in zebrafish using morpholinos. b2.1-depleted embryos developed compromised cardiac function by 48 hr postfertilization, which was ultimately lethal. b2.1 contractility defects were mimicked by pharmacological depression of LTCC, and rescued by LTCC stimulation, suggesting b2.1 phenotypes are at least in part LTCC-dependent. Morphological studies indicated that b2.1 contributes to heart size by regulating the rate of ventricle cell proliferation, and by modulating the transition of outer curvature cells to an elongated cell shape during chamber ballooning. In addition, b2.1-depleted cardiomyocytes failed to accumulate N-cadherin at the membrane, and dissociated easily from neighboring myocytes under stress. Conclusions: Hence, we propose that b2.1 may also function in the heart as a MAGUK scaffolding unit to maintain N-cadherin-based adherens junctions and heart tube integrity. Developmental Dynamics 241:648-662, 2012. V C 2012 Wiley Periodicals Inc.

The original article to which this Erratum refers was published in Developmental Dynamics 241:648-662.The cacnb2.1-MO1 morpholino sequence cited in the original article (5 0 -CCACCAGTCATTGTTAAACTTCTGT-3 0 ) is incorrect. The correct cacnb2.1-MO1 sequnence is: 5 0 -ATGCAACAGACACCTTGAGTAAAAG-3 0 . The authors regret this error.

Background Nanopore sequencing technology has revolutionized the field of genome biology with its ability to generate extra-long reads that can resolve regions of the genome that were previously inaccessible to short-read sequencing platforms. Over 50% of the zebrafish genome consists of difficult to map, highly repetitive, low complexity elements that pose inherent problems for short-read sequencers and assemblers. Results We used long-read nanopore sequencing to generate a de novo assembly of the zebrafish genome and compared our assembly to the current reference genome, GRCz11. The new assembly identified 1697 novel insertions and deletions over one kilobase in length and placed 106 previously unlocalized scaffolds. We also discovered additional sites of retrotransposon integration previously unreported in GRCz11 and observed the expression of these transposable elements in adult zebrafish under physiologic conditions, implying they have active mobility in the zebrafish genome and contribute to the ever-changing genomic landscape. Conclusions We used nanopore sequencing to improve upon and resolve the issues plaguing the current zebrafish reference assembly, GRCz11. Zebrafish is a prominent model of human disease, and our corrected assembly will be useful for studies relying on interspecies comparisons and precise linkage of genetic events to disease phenotypes.