High resolution annotation of zebrafish transcriptome using long-read sequencing.

With the emergence of zebrafish as an important model organism, a concerted effort has been made to study its transcriptome. This effort is limited, however, by gaps in zebrafish annotation, which are especially pronounced concerning transcripts dynamically expressed during zygotic genome activation (ZGA). To date, short-read sequencing has been the principal technology for zebrafish transcriptome annotation. In part because these sequence reads are too short for assembly methods to resolve the full complexity of the transcriptome, the current annotation is rudimentary. By providing direct observation of full-length transcripts, recently refined long-read sequencing platforms can dramatically improve annotation coverage and accuracy. Here, we leveraged the SMRT platform to study the transcriptome of zebrafish embryos before and after ZGA. Our analysis revealed additional novelty and complexity in the zebrafish transcriptome, identifying 2539 high-confidence novel transcripts that originated from previously unannotated loci and 1835 high-confidence new isoforms in previously annotated genes. We validated these findings using a suite of computational approaches including structural prediction, sequence homology, and functional conservation analyses, as well as by confirmatory transcript quantification with short-read sequencing data. Our analyses provided insight into new homologs and paralogs of functionally important proteins and noncoding RNAs, isoform switching occurrences, and different classes of novel splicing events. Several novel isoforms representing distinct splicing events were validated through PCR experiments, including the discovery and validation of a novel 8-kb transcript spanning multiple mir-430 elements, an important driver of early development. Our study provides a significantly improved zebrafish transcriptome annotation resource.

Zebrafish Discoveries in Cancer Epigenetics.

The cancer epigenome is fundamentally different than that of normal cells. How these differences arise in and contribute to carcinogenesis is not known, and studies using model organisms such as zebrafish provide an opportunity to address these important questions. Modifications of histones and DNA comprise the complex epigenome, and these influence chromatin structure, genome stability and gene expression, all of which are fundamental to the cellular changes that cause cancer. The cancer genome atlas covers the wide spectrum of genetic changes associated with nearly every cancer type, however, this catalog is currently uni-dimensional. As the pattern of epigenetic marks and chromatin structure in cancer cells is described and overlaid on the mutational landscape, the map of the cancer genome becomes multi-dimensional and highly complex. Two major questions remain in the field: (1) how the epigenome becomes repatterned in cancer and (2) which of these changes are cancer-causing. Zebrafish provide a tractable in vivo system to monitor the epigenome during transformation and to identify epigenetic drivers of cancer. In this chapter, we review principles of cancer epigenetics and discuss recent work using zebrafish whereby epigenetic modifiers were established as cancer driver genes, thus providing novel insights into the mechanisms of epigenetic reprogramming in cancer.

UHRF1 regulation of Dnmt1 is required for pre-gastrula zebrafish development.

Landmark epigenetic events underlie early embryonic development, yet how epigenetic modifiers are regulated to achieve rapid epigenome re-patterning is not known. Uhrf1 and DNA methyltransferase 1 (Dnmt1) are known to largely mediate maintenance DNA methylation and Uhrf1 is also required for both Dnmt1 localization and stability. Here, we investigate how these two key epigenetic modifiers regulate early zebrafish development and characterize the developmental consequences of disrupting their homeostatic relationship. Unlike Uhrf1 knockdown, which causes developmental arrest and death prior to gastrulation, overexpression of human UHRF1 (WT-UHRF1) caused asymmetric epiboly, inefficient gastrulation and multi-systemic defects. UHRF1 phosphorylation was previously demonstrated as essential for zebrafish embryogenesis, and we found that penetrance of the asymmetric epiboly phenotype was significantly increased in embryos injected with mRNA encoding non-phosphorylatable UHRF1 (UHRF1(S661A)). Surprisingly, both WT-UHRF1 and UHRF1(S661A) overexpression caused DNA hypomethylation. However, since other approaches that caused an equivalent degree of DNA hypomethylation did not cause the asymmetric epiboly phenotype, we conclude that bulk DNA methylation is not the primary mechanism. Instead, UHRF1(S661A) overexpression resulted in accumulation of Dnmt1 protein and the overexpression of both WT and a catalytically inactive Dnmt1 phenocopied the assymetric epiboly phenotype. Dnmt1 knockdown suppressed the phenotype caused by UHRF1(S661A) overexpression, and Uhrf1 knockdown suppressed the effect of Dnmt1 overexpression. Therefore, we conclude that the interaction between these two proteins is the mechanism underlying the gastrulation defects. This indicates that Dnmt1 stability requires UHRF1 phosphorylation and that crosstalk between the proteins is essential for the function of these two important epigenetic regulators during gastrulation.

DNA hypomethylation induces a DNA replication-associated cell cycle arrest to block hepatic outgrowth in uhrf1 mutant zebrafish embryos.

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 re-replicating and arrested hepatocytes. Importantly, the DNA re-replication 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.

UHRF1 phosphorylation by cyclin A2/cyclin-dependent kinase 2 is required for zebrafish embryogenesis.

Ubiquitin-like, containing PHD and RING finger domains 1 (uhrf1) is regulated at the transcriptional level during the cell cycle and in developing zebrafish embryos. We identify phosphorylation as a novel means of regulating UHRF1 and demonstrate that Uhrf1 phosphorylation is required for gastrulation in zebrafish. Human UHRF1 contains a conserved cyclin-dependent kinase 2 (CDK2) phosphorylation site at Ser-661 that is phosphorylated in vitro by CDK2 partnered with cyclin A2 (CCNA2), but not cyclin E. An antibody specific for phospho-Ser-661 recognizes UHRF1 in both mammalian cancer cells and in nontransformed zebrafish cells, but not in zebrafish bearing a mutation in ccna2. Depleting Uhrf1 from zebrafish embryos by morpholino injection causes arrest before gastrulation and early embryonic death. This phenotype is rescued by wild-type UHRF1, but not by UHRF1 in which the phospho-acceptor site is mutated, demonstrating that UHRF1 phosphorylation is essential for embryogenesis. UHRF1 was detected in the nucleus and cytoplasm, whereas nonphosphorylatable UHRF1 is unable to localize to the cytoplasm, suggesting the importance of localization in UHRF1 function. Together, these data point to an essential role for UHRF1 phosphorylation by CDK/CCNA2 during early vertebrate development.