The emerging H3K9me3 chromatin landscape during zebrafish embryogenesis.

The structural organization of eukaryotic genomes is contingent upon the fractionation of DNA into transcriptionally permissive euchromatin and repressive heterochromatin. However, we have a limited understanding of how these distinct states are first established during animal embryogenesis. Histone 3 lysine 9 trimethylation (H3K9me3) is critical to heterochromatin formation, and bulk establishment of this mark is thought to help drive large-scale remodeling of an initially naive chromatin state during animal embryogenesis.

The emerging H3K9me3 chromatin landscape during zebrafish embryogenesis.

The structural organization of eukaryotic genomes is contingent upon the fractionation of DNA into transcriptionally permissive euchromatin and repressive heterochromatin. However, we have a limited understanding of how these distinct states are first established during animal embryogenesis. Histone 3 lysine 9 trimethylation (H3K9me3) is critical to heterochromatin formation and bulk establishment of this mark is thought to help drive large-scale remodeling of an initially naive chromatin state during animal embryogenesis.

Uncovering Regulators of Heterochromatin Mediated Silencing Using a Zebrafish Transgenic Reporter.

Heterochromatin formation and maintenance is critical for the repression of transcription from repetitive sequences. However, tools for monitoring heterochromatin mediated repression of repeats in the context of vertebrate development have been lacking. Here we demonstrate that a large concatemeric transgene integration containing the dsRed fluorescent reporter under the control of a ubiquitous promoter recapitulates molecular hallmarks of heterochromatic silencing, and that expression from the transgene array can be reactivated by depletion of known regulators of heterochromatin.

Identification of chromatin states during zebrafish gastrulation using CUT&RUN and CUT&Tag.

Background: Cell fate decisions are governed by interactions between sequence-specific transcription factors and a dynamic chromatin landscape. Zebrafish offer a powerful system for probing the mechanisms that drive these cell fate choices, especially in the context of early embryogenesis. However, technical challenges associated with conventional methods for chromatin profiling have slowed progress toward understanding the exact relationships between chromatin changes, transcription factor binding, and cellular differentiation during zebrafish embryogenesis.

Chromatin dynamics at the maternal to zygotic transition: recent advances from the zebrafish model.

Early animal development is characterized by intense reorganization of the embryonic genome, including large-scale changes in chromatin structure and in the DNA and histone modifications that help shape this structure. Particularly profound shifts in the chromatin landscape are associated with the maternal-to-zygotic transition, when the zygotic genome is first transcribed and maternally loaded transcripts are degraded. The accessibility of the early zebrafish embryo facilitates the interrogation of chromatin during this critical window of development, making it an important model for early chromatin regulation.

TEADs, Yap, Taz, Vgll4s transcription factors control the establishment of Left-Right asymmetry in zebrafish.

In many vertebrates, establishment of Left-Right (LR) asymmetry results from the activity of a ciliated organ functioning as the LR Organizer (LRO). While regulation of the formation of this structure by major signaling pathways has been described, the transcriptional control of LRO formation is poorly understood. Using the zebrafish model, we show that the transcription factors and cofactors mediating or regulating the transcriptional outcome of the Hippo signaling pathway play a pivotal role in controlling the expression of genes essential to the formation of the LRO including ligands and receptors of signaling pathways involved in this process and most genes required for motile ciliogenesis.

DNA Methylation: Shared and Divergent Features across Eukaryotes.

Chemical modification of nucleotide bases in DNA provides one mechanism for conveying information in addition to the genetic code. 5-methylcytosine (5mC) represents the most common chemically modified base in eukaryotic genomes. Sometimes referred to simply as DNA methylation, in eukaryotes 5mC is most prevalent at CpG dinucleotides and is frequently associated with transcriptional repression of transposable elements.

The maternal to zygotic transition regulates genome-wide heterochromatin establishment in the zebrafish embryo.

The segregation of eukaryotic genomes into euchromatin and heterochromatin represents a fundamental and poorly understood process. Here, we demonstrate that genome-wide establishment of heterochromatin is triggered by the maternal to zygotic transition (MZT) during zebrafish embryogenesis. We find that prior to MZT, zebrafish lack hallmarks of heterochromatin including histone H3 lysine 9 trimethylation (H3K9me3) and condensed chromatin ultrastructure.

TETs Regulate Proepicardial Cell Migration through Extracellular Matrix Organization during Zebrafish Cardiogenesis.

Ten-eleven translocation (Tet) enzymes (Tet1/2/3) mediate 5-methylcytosine (5mC) hydroxylation, which can facilitate DNA demethylation and thereby impact gene expression. Studied mostly for how mutant isoforms impact cancer, the normal roles for Tet enzymes during organogenesis are largely unknown. By analyzing compound mutant zebrafish, we discovered a requirement for Tet2/3 activity in the embryonic heart for recruitment of epicardial progenitors, associated with development of the atrial-ventricular canal (AVC).

Pericentromeric hypomethylation elicits an interferon response in an animal model of ICF syndrome.

Unlabelled: Pericentromeric satellite repeats are enriched in 5-methylcytosine (5mC). Loss of 5mC at these sequences is common in cancer and is a hallmark of Immunodeficiency, Centromere and Facial abnormalities (ICF) syndrome. While the general importance of 5mC is well-established, the specific functions of 5mC at pericentromeres are less clear.

OGT binds a conserved C-terminal domain of TET1 to regulate TET1 activity and function in development.

TET enzymes convert 5-methylcytosine to 5-hydroxymethylcytosine and higher oxidized derivatives. TETs stably associate with and are post-translationally modified by the nutrient-sensing enzyme OGT, suggesting a connection between metabolism and the epigenome. Here, we show for the first time that modification by OGT enhances TET1 activity in vitro.

Overlapping Requirements for Tet2 and Tet3 in Normal Development and Hematopoietic Stem Cell Emergence.

The Tet family of methylcytosine dioxygenases (Tet1, Tet2, and Tet3) convert 5-methylcytosine to 5-hydroxymethylcytosine. To date, functional overlap among Tet family members has not been examined systematically in the context of embryonic development. To clarify the potential for overlap among Tet enzymes during development, we mutated the zebrafish orthologs of Tet1, Tet2, and Tet3 and examined single-, double-, and triple-mutant genotypes.

Epigenetic control of intestinal barrier function and inflammation in zebrafish.

The intestinal epithelium forms a barrier protecting the organism from microbes and other proinflammatory stimuli. The integrity of this barrier and the proper response to infection requires precise regulation of powerful immune homing signals such as tumor necrosis factor (TNF). Dysregulation of TNF leads to inflammatory bowel diseases (IBD), but the mechanism controlling the expression of this potent cytokine and the events that trigger the onset of chronic inflammation are unknown.

Adoption of the Q transcriptional regulatory system for zebrafish transgenesis.

The Gal4-UAS regulatory system of yeast is widely used to modulate gene expression in Drosophila; however, there are limitations to its usefulness in transgenic zebrafish, owing to progressive methylation and silencing of the CpG-rich multicopy upstream activation sequence. Although a modified, less repetitive UAS construct may overcome this problem, it is highly desirable to have additional transcriptional regulatory systems that can be applied independently or in combination with the Gal4/UAS system for intersectional gene expression. The Q transcriptional regulatory system of Neurospora crassa functions similarly to Gal4/UAS.

CEP162 is an axoneme-recognition protein promoting ciliary transition zone assembly at the cilia base.

The transition zone is a specialized compartment found at the base of cilia, adjacent to the centriole distal end, where axonemal microtubules are heavily crosslinked to the surrounding membrane to form a barrier that gates the ciliary compartment. A number of ciliopathy molecules have been found to associate with the transition zone, but factors that directly recognize axonemal microtubules to specify transition zone assembly at the cilia base remain unclear. Here, through quantitative centrosome proteomics, we identify an axoneme-associated protein, CEP162 (KIAA1009), tethered specifically at centriole distal ends to promote transition zone assembly.

ΦC31 integrase mediates efficient cassette exchange in the zebrafish germline.

Site-specific recombinases (SSRs) are powerful tools for genome manipulation, used in diverse organisms including Drosophila melanogaster, mouse, Arabidopsis, zebrafish, and human cultured cells. The integrase from the bacteriophage ΦC31 belongs to the large serine family of integrases, and in contrast to other widely used SSRs such as Cre and Flp, recombination is directional and therefore irreversible. We have developed a vector system for recombinase-mediated cassette exchange (RMCE) in the zebrafish, allowing swapping of the coding sequence in an integrated transgene.

DNA methylation in zebrafish.

DNA methylation is crucial for normal development and cellular differentiation in many large-genome eukaryotes. The small tropical freshwater fish Danio rerio (zebrafish) has recently emerged as a powerful system for the study of DNA methylation, especially in the context of development. This review summarizes our current knowledge of DNA methylation in zebrafish and provides evidence for the general conservation of this system with mammals.

Transgenerational analysis of transcriptional silencing in zebrafish.

The yeast Gal4/UAS transcriptional activation system is a powerful tool for regulating gene expression in Drosophila and has been increasing in popularity for developmental studies in zebrafish. It is also useful for studying the basis of de novo transcriptional silencing. Fluorescent reporter genes under the control of multiple tandem copies of the upstream activator sequence (UAS) often show evidence of variegated expression and DNA methylation in transgenic zebrafish embryos.

Conservation and divergence of methylation patterning in plants and animals.

Cytosine DNA methylation is a heritable epigenetic mark present in many eukaryotic organisms. Although DNA methylation likely has a conserved role in gene silencing, the levels and patterns of DNA methylation appear to vary drastically among different organisms. Here we used shotgun genomic bisulfite sequencing (BS-Seq) to compare DNA methylation in eight diverse plant and animal genomes.

Loss of Dnmt1 catalytic activity reveals multiple roles for DNA methylation during pancreas development and regeneration.

Developmental mechanisms regulating gene expression and the stable acquisition of cell fate direct cytodifferentiation during organogenesis. Moreover, it is likely that such mechanisms could be exploited to repair or regenerate damaged organs. DNA methyltransferases (Dnmts) are enzymes critical for epigenetic regulation, and are used in concert with histone methylation and acetylation to regulate gene expression and maintain genomic integrity and chromosome structure.

Transcriptional silencing and reactivation in transgenic zebrafish.

Epigenetic regulation of transcriptional silencing is essential for normal development. Despite its importance, in vivo systems for examining gene silencing at cellular resolution have been lacking in developing vertebrates. We describe a transgenic approach that allows monitoring of an epigenetically regulated fluorescent reporter in developing zebrafish and their progeny.

Gal4/UAS transgenic tools and their application to zebrafish.

The ability to regulate gene expression in a cell-specific and temporally restricted manner provides a powerful means to test gene function, bypass the action of lethal genes, label subsets of cells for developmental studies, monitor subcellular structures, and target tissues for selective ablation or physiological analyses. The galactose-inducible system of yeast, mediated by the transcriptional activator Gal4 and its consensus UAS binding site, has proven to be a highly successful and versatile system for controlling transcriptional activation in Drosophila. It has also been used effectively, albeit in a more limited manner, in the mouse.

Transactivation from Gal4-VP16 transgenic insertions for tissue-specific cell labeling and ablation in zebrafish.

Prior studies with transgenic zebrafish confirmed the functionality of the transcription factor Gal4 to drive expression of other genes under the regulation of upstream activator sequences (UAS). However, widespread application of this powerful binary system has been limited, in part, by relatively inefficient techniques for establishing transgenic zebrafish and by the inadequacy of Gal4 to effect high levels of expression from UAS-regulated genes. We have used the Tol2 transposition system to distribute a self-reporting gene/enhancer trap vector efficiently throughout the zebrafish genome.

Methylation of tRNAAsp by the DNA methyltransferase homolog Dnmt2.

The sequence and the structure of DNA methyltransferase-2 (Dnmt2) bear close affinities to authentic DNA cytosine methyltransferases. A combined genetic and biochemical approach revealed that human DNMT2 did not methylate DNA but instead methylated a small RNA; mass spectrometry showed that this RNA is aspartic acid transfer RNA (tRNA(Asp)) and that DNMT2 specifically methylated cytosine 38 in the anticodon loop. The function of DNMT2 is highly conserved, and human DNMT2 protein restored methylation in vitro to tRNA(Asp) from Dnmt2-deficient strains of mouse, Arabidopsis thaliana, and Drosophila melanogaster in a manner that was dependent on preexisting patterns of modified nucleosides.

Eukaryotic cytosine methyltransferases.

Large-genome eukaryotes use heritable cytosine methylation to silence promoters, especially those associated with transposons and imprinted genes. Cytosine methylation does not reinforce or replace ancestral gene regulation pathways but instead endows methylated genomes with the ability to repress specific promoters in a manner that is buffered against changes in the internal and external environment. Recent studies have shown that the targeting of de novo methylation depends on multiple inputs; these include the interaction of repeated sequences, local states of histone lysine methylation, small RNAs and components of the RNAi pathway, and divergent and catalytically inert cytosine methyltransferase homologues that have acquired regulatory roles.