Abstract
5-methylcytosine (5mC) is a gene-regulatory mark associated with transcriptional repression. 5mC can be erased through the catalytic action of Ten-eleven translocation (TET) methylcytosine dioxygenases (TET1, TET2, TET3), which oxidize 5mC resulting in its removal from the genome. In vertebrates, TET enzymes facilitate DNA demethylation of regulatory regions linked to genes involved in developmental processes. Consequently, TET ablation leads to severe morphological defects and developmental arrest. Here we describe a system that can facilitate the study of relationships between TET enzymes, 5mC, and embryo development. We provide detailed descriptions for the generation of F0 zebrafish tet1/2/3 knockouts using CRISPR/Cas9 technology and elaborate on the strategies to assess the impact of TET loss by reduced representation bisulfite sequencing (RRBS).
Highlights
5-methylcytosine (5mC) is a gene-regulatory mark associated with transcriptional repression. 5mC can be erased through the catalytic action of Ten-eleven translocation (TET) methylcytosine dioxygenases (TET1, TET2, TET3), which oxidize 5mC resulting in its removal from the genome
5-methylcytosine (5mC) is a chemical modification of vertebrate genomes frequently associated with transcriptional repression [1]. 5mC is generated by the transfer of a methyl group from S-adenosylmethionine (SAM) to the fifth carbon of the cytosine pyrimidine ring, and this reaction is catalyzed by DNA methyltransferases (DNMTs)
In vitro studies have demonstrated that 5fC and 5caC can be removed from the DNA and replaced with unmethylated cytosines by thymine DNA glycosylase (TDG) and base-excision repair machinery (BER) [8, 9]
Summary
5-methylcytosine (5mC) is a chemical modification of vertebrate genomes frequently associated with transcriptional repression [1]. 5mC is generated by the transfer of a methyl group from S-adenosylmethionine (SAM) to the fifth carbon of the cytosine pyrimidine ring, and this reaction is catalyzed by DNA methyltransferases (DNMTs). TALEN-induced zebrafish tet TKOs were characterized by brain abnormalities, smaller eyes, altered pigmentation, trunk curvature and did not survive beyond the larval period [13] These animal studies indicate that TET proteins are dispensable for pluripotency but are required for organ development and body plan formation. Whole genome bisulfite sequencing (WGBS) of mouse, zebrafish, and frog embryos revealed TET-mediated demethylation of developmental enhancers during the vertebrate phylotypic period [12]. WGBS analysis of tet1/2/3 zebrafish morphants unraveled that the loss of Tet proteins reduces extent of demethylation, resulting in hypermethylation of these regions [12] This suggests that the activation of key developmental pathways could be driven by active 5mC demethylation. The RNA is used for qPCR analyses, whereas the DNA is used for both amplicon sequencing, to validate knockout efficiency, and for RRBS library construction This system allows for rapid and cost-efficient generation of data, overcoming specificity issues associated with
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