Abstract
Although DNA methylation is critical for proper embryonic and tissue-specific development, how different DNA methyltransferases affect tissue-specific development and their targets remains unknown. We address this issue in zebrafish through antisense-based morpholino knockdown of Dnmt3 and Dnmt1. Our data reveal that Dnmt3 is required for proper neurogenesis, and its absence results in profound defects in brain and retina. Interestingly, other organs such as intestine remain unaffected suggesting tissue-specific requirements of Dnmt3. Further, comparison of Dnmt1 knockdown phenotypes with those of Dnmt3 suggested that these two families have distinct functions. Consistent with this idea, Dnmt1 failed to complement Dnmt3 deficiency, and Dnmt3 failed to complement Dnmt1 deficiency. Downstream of Dnmt3 we identify a neurogenesis regulator, lef1, as a Dnmt3-specific target gene that is demethylated and up-regulated in dnmt3 morphants. Knockdown of lef1 rescued neurogenesis defects resulting from Dnmt3 absence. Mechanistically, we show cooperation between Dnmt3 and an H3K9 methyltransferase G9a in regulating lef1. Further, like Dnmt1-Suv39h1 cooperativity, Dnmt3 and G9a seemed to function together for tissue-specific development. G9a knockdown, but not Suv39h1 loss, phenocopied dnmt3 morphants and G9a overexpression provided a striking rescue of dnmt3 morphant phenotypes, whereas Suv39h1 overexpression failed, supporting the notion of specific DNMT-histone methyltransferase networks. Consistent with this model, H3K9me3 levels on the lef1 promoter were reduced in both dnmt3 and g9a morphants, and its knockdown rescued neurogenesis defects in g9a morphants. We propose a model wherein specific DNMT-histone methyltransferase networks are utilized to silence critical regulators of cell fate in a tissue-specific manner.
Highlights
It has been long hypothesized that the process of DNA methylation is utilized for initiating or maintaining gene silencing during this tissue-specific and temporal transcriptional regulation [1, 2]
Does Dnmt3 function in a tissue-specific manner during zebrafish development, and if so, are these functions distinct from Dnmt1? does Dnmt3 depend on a repressive histone methyltransferases (HMTs) for its function and if so, which HMT? Are there regulatory genes that are silenced by DNA methylation and depend on specific DNA methyltransferase? we used an antisense morpholino knockdown approach to show that Dnmt3 is required for proper development of specific organs, such as brain and retina
We show that Dnmt1 and Dnmt3 have largely distinct tissue-specific and temporal functions during zebrafish development
Summary
Zebrafish Morpholino, mRNA, and Plasmid Injections—Zebrafish stocks and embryo culture were performed as described previously [13]. Embryos were injected with dnmt Mo2 (8 ng) or g9a Mo1 (6 ng) at one cell stage, and extracts were made at 24 hpf. C, whole mount in situ analysis of ngn-1, ascl1a, and ascl1b expression in dnmt morpholino alone or with p53 morpholino-, dnmt morpholino-, or control morpholino-injected embryos at 30 hpf. Whereas ngn-1 expression was normal in dnmt and dnmt morphants, ascl1a and ascl1b were selectively is absent This defect can be complemented by co-injection of the wild-type (Dnmt3WT) but not by a catalytically inactive derivative (Dnmt3C1240S). HEK293 cells were transfected with Dnmt3WT and Dnmt3C1240S, and Western blots were performed using antibodies against His tag Both of these show equal expression of these derivatives. Embryos tail was up-regulated in the notochord in the tail of were stored in glycerol and pictures were taken
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