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RNAi-mediated gene silencing in zebrafish triggered by convergent transcription.

Andrews OE, Cha DJ, Wei C, Patton JG - Sci Rep (2014)

Bottom Line: We found that introduction of transgenes containing convergent transcription units in zebrafish embryos induced stable transcriptional gene silencing (TGS) in cis and trans for reporter (mCherry) and endogenous (One-Eyed Pinhead (OEP) and miR-27a/b) genes.By ChIP analyses, increased silencing was accompanied by enrichment of the heterochromatin mark H3K9me3 in the two convergently arranged promoters and in the intervening reading frame.Our work demonstrates that convergent transcription can induce gene silencing in zebrafish providing another tool to create specific temporal and spatial control of gene expression.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biological Sciences, Vanderbilt University, Nashville, TN [2].

ABSTRACT
RNAi based strategies to induce gene silencing are commonly employed in numerous model organisms but have not been extensively used in zebrafish. We found that introduction of transgenes containing convergent transcription units in zebrafish embryos induced stable transcriptional gene silencing (TGS) in cis and trans for reporter (mCherry) and endogenous (One-Eyed Pinhead (OEP) and miR-27a/b) genes. Convergent transcription enabled detection of both sense and antisense transcripts and silencing was suppressed upon Dicer knockdown, indicating processing of double stranded RNA. By ChIP analyses, increased silencing was accompanied by enrichment of the heterochromatin mark H3K9me3 in the two convergently arranged promoters and in the intervening reading frame. Our work demonstrates that convergent transcription can induce gene silencing in zebrafish providing another tool to create specific temporal and spatial control of gene expression.

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Silencing of One Eyed Pinhead.Convergent silencing of the zebrafish One Eyed Pinhead (OEP) gene with the β-actin promoter driving sense transcription and an inverted CMV promoter driving antisense transcription. The OEP open reading frame was directly cloned between the two promoters. (B–G) Compared to uninjected control embryos (UICs), CT-OEP injected embryos phenocopied OEP mutants with curved body axis, cyclopia (black arrow in C), and reduced notochord (red arrows in F compared to G). In 12% of the CT-OEP embryos, we observed a more severe phenotype with complete loss of eye development (red asterisk in E). (H) Graph showing % transient transgenics showing WT, classic OEP, or more severe phenotypes. n = 167. (I). Silencing of OEP is Dicer dependent. Embryos were injected as indicated in the absence or presence of Dicer MO or a control mismatch morpholino. **p < 0.005.
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f3: Silencing of One Eyed Pinhead.Convergent silencing of the zebrafish One Eyed Pinhead (OEP) gene with the β-actin promoter driving sense transcription and an inverted CMV promoter driving antisense transcription. The OEP open reading frame was directly cloned between the two promoters. (B–G) Compared to uninjected control embryos (UICs), CT-OEP injected embryos phenocopied OEP mutants with curved body axis, cyclopia (black arrow in C), and reduced notochord (red arrows in F compared to G). In 12% of the CT-OEP embryos, we observed a more severe phenotype with complete loss of eye development (red asterisk in E). (H) Graph showing % transient transgenics showing WT, classic OEP, or more severe phenotypes. n = 167. (I). Silencing of OEP is Dicer dependent. Embryos were injected as indicated in the absence or presence of Dicer MO or a control mismatch morpholino. **p < 0.005.

Mentions: To address the ability of convergent transcription to silence genes in trans, we generated a convergent construct targeting the endogenous One-Eyed Pinhead (OEP) gene31 (Fig. 3A). We chose this gene because the OEP phenotype is well characterized and easy to score. As shown in Fig. 3B–G and quantified in Fig. 3H, 43% of transient transgenic animals injected with CT-OEP displayed cyclopia, strong ventral curvature of the body axis, and a reduced/misshapen notochord, consistent with OEP mutants and morpholino knockdown of OEP3132. More severe defects were observed in 12% of the embryos with eyeless, headless, or severely reduced body axis. Interestingly, the more severe phenotypes resemble those observed with high concentrations of OEP MOs which were proposed to be due to the loss of both maternal and zygotic OEP expression32. In our experiments, the more severe effects would not be due to destruction of maternal transcripts but are consistent with increased silencing of the endogenous OEP gene.


RNAi-mediated gene silencing in zebrafish triggered by convergent transcription.

Andrews OE, Cha DJ, Wei C, Patton JG - Sci Rep (2014)

Silencing of One Eyed Pinhead.Convergent silencing of the zebrafish One Eyed Pinhead (OEP) gene with the β-actin promoter driving sense transcription and an inverted CMV promoter driving antisense transcription. The OEP open reading frame was directly cloned between the two promoters. (B–G) Compared to uninjected control embryos (UICs), CT-OEP injected embryos phenocopied OEP mutants with curved body axis, cyclopia (black arrow in C), and reduced notochord (red arrows in F compared to G). In 12% of the CT-OEP embryos, we observed a more severe phenotype with complete loss of eye development (red asterisk in E). (H) Graph showing % transient transgenics showing WT, classic OEP, or more severe phenotypes. n = 167. (I). Silencing of OEP is Dicer dependent. Embryos were injected as indicated in the absence or presence of Dicer MO or a control mismatch morpholino. **p < 0.005.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048883&req=5

f3: Silencing of One Eyed Pinhead.Convergent silencing of the zebrafish One Eyed Pinhead (OEP) gene with the β-actin promoter driving sense transcription and an inverted CMV promoter driving antisense transcription. The OEP open reading frame was directly cloned between the two promoters. (B–G) Compared to uninjected control embryos (UICs), CT-OEP injected embryos phenocopied OEP mutants with curved body axis, cyclopia (black arrow in C), and reduced notochord (red arrows in F compared to G). In 12% of the CT-OEP embryos, we observed a more severe phenotype with complete loss of eye development (red asterisk in E). (H) Graph showing % transient transgenics showing WT, classic OEP, or more severe phenotypes. n = 167. (I). Silencing of OEP is Dicer dependent. Embryos were injected as indicated in the absence or presence of Dicer MO or a control mismatch morpholino. **p < 0.005.
Mentions: To address the ability of convergent transcription to silence genes in trans, we generated a convergent construct targeting the endogenous One-Eyed Pinhead (OEP) gene31 (Fig. 3A). We chose this gene because the OEP phenotype is well characterized and easy to score. As shown in Fig. 3B–G and quantified in Fig. 3H, 43% of transient transgenic animals injected with CT-OEP displayed cyclopia, strong ventral curvature of the body axis, and a reduced/misshapen notochord, consistent with OEP mutants and morpholino knockdown of OEP3132. More severe defects were observed in 12% of the embryos with eyeless, headless, or severely reduced body axis. Interestingly, the more severe phenotypes resemble those observed with high concentrations of OEP MOs which were proposed to be due to the loss of both maternal and zygotic OEP expression32. In our experiments, the more severe effects would not be due to destruction of maternal transcripts but are consistent with increased silencing of the endogenous OEP gene.

Bottom Line: We found that introduction of transgenes containing convergent transcription units in zebrafish embryos induced stable transcriptional gene silencing (TGS) in cis and trans for reporter (mCherry) and endogenous (One-Eyed Pinhead (OEP) and miR-27a/b) genes.By ChIP analyses, increased silencing was accompanied by enrichment of the heterochromatin mark H3K9me3 in the two convergently arranged promoters and in the intervening reading frame.Our work demonstrates that convergent transcription can induce gene silencing in zebrafish providing another tool to create specific temporal and spatial control of gene expression.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biological Sciences, Vanderbilt University, Nashville, TN [2].

ABSTRACT
RNAi based strategies to induce gene silencing are commonly employed in numerous model organisms but have not been extensively used in zebrafish. We found that introduction of transgenes containing convergent transcription units in zebrafish embryos induced stable transcriptional gene silencing (TGS) in cis and trans for reporter (mCherry) and endogenous (One-Eyed Pinhead (OEP) and miR-27a/b) genes. Convergent transcription enabled detection of both sense and antisense transcripts and silencing was suppressed upon Dicer knockdown, indicating processing of double stranded RNA. By ChIP analyses, increased silencing was accompanied by enrichment of the heterochromatin mark H3K9me3 in the two convergently arranged promoters and in the intervening reading frame. Our work demonstrates that convergent transcription can induce gene silencing in zebrafish providing another tool to create specific temporal and spatial control of gene expression.

Show MeSH
Related in: MedlinePlus