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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 miR-27a/b.(A) Convergent silencing of the miR-27a/b genes with ubiquitin promoters driving both sense and antisense transcription. A synthetic DNA sequence encompassing precursor sequences for both miR-27a and miR-27b were directly cloned between the two promoters. (B) Compared to control embryos injected with dye only (DIC), lateral and ventral views show that CT-miR-27a/b injected embryos phenocopy miR-27a/b morphants (see Supplemental Figure 4) with defects in pharyngeal arch morphogenesis, craniofacial defects, and inhibition of pectoral fin outgrowth as shown by decreased staining of ECM (cartilage) with alcian blue. (C) Compared to DICs, craniofacial defects observed with alcian blue staining were observed in 8–9.5% of CT-miR-27a/b embryos using two different plasmid clones of the construct in (A). n = 191 for DIC, n = 458 for CT-miR-27a/b clone 1, and n = 272 for CT-miR-27a/b clone 2. All images are at 4 dpf.
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f4: Silencing of miR-27a/b.(A) Convergent silencing of the miR-27a/b genes with ubiquitin promoters driving both sense and antisense transcription. A synthetic DNA sequence encompassing precursor sequences for both miR-27a and miR-27b were directly cloned between the two promoters. (B) Compared to control embryos injected with dye only (DIC), lateral and ventral views show that CT-miR-27a/b injected embryos phenocopy miR-27a/b morphants (see Supplemental Figure 4) with defects in pharyngeal arch morphogenesis, craniofacial defects, and inhibition of pectoral fin outgrowth as shown by decreased staining of ECM (cartilage) with alcian blue. (C) Compared to DICs, craniofacial defects observed with alcian blue staining were observed in 8–9.5% of CT-miR-27a/b embryos using two different plasmid clones of the construct in (A). n = 191 for DIC, n = 458 for CT-miR-27a/b clone 1, and n = 272 for CT-miR-27a/b clone 2. All images are at 4 dpf.

Mentions: To further demonstrate the utility of targeting endogenous genes with convergent transcription, we decided to target a miRNA gene. We chose miR-27a/b because morpholino and CRISPR knockdown of miR-27a/b results in dramatic craniofacial defects and impairment of pectoral fin outgrowth that can be readily detected both visually, and by staining extracellular matrix (ECM) and cartilage with alcian blue (Kara et al, manuscript in preparation). Tol2 based CT and non-CT constructs were generated with identical convergently arranged zebrafish Ubiquitin promoters flanking a fusion of DNA sequences encoding the precursors of both miR-27a and miR-27b (Fig. 4A). We chose to use the Ubiquitin promoter as a means to test the utility of multiple promoters and to begin to address questions related to differential promoter strength. Although the frequency of the defect was lower than what we observed with mCherry and OEP convergent silencing, we found that transgenic animals containing the CT construct showed a nearly identical phenotype to that produced upon morpholino knockdown (Supp Fig. 4) of miR-27a/b (Fig. 4B). This included loss of craniofacial structures, loss of upper and lower jaws, and impaired pectoral fin outgrowth. Only ~8–10% of transgenic animals displayed the phenotype but this was significantly higher than that compared to DICs where much less than 1% of the embryos showed some form of jaw defect or slight changes in alcian blue staining patterns. While the reasons for less efficient silencing of the miR-27a/b genes are not clear, the results indicate that convergent transcription can be used to silence noncoding RNAs in trans.


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

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

Silencing of miR-27a/b.(A) Convergent silencing of the miR-27a/b genes with ubiquitin promoters driving both sense and antisense transcription. A synthetic DNA sequence encompassing precursor sequences for both miR-27a and miR-27b were directly cloned between the two promoters. (B) Compared to control embryos injected with dye only (DIC), lateral and ventral views show that CT-miR-27a/b injected embryos phenocopy miR-27a/b morphants (see Supplemental Figure 4) with defects in pharyngeal arch morphogenesis, craniofacial defects, and inhibition of pectoral fin outgrowth as shown by decreased staining of ECM (cartilage) with alcian blue. (C) Compared to DICs, craniofacial defects observed with alcian blue staining were observed in 8–9.5% of CT-miR-27a/b embryos using two different plasmid clones of the construct in (A). n = 191 for DIC, n = 458 for CT-miR-27a/b clone 1, and n = 272 for CT-miR-27a/b clone 2. All images are at 4 dpf.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4048883&req=5

f4: Silencing of miR-27a/b.(A) Convergent silencing of the miR-27a/b genes with ubiquitin promoters driving both sense and antisense transcription. A synthetic DNA sequence encompassing precursor sequences for both miR-27a and miR-27b were directly cloned between the two promoters. (B) Compared to control embryos injected with dye only (DIC), lateral and ventral views show that CT-miR-27a/b injected embryos phenocopy miR-27a/b morphants (see Supplemental Figure 4) with defects in pharyngeal arch morphogenesis, craniofacial defects, and inhibition of pectoral fin outgrowth as shown by decreased staining of ECM (cartilage) with alcian blue. (C) Compared to DICs, craniofacial defects observed with alcian blue staining were observed in 8–9.5% of CT-miR-27a/b embryos using two different plasmid clones of the construct in (A). n = 191 for DIC, n = 458 for CT-miR-27a/b clone 1, and n = 272 for CT-miR-27a/b clone 2. All images are at 4 dpf.
Mentions: To further demonstrate the utility of targeting endogenous genes with convergent transcription, we decided to target a miRNA gene. We chose miR-27a/b because morpholino and CRISPR knockdown of miR-27a/b results in dramatic craniofacial defects and impairment of pectoral fin outgrowth that can be readily detected both visually, and by staining extracellular matrix (ECM) and cartilage with alcian blue (Kara et al, manuscript in preparation). Tol2 based CT and non-CT constructs were generated with identical convergently arranged zebrafish Ubiquitin promoters flanking a fusion of DNA sequences encoding the precursors of both miR-27a and miR-27b (Fig. 4A). We chose to use the Ubiquitin promoter as a means to test the utility of multiple promoters and to begin to address questions related to differential promoter strength. Although the frequency of the defect was lower than what we observed with mCherry and OEP convergent silencing, we found that transgenic animals containing the CT construct showed a nearly identical phenotype to that produced upon morpholino knockdown (Supp Fig. 4) of miR-27a/b (Fig. 4B). This included loss of craniofacial structures, loss of upper and lower jaws, and impaired pectoral fin outgrowth. Only ~8–10% of transgenic animals displayed the phenotype but this was significantly higher than that compared to DICs where much less than 1% of the embryos showed some form of jaw defect or slight changes in alcian blue staining patterns. While the reasons for less efficient silencing of the miR-27a/b genes are not clear, the results indicate that convergent transcription can be used to silence noncoding RNAs in trans.

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