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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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mCherry silencing is Dicer dependent and results in increased levels of H3K9me3 chromatin modification.(A) Detection of sense and antisense mCherry transcripts via qPCR analysis in F2 embryos from 3 different CT-mCherry F1 lines. (B) Increased mCherry sense mRNA levels upon co-injection of a Dicer MO compared to control (ctrl) MO injected transient transgenics at 54 hpf. (C) Rescue of mCherry protein levels upon co-injection of Dicer MO. Protein lysates were prepared from embryos as in (B) and Western blots were prepared with antibodies against mCherry and α-tubulin. The full gel is shown in Supplemental Fig. 1. For comparison, protein levels in a non-CT embryo are as shown. (D) ChIP-qPCR showing significant enrichment of H3K9me3 levels on convergent chromatin from CT-mCherry F2 embryos (F1 line 9119). Enrichment was determined compared to negative IgG control at 5 dpf. ChIP values and standard deviations are shown from three independent biological experiments. ***p < .001 based on unpaired, two-tailed distribution Student's t-test.
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f2: mCherry silencing is Dicer dependent and results in increased levels of H3K9me3 chromatin modification.(A) Detection of sense and antisense mCherry transcripts via qPCR analysis in F2 embryos from 3 different CT-mCherry F1 lines. (B) Increased mCherry sense mRNA levels upon co-injection of a Dicer MO compared to control (ctrl) MO injected transient transgenics at 54 hpf. (C) Rescue of mCherry protein levels upon co-injection of Dicer MO. Protein lysates were prepared from embryos as in (B) and Western blots were prepared with antibodies against mCherry and α-tubulin. The full gel is shown in Supplemental Fig. 1. For comparison, protein levels in a non-CT embryo are as shown. (D) ChIP-qPCR showing significant enrichment of H3K9me3 levels on convergent chromatin from CT-mCherry F2 embryos (F1 line 9119). Enrichment was determined compared to negative IgG control at 5 dpf. ChIP values and standard deviations are shown from three independent biological experiments. ***p < .001 based on unpaired, two-tailed distribution Student's t-test.

Mentions: In order to determine whether the mechanism of mCherry silencing was as predicted based on convergent silencing in S. pombe25, we designed primers to amplify sense and antisense mCherry transcripts. We reasoned that convergent transcription should produce both sense and antisense transcripts. As expected, the levels of sense mCherry transcripts in non-CT F2 embryos were abundant, 30-fold more than the levels detected in CT-mCherry animals, whereas the levels of antisense transcripts were at or just barely above background levels. In contrast, RNA isolation and qPCR analyses from F2 embryos derived from multiple F1s confirmed elevated levels of both sense and antisense mCherry transcripts with slight variation between lines (Fig. 2A). These data argue against the idea that the loss of mCherry is due to simple steric interference from colliding convergent polymerases.


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

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

mCherry silencing is Dicer dependent and results in increased levels of H3K9me3 chromatin modification.(A) Detection of sense and antisense mCherry transcripts via qPCR analysis in F2 embryos from 3 different CT-mCherry F1 lines. (B) Increased mCherry sense mRNA levels upon co-injection of a Dicer MO compared to control (ctrl) MO injected transient transgenics at 54 hpf. (C) Rescue of mCherry protein levels upon co-injection of Dicer MO. Protein lysates were prepared from embryos as in (B) and Western blots were prepared with antibodies against mCherry and α-tubulin. The full gel is shown in Supplemental Fig. 1. For comparison, protein levels in a non-CT embryo are as shown. (D) ChIP-qPCR showing significant enrichment of H3K9me3 levels on convergent chromatin from CT-mCherry F2 embryos (F1 line 9119). Enrichment was determined compared to negative IgG control at 5 dpf. ChIP values and standard deviations are shown from three independent biological experiments. ***p < .001 based on unpaired, two-tailed distribution Student's t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f2: mCherry silencing is Dicer dependent and results in increased levels of H3K9me3 chromatin modification.(A) Detection of sense and antisense mCherry transcripts via qPCR analysis in F2 embryos from 3 different CT-mCherry F1 lines. (B) Increased mCherry sense mRNA levels upon co-injection of a Dicer MO compared to control (ctrl) MO injected transient transgenics at 54 hpf. (C) Rescue of mCherry protein levels upon co-injection of Dicer MO. Protein lysates were prepared from embryos as in (B) and Western blots were prepared with antibodies against mCherry and α-tubulin. The full gel is shown in Supplemental Fig. 1. For comparison, protein levels in a non-CT embryo are as shown. (D) ChIP-qPCR showing significant enrichment of H3K9me3 levels on convergent chromatin from CT-mCherry F2 embryos (F1 line 9119). Enrichment was determined compared to negative IgG control at 5 dpf. ChIP values and standard deviations are shown from three independent biological experiments. ***p < .001 based on unpaired, two-tailed distribution Student's t-test.
Mentions: In order to determine whether the mechanism of mCherry silencing was as predicted based on convergent silencing in S. pombe25, we designed primers to amplify sense and antisense mCherry transcripts. We reasoned that convergent transcription should produce both sense and antisense transcripts. As expected, the levels of sense mCherry transcripts in non-CT F2 embryos were abundant, 30-fold more than the levels detected in CT-mCherry animals, whereas the levels of antisense transcripts were at or just barely above background levels. In contrast, RNA isolation and qPCR analyses from F2 embryos derived from multiple F1s confirmed elevated levels of both sense and antisense mCherry transcripts with slight variation between lines (Fig. 2A). These data argue against the idea that the loss of mCherry is due to simple steric interference from colliding convergent polymerases.

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