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The Tnt1 retrotransposon escapes silencing in tobacco, its natural host.

Hernández-Pinzón I, Cifuentes M, Hénaff E, Santiago N, Espinás ML, Casacuberta JM - PLoS ONE (2012)

Bottom Line: Transcriptional gene silencing is a general and highly effective control of retrotransposon expression.Silencing of Tnt1-containing transgenes correlates with high DNA methylation and the inability to incorporate H2A.Z into their promoters, whereas the endogenous Tnt1 elements remain partially methylated at asymmetrical positions and incorporate H2A.Z upon induction.Our results show that the promoter of Tnt1 is a target of silencing in tobacco, but also that endogenous Tnt1 elements can escape this control and be expressed in their natural host.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Center for Research in Agricultural Genomics, CRAG, CSIC-IRTA-UAB, Barcelona, Spain.

ABSTRACT
Retrotransposons' high capacity for mutagenesis is a threat that genomes need to control tightly. Transcriptional gene silencing is a general and highly effective control of retrotransposon expression. Yet, some retrotransposons manage to transpose and proliferate in plant genomes, suggesting that, as shown for plant viruses, retrotransposons can escape silencing. However no evidence of retrotransposon silencing escape has been reported. Here we analyze the silencing control of the tobacco Tnt1 retrotransposon and report that even though constructs driven by the Tnt1 promoter become silenced when stably integrated in tobacco, the endogenous Tnt1 elements remain active. Silencing of Tnt1-containing transgenes correlates with high DNA methylation and the inability to incorporate H2A.Z into their promoters, whereas the endogenous Tnt1 elements remain partially methylated at asymmetrical positions and incorporate H2A.Z upon induction. Our results show that the promoter of Tnt1 is a target of silencing in tobacco, but also that endogenous Tnt1 elements can escape this control and be expressed in their natural host.

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LTR-GFP-LTR transgenes are highly methylated in both symmetrical and asymmetrical contexts.The 5′ region of the LTR-GFP-LTR transgene, including the 5′ LTR, was amplified and cloned from bisulfite converted DNA from R10-treated (+ R10) as well as non-treated leaves of two independent transgenic lines. At least 10 clones were sequenced from each transgenic line (only one sequence is shown when the same sequence was obtained several times). The methylation state of each cytosine is shown as open (not methylated) or closed (methylated) circle. The sequence context of each cytosine is shown by the color of the circle (red, CG; blue, CHG; green, CHH). The different regions of the transgene are shown. The position of the BII boxes, known to bind defense-induced DNA binding factors [59] are indicated by red lines.
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pone-0033816-g003: LTR-GFP-LTR transgenes are highly methylated in both symmetrical and asymmetrical contexts.The 5′ region of the LTR-GFP-LTR transgene, including the 5′ LTR, was amplified and cloned from bisulfite converted DNA from R10-treated (+ R10) as well as non-treated leaves of two independent transgenic lines. At least 10 clones were sequenced from each transgenic line (only one sequence is shown when the same sequence was obtained several times). The methylation state of each cytosine is shown as open (not methylated) or closed (methylated) circle. The sequence context of each cytosine is shown by the color of the circle (red, CG; blue, CHG; green, CHH). The different regions of the transgene are shown. The position of the BII boxes, known to bind defense-induced DNA binding factors [59] are indicated by red lines.

Mentions: In plants TGS is usually accompanied by DNA methylation of the silenced promoters, in particular in the case of transposable elements [40]. We have therefore analyzed the level of methylation of the promoter sequences within the silenced LTR-GFP-LTR plants by bisulfite sequencing. Our results showed that although the T-DNA and the GFP sequences present in the transgene are essentially free of methylation, the transgene sequences showing sequence identity to the endogenous Tnt1 elements (i.e. the LTR sequences) are heavily methylated in both symmetrical and asymmetrical methylation contexts (Figure 3). The degree of methylation is similar in leaves treated and non-treated with R10. The degree of methylation is also similar in the 5′ and the 3′ LTRs (compare Figure 3 with Figure S5). Our analysis shows that more than 80% of the cytosines located in a symmetrical context are methylated while methylation at asymmetrical positions ranges from 70 to 77%.


The Tnt1 retrotransposon escapes silencing in tobacco, its natural host.

Hernández-Pinzón I, Cifuentes M, Hénaff E, Santiago N, Espinás ML, Casacuberta JM - PLoS ONE (2012)

LTR-GFP-LTR transgenes are highly methylated in both symmetrical and asymmetrical contexts.The 5′ region of the LTR-GFP-LTR transgene, including the 5′ LTR, was amplified and cloned from bisulfite converted DNA from R10-treated (+ R10) as well as non-treated leaves of two independent transgenic lines. At least 10 clones were sequenced from each transgenic line (only one sequence is shown when the same sequence was obtained several times). The methylation state of each cytosine is shown as open (not methylated) or closed (methylated) circle. The sequence context of each cytosine is shown by the color of the circle (red, CG; blue, CHG; green, CHH). The different regions of the transgene are shown. The position of the BII boxes, known to bind defense-induced DNA binding factors [59] are indicated by red lines.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3316501&req=5

pone-0033816-g003: LTR-GFP-LTR transgenes are highly methylated in both symmetrical and asymmetrical contexts.The 5′ region of the LTR-GFP-LTR transgene, including the 5′ LTR, was amplified and cloned from bisulfite converted DNA from R10-treated (+ R10) as well as non-treated leaves of two independent transgenic lines. At least 10 clones were sequenced from each transgenic line (only one sequence is shown when the same sequence was obtained several times). The methylation state of each cytosine is shown as open (not methylated) or closed (methylated) circle. The sequence context of each cytosine is shown by the color of the circle (red, CG; blue, CHG; green, CHH). The different regions of the transgene are shown. The position of the BII boxes, known to bind defense-induced DNA binding factors [59] are indicated by red lines.
Mentions: In plants TGS is usually accompanied by DNA methylation of the silenced promoters, in particular in the case of transposable elements [40]. We have therefore analyzed the level of methylation of the promoter sequences within the silenced LTR-GFP-LTR plants by bisulfite sequencing. Our results showed that although the T-DNA and the GFP sequences present in the transgene are essentially free of methylation, the transgene sequences showing sequence identity to the endogenous Tnt1 elements (i.e. the LTR sequences) are heavily methylated in both symmetrical and asymmetrical methylation contexts (Figure 3). The degree of methylation is similar in leaves treated and non-treated with R10. The degree of methylation is also similar in the 5′ and the 3′ LTRs (compare Figure 3 with Figure S5). Our analysis shows that more than 80% of the cytosines located in a symmetrical context are methylated while methylation at asymmetrical positions ranges from 70 to 77%.

Bottom Line: Transcriptional gene silencing is a general and highly effective control of retrotransposon expression.Silencing of Tnt1-containing transgenes correlates with high DNA methylation and the inability to incorporate H2A.Z into their promoters, whereas the endogenous Tnt1 elements remain partially methylated at asymmetrical positions and incorporate H2A.Z upon induction.Our results show that the promoter of Tnt1 is a target of silencing in tobacco, but also that endogenous Tnt1 elements can escape this control and be expressed in their natural host.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Center for Research in Agricultural Genomics, CRAG, CSIC-IRTA-UAB, Barcelona, Spain.

ABSTRACT
Retrotransposons' high capacity for mutagenesis is a threat that genomes need to control tightly. Transcriptional gene silencing is a general and highly effective control of retrotransposon expression. Yet, some retrotransposons manage to transpose and proliferate in plant genomes, suggesting that, as shown for plant viruses, retrotransposons can escape silencing. However no evidence of retrotransposon silencing escape has been reported. Here we analyze the silencing control of the tobacco Tnt1 retrotransposon and report that even though constructs driven by the Tnt1 promoter become silenced when stably integrated in tobacco, the endogenous Tnt1 elements remain active. Silencing of Tnt1-containing transgenes correlates with high DNA methylation and the inability to incorporate H2A.Z into their promoters, whereas the endogenous Tnt1 elements remain partially methylated at asymmetrical positions and incorporate H2A.Z upon induction. Our results show that the promoter of Tnt1 is a target of silencing in tobacco, but also that endogenous Tnt1 elements can escape this control and be expressed in their natural host.

Show MeSH
Related in: MedlinePlus