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A position effect on the heritability of epigenetic silencing.

Singh J, Freeling M, Lisch D - PLoS Genet. (2008)

Bottom Line: In animals and yeast, position effects have been well documented.In contrast, there are few examples of position effects in plants, and there are no documented examples in either plants or animals for positions that are associated with the reversal of previously established silenced states.To our knowledge, this is the first documented example of a position effect that is associated with the reversal of epigenetic silencing.

View Article: PubMed Central - PubMed

Affiliation: Plant Science Department, McGill University, Macdonald Campus, Ste Anne de Bellevue, Quebec, Canada.

ABSTRACT
In animals and yeast, position effects have been well documented. In animals, the best example of this process is Position Effect Variegation (PEV) in Drosophila melanogaster. In PEV, when genes are moved into close proximity to constitutive heterochromatin, their expression can become unstable, resulting in variegated patches of gene expression. This process is regulated by a variety of proteins implicated in both chromatin remodeling and RNAi-based silencing. A similar phenomenon is observed when transgenes are inserted into heterochromatic regions in fission yeast. In contrast, there are few examples of position effects in plants, and there are no documented examples in either plants or animals for positions that are associated with the reversal of previously established silenced states. MuDR transposons in maize can be heritably silenced by a naturally occurring rearranged version of MuDR. This element, Muk, produces a long hairpin RNA molecule that can trigger DNA methylation and heritable silencing of one or many MuDR elements. In most cases, MuDR elements remain inactive even after Muk segregates away. Thus, Muk-induced silencing involves a directed and heritable change in gene activity in the absence of changes in DNA sequence. Using classical genetic analysis, we have identified an exceptional position at which MuDR element silencing is unstable. Muk effectively silences the MuDR element at this position. However, after Muk is segregated away, element activity is restored. This restoration is accompanied by a reversal of DNA methylation. To our knowledge, this is the first documented example of a position effect that is associated with the reversal of epigenetic silencing. This observation suggests that there are cis-acting sequences that alter the propensity of an epigenetically silenced gene to remain inactive. This raises the interesting possibility that an important feature of local chromatin environments may be the capacity to erase previously established epigenetic marks.

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Genetic and Southern blot analysis of a family segregating for active MuDR(p5) and MuDR(p4) elements.A) XhoI digests of a family segregating for MuDR(p5) and MuDR(p4), in which the female parent carried unmethylated MuDR(p5) and MuDR(p4) following the loss of Muk. Kernels were separated into classes based on somatic excision frequency, planted, and the resulting progeny plants were subjected to Southern blot analysis. B) Summarized frequency of spotted kernels in progeny of test crosses of the plants analyzed in panel A.
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pgen-1000216-g006: Genetic and Southern blot analysis of a family segregating for active MuDR(p5) and MuDR(p4) elements.A) XhoI digests of a family segregating for MuDR(p5) and MuDR(p4), in which the female parent carried unmethylated MuDR(p5) and MuDR(p4) following the loss of Muk. Kernels were separated into classes based on somatic excision frequency, planted, and the resulting progeny plants were subjected to Southern blot analysis. B) Summarized frequency of spotted kernels in progeny of test crosses of the plants analyzed in panel A.

Mentions: After the loss of Muk, MuDR(p4) never reactivated when it was by itself (Table 1). However, MuDR(p4) did become heritably reactivated in the presence of a reactivated MuDR(p5) element, but only when MuDR(p5) was fully active in the generation immediately following the loss of Muk. Plants that carried an active MuDR(p5) element (as judged by hypomethylation of HinfI sites in both MuDR(p5) and Mu1), also carried unmethylated MuDR(p4) elements (Figure 4B, lanes 7 and 8). When these plants were test crossed, they gave rise to an average of 77% spotted progeny, consistent with the independent segregation of two active MuDR elements (Table 1). To test this hypothesis, kernels from one of these families were planted and the resulting plants were subjected to Southern blot analysis (Figure 6A) and were test crossed (Table 3). In this family, both MuDR(p5) and MuDR(p4) cosegregated with Mu activity. All spotted kernels in this family carried either MuDR(p5), MuDR(p4) or both, while none of the pale kernels had either. Plants carrying either MuDR(p5) or MuDR(p4) gave rise to an average of 50% and 48% spotted kernels respectively. Plants that carried both elements gave rise to an average of 78% spotted kernels, consistent with the independent assortment of two unlinked active MuDR elements (Table 3). Those that carried neither element did not give rise to any spotted kernels (data not shown). The elements also showed a positive dosage effect; the most heavily spotted kernels carried both elements (7/7) while the more moderate or weakly spotted kernels (19/19) carried a single MuDR element. These data demonstrate that both elements were active in this family, and that they were the only active elements present. Since MuDR(p4) alone never exhibited reactivation in this family (Table 1) or any other we have examined (see below), we suggest that MuDR(p4) required the presence of active MuDR(p5) to become reactivated.


A position effect on the heritability of epigenetic silencing.

Singh J, Freeling M, Lisch D - PLoS Genet. (2008)

Genetic and Southern blot analysis of a family segregating for active MuDR(p5) and MuDR(p4) elements.A) XhoI digests of a family segregating for MuDR(p5) and MuDR(p4), in which the female parent carried unmethylated MuDR(p5) and MuDR(p4) following the loss of Muk. Kernels were separated into classes based on somatic excision frequency, planted, and the resulting progeny plants were subjected to Southern blot analysis. B) Summarized frequency of spotted kernels in progeny of test crosses of the plants analyzed in panel A.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2563033&req=5

pgen-1000216-g006: Genetic and Southern blot analysis of a family segregating for active MuDR(p5) and MuDR(p4) elements.A) XhoI digests of a family segregating for MuDR(p5) and MuDR(p4), in which the female parent carried unmethylated MuDR(p5) and MuDR(p4) following the loss of Muk. Kernels were separated into classes based on somatic excision frequency, planted, and the resulting progeny plants were subjected to Southern blot analysis. B) Summarized frequency of spotted kernels in progeny of test crosses of the plants analyzed in panel A.
Mentions: After the loss of Muk, MuDR(p4) never reactivated when it was by itself (Table 1). However, MuDR(p4) did become heritably reactivated in the presence of a reactivated MuDR(p5) element, but only when MuDR(p5) was fully active in the generation immediately following the loss of Muk. Plants that carried an active MuDR(p5) element (as judged by hypomethylation of HinfI sites in both MuDR(p5) and Mu1), also carried unmethylated MuDR(p4) elements (Figure 4B, lanes 7 and 8). When these plants were test crossed, they gave rise to an average of 77% spotted progeny, consistent with the independent segregation of two active MuDR elements (Table 1). To test this hypothesis, kernels from one of these families were planted and the resulting plants were subjected to Southern blot analysis (Figure 6A) and were test crossed (Table 3). In this family, both MuDR(p5) and MuDR(p4) cosegregated with Mu activity. All spotted kernels in this family carried either MuDR(p5), MuDR(p4) or both, while none of the pale kernels had either. Plants carrying either MuDR(p5) or MuDR(p4) gave rise to an average of 50% and 48% spotted kernels respectively. Plants that carried both elements gave rise to an average of 78% spotted kernels, consistent with the independent assortment of two unlinked active MuDR elements (Table 3). Those that carried neither element did not give rise to any spotted kernels (data not shown). The elements also showed a positive dosage effect; the most heavily spotted kernels carried both elements (7/7) while the more moderate or weakly spotted kernels (19/19) carried a single MuDR element. These data demonstrate that both elements were active in this family, and that they were the only active elements present. Since MuDR(p4) alone never exhibited reactivation in this family (Table 1) or any other we have examined (see below), we suggest that MuDR(p4) required the presence of active MuDR(p5) to become reactivated.

Bottom Line: In animals and yeast, position effects have been well documented.In contrast, there are few examples of position effects in plants, and there are no documented examples in either plants or animals for positions that are associated with the reversal of previously established silenced states.To our knowledge, this is the first documented example of a position effect that is associated with the reversal of epigenetic silencing.

View Article: PubMed Central - PubMed

Affiliation: Plant Science Department, McGill University, Macdonald Campus, Ste Anne de Bellevue, Quebec, Canada.

ABSTRACT
In animals and yeast, position effects have been well documented. In animals, the best example of this process is Position Effect Variegation (PEV) in Drosophila melanogaster. In PEV, when genes are moved into close proximity to constitutive heterochromatin, their expression can become unstable, resulting in variegated patches of gene expression. This process is regulated by a variety of proteins implicated in both chromatin remodeling and RNAi-based silencing. A similar phenomenon is observed when transgenes are inserted into heterochromatic regions in fission yeast. In contrast, there are few examples of position effects in plants, and there are no documented examples in either plants or animals for positions that are associated with the reversal of previously established silenced states. MuDR transposons in maize can be heritably silenced by a naturally occurring rearranged version of MuDR. This element, Muk, produces a long hairpin RNA molecule that can trigger DNA methylation and heritable silencing of one or many MuDR elements. In most cases, MuDR elements remain inactive even after Muk segregates away. Thus, Muk-induced silencing involves a directed and heritable change in gene activity in the absence of changes in DNA sequence. Using classical genetic analysis, we have identified an exceptional position at which MuDR element silencing is unstable. Muk effectively silences the MuDR element at this position. However, after Muk is segregated away, element activity is restored. This restoration is accompanied by a reversal of DNA methylation. To our knowledge, this is the first documented example of a position effect that is associated with the reversal of epigenetic silencing. This observation suggests that there are cis-acting sequences that alter the propensity of an epigenetically silenced gene to remain inactive. This raises the interesting possibility that an important feature of local chromatin environments may be the capacity to erase previously established epigenetic marks.

Show MeSH
Related in: MedlinePlus