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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 MuDR(p5), MuDR(p4) and Muk.A) Graphic depiction of summarized frequency of spotted progeny kernels derived from different classes of individuals depicted in Figure 3. For each class, the relevant genotypes are as indicated. “meth” refers to the methylation status of Mu1 elements of each class, as determined in Figure 3. B) Southern blot analysis of representative individuals from each class depicted in panel A. Samples were digested with HinfI and probed with a fragment including all of the MuDR TIR. The relevant fragments are as indicated by the red arrows. The additional fragments visible on this blot represent hMuDR elements that do not cosegregate in this family with activity or a lack thereof. C) Restriction map of the region around one of the terminal inverted repeat flanking the MuDR elements. The indicated sizes are those expected if the HinfI site in the TIR is methylated or unmethylated at the two positions based on available sequence. Because Muk has an identical TIR to MuDR and is methylated at the HinfI, it can also be seen as a unique fragment of the indicated size. D) An example of a plant in which reactivation of MuDR(p5) was delayed. Because the reporter a1-mum2 allele is suppressible, the green sectors represent tissue in which MuDR(p5) has been reactivated during somatic development.
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pgen-1000216-g004: Genetic and Southern blot analysis of a family segregating for MuDR(p5), MuDR(p4) and Muk.A) Graphic depiction of summarized frequency of spotted progeny kernels derived from different classes of individuals depicted in Figure 3. For each class, the relevant genotypes are as indicated. “meth” refers to the methylation status of Mu1 elements of each class, as determined in Figure 3. B) Southern blot analysis of representative individuals from each class depicted in panel A. Samples were digested with HinfI and probed with a fragment including all of the MuDR TIR. The relevant fragments are as indicated by the red arrows. The additional fragments visible on this blot represent hMuDR elements that do not cosegregate in this family with activity or a lack thereof. C) Restriction map of the region around one of the terminal inverted repeat flanking the MuDR elements. The indicated sizes are those expected if the HinfI site in the TIR is methylated or unmethylated at the two positions based on available sequence. Because Muk has an identical TIR to MuDR and is methylated at the HinfI, it can also be seen as a unique fragment of the indicated size. D) An example of a plant in which reactivation of MuDR(p5) was delayed. Because the reporter a1-mum2 allele is suppressible, the green sectors represent tissue in which MuDR(p5) has been reactivated during somatic development.

Mentions: Active MuDR elements, regardless of their position would be expected to yield a fragment of 445 bp when digested with HinfI. This size is consistent with a lack of methylation of both the HinfI site within the TIR adjoining mudrA (TIRA) of MuDR elements and of a second site within the first intron of mudrA. Methylation of the TIR HinfI site of TIRA of MuDR elements will yield larger fragments whose size depends on the MuDR insertion sites. Based on the sequence of DNA flanking MuDR(p4) and MuDR(p5), if the TIR HinfI site (but not the internal HinfI site) is methylated the expected fragment sizes are 648 bp and 1003 bp respectively. Similarly, the expected fragment size if the TIR HinfI site is methylated in Mu killer is 500 bp. In each case the expected fragment sizes were observed (Figure 4B).


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 MuDR(p5), MuDR(p4) and Muk.A) Graphic depiction of summarized frequency of spotted progeny kernels derived from different classes of individuals depicted in Figure 3. For each class, the relevant genotypes are as indicated. “meth” refers to the methylation status of Mu1 elements of each class, as determined in Figure 3. B) Southern blot analysis of representative individuals from each class depicted in panel A. Samples were digested with HinfI and probed with a fragment including all of the MuDR TIR. The relevant fragments are as indicated by the red arrows. The additional fragments visible on this blot represent hMuDR elements that do not cosegregate in this family with activity or a lack thereof. C) Restriction map of the region around one of the terminal inverted repeat flanking the MuDR elements. The indicated sizes are those expected if the HinfI site in the TIR is methylated or unmethylated at the two positions based on available sequence. Because Muk has an identical TIR to MuDR and is methylated at the HinfI, it can also be seen as a unique fragment of the indicated size. D) An example of a plant in which reactivation of MuDR(p5) was delayed. Because the reporter a1-mum2 allele is suppressible, the green sectors represent tissue in which MuDR(p5) has been reactivated during somatic development.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1000216-g004: Genetic and Southern blot analysis of a family segregating for MuDR(p5), MuDR(p4) and Muk.A) Graphic depiction of summarized frequency of spotted progeny kernels derived from different classes of individuals depicted in Figure 3. For each class, the relevant genotypes are as indicated. “meth” refers to the methylation status of Mu1 elements of each class, as determined in Figure 3. B) Southern blot analysis of representative individuals from each class depicted in panel A. Samples were digested with HinfI and probed with a fragment including all of the MuDR TIR. The relevant fragments are as indicated by the red arrows. The additional fragments visible on this blot represent hMuDR elements that do not cosegregate in this family with activity or a lack thereof. C) Restriction map of the region around one of the terminal inverted repeat flanking the MuDR elements. The indicated sizes are those expected if the HinfI site in the TIR is methylated or unmethylated at the two positions based on available sequence. Because Muk has an identical TIR to MuDR and is methylated at the HinfI, it can also be seen as a unique fragment of the indicated size. D) An example of a plant in which reactivation of MuDR(p5) was delayed. Because the reporter a1-mum2 allele is suppressible, the green sectors represent tissue in which MuDR(p5) has been reactivated during somatic development.
Mentions: Active MuDR elements, regardless of their position would be expected to yield a fragment of 445 bp when digested with HinfI. This size is consistent with a lack of methylation of both the HinfI site within the TIR adjoining mudrA (TIRA) of MuDR elements and of a second site within the first intron of mudrA. Methylation of the TIR HinfI site of TIRA of MuDR elements will yield larger fragments whose size depends on the MuDR insertion sites. Based on the sequence of DNA flanking MuDR(p4) and MuDR(p5), if the TIR HinfI site (but not the internal HinfI site) is methylated the expected fragment sizes are 648 bp and 1003 bp respectively. Similarly, the expected fragment size if the TIR HinfI site is methylated in Mu killer is 500 bp. In each case the expected fragment sizes were observed (Figure 4B).

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