Limits...
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.

Show MeSH

Related in: MedlinePlus

Southern blot analysis of a family segregating for MuDR(p5), MuDR(p4) and Muk.Kernels were separated by somatic excision frequency and DNA was extracted from plants grown from those kernels. A) A SacI digest probed with a fragment of MuDR (probe B). The diagnostic 4.9 kb MuDR fragment is as indicated. The smaller Muk-specific fragment is as indicated, as is as the larger fragment that results from methylation of the SacI site in the Muk TIR (red arrow). B) An XhoI digest of the same samples probed with a second fragment of MuDR (probe A). Polymorphisms specific to MuDR at each of two positions are as indicated. C) A HinfI digest of the same samples probed with an internal fragment of Mu1 (probe C). Fragments corresponding to unmethylated and methylated Mu1 elements in this background are as indicated. D) A restriction map of MuDR with probe regions as indicated. The red arrows indicate TIRs. E) A restriction map of Mu1 at a1-mum2.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2563033&req=5

pgen-1000216-g003: Southern blot analysis of a family segregating for MuDR(p5), MuDR(p4) and Muk.Kernels were separated by somatic excision frequency and DNA was extracted from plants grown from those kernels. A) A SacI digest probed with a fragment of MuDR (probe B). The diagnostic 4.9 kb MuDR fragment is as indicated. The smaller Muk-specific fragment is as indicated, as is as the larger fragment that results from methylation of the SacI site in the Muk TIR (red arrow). B) An XhoI digest of the same samples probed with a second fragment of MuDR (probe A). Polymorphisms specific to MuDR at each of two positions are as indicated. C) A HinfI digest of the same samples probed with an internal fragment of Mu1 (probe C). Fragments corresponding to unmethylated and methylated Mu1 elements in this background are as indicated. D) A restriction map of MuDR with probe regions as indicated. The red arrows indicate TIRs. E) A restriction map of Mu1 at a1-mum2.

Mentions: A) An ear derived from a plant carrying MuDR(p3) that was crossed as a female to a plant that was homozygous for Muk. Because Muk does not alter somatic excision frequency in F1 aleurone, changes in excision frequency from low to high could be used to screen for new insertions of MuDR(p3), as is indicated. Kernels from this ear and the control test cross ear (MuDR(p3)×a1-mum2 tester, not shown) were separated by excision frequency and planted. B) Southern blot of DNA from plants grown from the test cross (lanes 1–9) and the cross to a Muk homozygote (lanes 11–14). In the top panel, the DNA was digested with EcoRI, used to distinguish MuDR elements at different positions based on size polymorphisms, and probed with an internal fragment of MuDR (probe B, Figure 3). The red arrows indicate new MuDR insertions. In the bottom panel, the DNA was digested with the methyl-sensitive enzyme HinfI and probed with an internal portion of Mu1 (probe C, Figure 3). The resulting fragments resulting from methylated and unmethylated HinfI sites in the end of the Mu1 element at the a1-mum2 reporter are as indicated. Following analysis of the DNA, each plant was then crossed to an a1-mum2 tester. The numbers below the blots indicate the percent frequency of spotted kernels arising from test crosses of plants in the lanes above them.


A position effect on the heritability of epigenetic silencing.

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

Southern blot analysis of a family segregating for MuDR(p5), MuDR(p4) and Muk.Kernels were separated by somatic excision frequency and DNA was extracted from plants grown from those kernels. A) A SacI digest probed with a fragment of MuDR (probe B). The diagnostic 4.9 kb MuDR fragment is as indicated. The smaller Muk-specific fragment is as indicated, as is as the larger fragment that results from methylation of the SacI site in the Muk TIR (red arrow). B) An XhoI digest of the same samples probed with a second fragment of MuDR (probe A). Polymorphisms specific to MuDR at each of two positions are as indicated. C) A HinfI digest of the same samples probed with an internal fragment of Mu1 (probe C). Fragments corresponding to unmethylated and methylated Mu1 elements in this background are as indicated. D) A restriction map of MuDR with probe regions as indicated. The red arrows indicate TIRs. E) A restriction map of Mu1 at a1-mum2.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1000216-g003: Southern blot analysis of a family segregating for MuDR(p5), MuDR(p4) and Muk.Kernels were separated by somatic excision frequency and DNA was extracted from plants grown from those kernels. A) A SacI digest probed with a fragment of MuDR (probe B). The diagnostic 4.9 kb MuDR fragment is as indicated. The smaller Muk-specific fragment is as indicated, as is as the larger fragment that results from methylation of the SacI site in the Muk TIR (red arrow). B) An XhoI digest of the same samples probed with a second fragment of MuDR (probe A). Polymorphisms specific to MuDR at each of two positions are as indicated. C) A HinfI digest of the same samples probed with an internal fragment of Mu1 (probe C). Fragments corresponding to unmethylated and methylated Mu1 elements in this background are as indicated. D) A restriction map of MuDR with probe regions as indicated. The red arrows indicate TIRs. E) A restriction map of Mu1 at a1-mum2.
Mentions: A) An ear derived from a plant carrying MuDR(p3) that was crossed as a female to a plant that was homozygous for Muk. Because Muk does not alter somatic excision frequency in F1 aleurone, changes in excision frequency from low to high could be used to screen for new insertions of MuDR(p3), as is indicated. Kernels from this ear and the control test cross ear (MuDR(p3)×a1-mum2 tester, not shown) were separated by excision frequency and planted. B) Southern blot of DNA from plants grown from the test cross (lanes 1–9) and the cross to a Muk homozygote (lanes 11–14). In the top panel, the DNA was digested with EcoRI, used to distinguish MuDR elements at different positions based on size polymorphisms, and probed with an internal fragment of MuDR (probe B, Figure 3). The red arrows indicate new MuDR insertions. In the bottom panel, the DNA was digested with the methyl-sensitive enzyme HinfI and probed with an internal portion of Mu1 (probe C, Figure 3). The resulting fragments resulting from methylated and unmethylated HinfI sites in the end of the Mu1 element at the a1-mum2 reporter are as indicated. Following analysis of the DNA, each plant was then crossed to an a1-mum2 tester. The numbers below the blots indicate the percent frequency of spotted kernels arising from test crosses of plants in the lanes above them.

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