A position effect on the heritability of epigenetic silencing

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.

Figure 1. The crossing schemes used to generate the families described in the text.

Tables and figures referring to particular families are as indicated. “p5” refers to MuDR(p5); “p4” refers to MuDR(p4). Percentages refer to the percent of spotted progeny kernels arising from a given cross.

Figure 2. An ear derived from a plant carrying MuDR(p3) and Southern blot of DNA from plants grown from the test cross and the cross to a Muk homozygote.

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.

Figure 3. 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.

Figure 4. 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.

Figure 5. Genetic and Southern blot analysis of families segregating for MuDR(p5) and Muk.

A) A HinfI digest of two families probed with an internal portion of Mu1. The first was derived from a cross between a plant carrying an active MuDR(p5) element and an a1-mum2 tester (lanes 1–12); the second was derived from a cross between the same plant carrying MuDR(p5) and a Muk homozygote. Methylated and unmethylated Mu1 elements at a1-mum2 are as indicated. Arrows indicate new insertions of Mu1 elements. B) DNA from representative individuals digested with HinfI and probed with the mudrA TIR. Fragments resulting from methylated and unmethylated HinfI sites within the TIR are as indicated, as is the fragment from Muk. Sample designations are the same as in panel A. C) Summarized frequencies of spotted kernels in progeny of test crosses of plants depicted in panel A.

Figure 6. 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.

Figure 7. Genetic and Southern blot analysis of a family derived from a plant that carried MuDR(p5) and MuDR(p4) in which reactivation was delayed and both elements were still methylated in the first generation following the loss of Muk.

A) XhoI digests of a family segregating for MuDR(p5) and MuDR(p4), in which the female parent carried methylated MuDR(p5) and MuDR(p4). 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 plants depicted in panel A.

Figure 8. A graphic representation of a lineage in which a plant carrying active MuDR(p5) and MuDR(p4) was crossed to a Muk homozygote, and resulting progeny plants were subsequently test crossed.

Percent figures refer to the summarized frequency of spotted progeny kernels derived from each cross.

Figure 9. A graphic representation of a lineage in which MuDR(p5) and a duplicate copy of that element were crossed to a Muk heterozygote.

Percent figures refer to the summarized frequency of spotted progeny kernels derived from each cross.

Figure 10. A representation of the region into which MuDR(p5) is inserted.

Sequences in yellow represent the target site duplication that was produced upon insertion. Sequences in green are the GA-rich sequences identified near the insertion. Sequences in red are presumed coding sequences. The rice homolog is the gene that best matches the Hemera gene in maize.