Copyright  1998 by the Society of America

Paramutation of the r1 Locus of Is Associated With Increased Cytosine Methylation

Elsbeth L. Walker Biology Department, University of Massachusetts, Amherst, Massachusetts 01003 Manuscript received September 25, 1997 Accepted for publication December 8, 1997

ABSTRACT In paramutation two of a interact so that one of the alleles is epigenetically silenced. The silenced state is then genetically transmissible for many generations. The large (220 kbp) multigenic complex R-r is paramutable: its level of expression is changed during paramutation. R-r was found to exhibit increases in its level of cytosine methylation (C-methylation) following paramutation. These C-methylation changes are localized to the 5Ј portions of the two in the complex that are most sensitive to paramutation. These methylation changes flank a small region called ␴ that is thought to have been derived from a transposon named doppia. A mutant derivative of R-r that has a deletion of the ␴ region fails to become methylated under conditions in which R-r is heavily methylated. This suggests that the presence of ␴ sequences at the locus is required for the methylation changes that are observed following paramutation.

ARAMUTATION is aregularly occurring, directed, pigmentation in which not all aleurone cells are colored. Pand heritable alteration of resulting The paramutant R-r complex is referred to as R-rЈ,to from the interaction of two alleles. One is referred distinguish it from nonparamutant R-r. Interestingly, to as paramutable: its expression changes following par- the effect on aleurone pigmentation is only observed if amutation. The other allele is paramutagenic: it causes R-rЈ is transmitted through male gametes (pollen). the change in the paramutable allele. When the paramu- When R-rЈ is transmitted through the female, the nor- table/paramutagenic heterozygote is crossed to allow mal dark uniform pigmentation pattern is observed. segregation of the two alleles, virtually 100% of the This dependence on the mode of transmission is not paramutable alleles transmitted display a decrease in due to a simple dosage effect; two paternally transmitted expression. This decreased expression level (the para- copies of R-rЈ still confer a paramutant in mutant phenotype) persists through many generations. aleurone (Kermicle 1970). Nonparamutant R-r also ex- Paramutation was first described in 1956 (Brink 1956), hibits mild silencing on male transmission. R-rЈ is char- but a mechanism to explain how one allele can heritably acterized as being metastable; under certain conditions, affect the expression of another has remained obscure. it reverts toward the standard full color phenotype One of the best studied examples of paramutation is (Brink 1956; Brink 1958; Styles and Brink 1966; the r1 locus of Zea mays (Brink 1973; Kermicle 1996). Styles and Brink 1968). r1 genes encode helix-loop-helix proteins that are capa- The paramutable r1 complex, R-r, consists of four du- ble of directing transcription of the structural genes in plicated copies or partial copies of the r1 transcription the biosynthetic pathway (Goff et al. 1990; unit (Walker et al. 1995). These four copies, referred Ludwig et al. 1989). Thus, dominant R1 genes confer to as components, are designated P, q, S1, and S2. The red or purple anthocyanin pigmentation to the tissues S1 and S2 components are complete genes and are in which they are expressed. Paramutation at r1 is illus- responsible for pigmentation of the aleurone layer of trated in Figure 1. The crosses involve two r1 complexes, the seed. The q component is nonfunctional because it the paramutable R-r complex, and the paramutagenic lacks downstream coding sequences. The P component R-marbled (R-mb) complex. In the aleurone layer of the is the third complete r1 gene, and is active in several seed, the R-r complex normally confers full coloration. tissues in the plant including the coleoptile, the roots, The R-mb complex confers a marbled pattern of pigmen- and the anther walls. The three complete genes of the tation to aleurone. Following outcrossing of the hetero- complex are not equally subject to paramutation: the S zygote to a recessive colorless, r, tester strain, kernels genes (S1 and S2) show a large decrease in the amount receiving the R-r complex show a mottled pattern of of pigment they confer following paramutation, while the P gene shows a relatively small decrease following heterozygosity with a paramutagenic r1 complex (Brink Address for correspondence: Biology Department, Morrill Science Cen- Mikula Brown ter, University of Massachusetts, Amherst, MA 01003. and 1958; 1966). E-mail: [email protected] The S genes are arranged in an inverted head-to-head

Genetics 148: 1973–1981 (April, 1998) 1974 E. L. Walker

paramutation (Coe 1966; Hollick et al. 1995; Pat- terson et al. 1995). In petunia, a transgenic copy of the maize A1 gene can spontaneously become paramuta- genic (Meyer et al. 1993). Paramutation-like phenom- ena include a broad class of “homology-dependent ” events associated with meiotically stable re- ductions in gene expression (reviewed in Matzke et al. 1996; Meyer and Saedler 1996). Certain examples are particularly similar to paramutation since homologous gene copies interact in a way that heritably alters the expression of one of the copies, although the silencing and silenced genes are nonallelic. Examples of this type of interaction include the transgenic H locus in which an inactive transgene locus has the ability to silence partially homologous transgenes introduced into the same genome by genetic crossing (Matzke et al. 1994), and inactivation of endogenous genes that appears to occur among members of the phosphoribosylanthrani- Figure 1.—Paramutation of r1. The series of crosses shown late synthase (PAI) gene family of the Wassilewskija involves three alleles of r1, the R-r complex (paramutable strain of Arabidopsis (Bender and Fink 1995). allele), the R-mb complex (paramutagenic allele), and a reces- Three features stand out as being held in common sive tester allele, r. R-r confers solid purple pigmentation to by several of these systems. One is that multiple copies the aleurone layer of the seeds; R-mb confers a marbled pattern of pigmentation to the aleurone; the r tester allele confers no of the genes are often present in the same genome color to aleurone. Paramutation is observed following test (reviewed in Flavell 1994; Matzke et al. 1996; Meyer crossing of the R-r/R-mb heterozygote. Half of the kernels on and Saedler 1996). A second feature is that inactivation the test cross ear carry the R-mb allele, which, when transmitted tends to be associated with increased cytosine methyla- through the female, confers a marbled pattern of pigmenta- tion (C-methylation) of both silenced and silencing loci tion in aleurone, but when transmitted through the male Bender Fink Eggleston Matzke confers extremely infrequent sectors rendering the aleurones ( and 1995; et al. 1995; mostly colorless. The R-r allele transmitted from the R-r/R- et al. 1994; Meyer et al. 1993; also reviewed in Matzke mb heterozygote is referred to as being “paramutant” and is et al. 1996; Meyer and Saedler 1996). A final feature designated as R-rЈ to indicate this new epigenetic state. Male noted by some reviewers (Martienssen 1996a; Matzke transmission of R-rЈ results in a pale mottled pattern of pig- mentation in the aleurone, while female transmission of R-rЈ et al. 1996) is the possible involvement of transposable results in a normal solid color pattern of aleurone pigmenta- elements in some of these systems. Epigenetic inactiva- tion. tion of transposable elements is a well-known phenome- non (reviewed by Fedoroff 1996; Martienssen 1996b). Some paramutation systems, , and in maize, orientation and are separated by a 387-bp region called e.g. b1 pl1 do not share all of these features. Alleles of and ␴. The ␴ region appears to be a rearranged remnant b1 pl1 Cone that are active in paramutation are simple, containing of a transposable element of the CACTA family ( Chandler et al. 1993; Upadhyaya et al. 1985) called doppia only a single gene copy ( et al. 1996). Further- (Walker et al. 1995). At its ends, ␴ contains partial more, no C-methylation differences have been found Chandler copies of the terminal inverted repeats of the doppia during either b1 or pl1 paramutation ( et al. element. Internal to these inverted repeats are multiple 1996). Alleles of pl1 that are active in paramutation do Chandler copies of a subterminal repeated element of doppia, contain a doppia transposable element ( et al. Hollick and a second region consisting of sequences that have 1996; et al. 1995), but alleles of pl1 that are Coc- undergone extensive rearrangement and that may or inactive in paramutation also contain this element ( ciolone Cone may not have been derived from doppia. The ␴ region and 1993). No transposable element has functions as the promoter for both the S1 and S2 genes been reported near or within the b1 alleles that are of R-r (Walker et al. 1995). doppia sequences that in- active in paramutation. In contrast, the r1 paramutation clude one terminal inverted repeat element and multi- system shares all three of these features. Both the para- ple subterminal repeats are also found distal to q mutable R-r complex (Robbins et al. 1991; Walker et (Walker et al. 1995). No doppia sequences are found al. 1995) and the paramutagenic R-mb (E. L. Walker, at the P component. unpublished results) and R-stippled (R-st)(Eggleston Recently, a variety of paramutation and paramutation- et al. 1995) complexes contain multiple r1 genes. Epige- like phenomena has been observed that can involve netic silencing of r1 genes has been correlated with either endogenous genes or transgenes. In maize, two increased C-methylation (Eggleston et al. 1995; Ron- other genes, b1 and pl1, are known to be subject to chi et al. 1995). Finally, the R-r complex harbors at R1 Paramutation 1975 least two fragments of the transposable element, doppia ments at R-r do not themselves trigger C-methylation in (Walker et al. 1995). r1 paramutation. The question of whether the ␴ region, the multiple repeated genes, or both make R-r susceptible to paramu- tation has been partially addressed using a series of MATERIALS AND METHODS mutant derivative alleles of R-r that have lost the ␴ region Genetic stocks: All stocks were maintained in the W22 in- and varying amounts of flanking S1 and S2 sequences bred background. All stocks are homozygous dominant for (Kermicle 1996). In this experiment, a test of secondary the a1, a2, c1, c2, bz1, and bz2 genes necessary for anthocyanin synthesis in aleurone and homozygous recessive for the pl1 paramutation was used to assess the paramutability of and b1 genes (see Dooner et al. 1991 for descriptions). The these ␴-deleted alleles. Secondary paramutation refers R-r:standard allele used in this study confers strong pigmenta- to the phenomenon that paramutant r1 alleles become tion of the aleurone of the seed, the coleoptile, the roots and weakly paramutagenic; prior to paramutation, these al- leaf tip of seedlings, and to the roots and anthers of mature Stadler leles are not paramutagenic. In this test, the potentially plants, and has been described previously ( 1948; Walker et al. 1995). The R-mb allele confers strong pigmenta- paramutable ␴-deleted alleles were kept heterozygous tion to the aleurone and embryo in a “marbled” pattern of with a paramutagenic allele for three generations, then dark blotches on a colorless background, and also weakly outcrossed to a paramutationally neutral allele, then pigments the coleoptile (Weyers 1961). The isolation, charac- crossed to the paramutable R-r allele to determine if terization, and structure of derivative allele r-r:N1-3-1 has been Kermicle described previously (Kermicle and Axtell 1981; Walker they have acquired paramutagenicity. found et al. 1995). The recessive (r) tester allele used was r-g:Stadler ; that all but one of the ␴-deleted alleles failed to acquire it confers no pigmentation to any part of the plant or seed. paramutagenicity. Earlier tests on a similar, but molecu- Derivation of paramutant R-r: First-generation heterozy- larly undefined, set of alleles had found that none were gotes were generated in 1990, 1991, and 1993 by crossing able to acquire paramutagenicity (Brown 1966). Only homozygous R-r plants with homozygous R-mb plants. Second- generation heterozygotes were generated in 1991, 1992, and the r-r:N1-3-1 allele acquired any paramutagenicity, and 1994 by crossing each of these three lineages of first-genera- it was severely compromised in its ability to become tion heterozygotes with homozygous R-mb plants. Third-gener- paramutagenic relative to the standard paramutable al- ation heterozygotes were generated in 1992 and 1995 by cross- lele, R-r (Kermicle 1996). The r-r:N1-3-1 allele has the ing two independent second-generation heterozygote lineages smallest deletion of the alleles tested: it has a nearly with homozygous R-mb plants. For the studies presented here, at least two plants from each of the three second-generation precise deletion of ␴ with only 26 bp of the left end of heterozygote lineages and each of the two third-generation ␴ retained (Walker et al. 1995). The remainder of the heterozygote lineages were reciprocally crossed to a homozy- complex is intact. The finding that r-r:N1-3-1 has lost gous r tester strain. These crosses resulted in six families seg- ″ most of its ability to acquire secondary paramutagenicity regating r/R-r (second-generation paramutants) and four .(families segregating r/R-r ٞ (third-generation paramutants implies that the ␴ region has a role in causing paramuta- Homozygous R-r and heterozygous r/R-r stocks that serve as bility of R-r. The next smallest deletion is found in the controls for basal levels of methylation were grown at the same r-r:N1-3-2 allele which has a deletion that removes all time and under the same conditions (field or greenhouse) as but the right-most 18 bp of ␴ from the complex as well the paramutant stocks to which they were compared. as an additional 1578 bp of S1 (Walker et al. 1995). Preparation of DNA and genomic blotting: For analysis of methylation in first-generation heterozygotes, DNA was pre- Removal of this region takes away even the minimal pared from leaves of mature, flowering plants homozygous ability to acquire secondary paramutagenicity that was for R-r or heterozygous (first generation) for R-r/R-mb. For retained by r-r:N1-3-1. Presumably, in the r-r:N1-3-2 deriv- analysis of the standard pattern of methylation for R-rЈ, DNA ative, the remaining sequences necessary for acquisition samples were prepared from the third leaf of mature, flow- ering plants of genotype r/R-r″ (six families) and r/R-r ٞ (four of secondary paramutagenicity have been deleted. families) accordingto the protocol of Dellaporta (1994). For The aim of the experiments presented here is to exam- methylation analysis of the r-r :N1-3-1 allele, DNA was prepared ine whether r1 paramutation is associated with changes from leaves of three-week-old seedlings of second-generation in the level of C-methylation of the paramutable R-r r-r:N1-3-1Ј/R-mb heterozygotes and from leaves of three-week- Ј complex. Changes were detected and were mapped to old seedlings of second-generation R-r /R-mb heterozygotes. DNA samples were digested with a 5–10-fold excess of a non- a region of the complex within the highly paramutable methylation-sensitive restriction enzyme, and were then puri- S1 and S2 genes flanking the ␴ region. To examine fied by organic extraction and ethanol precipitation. Six ␮g whether ␴ has a role in eliciting these C-methylation of DNA (or 3 ␮g from homozygous stocks) were then digested changes, the r-r:N1-3-1 allele was used. This allele has a for 18–22 hr with a 5–10-fold excess of the methylation-sensi- tive restriction enzymes. Agarose gel electrophoresis and blot- deletion of most of the doppia-containing ␴ region, but ting were performed as described previously (Robbins et al. retains the multiple homologous genic elements of the 1991). Quantitation of the percentage of sites methylated was R-r complex (i.e., the P, q, S1, and S2 genes). The failure accomplished using ImageQuant (Molecular Dynamics, Inc., of r-r:N1-3-1 to become methylated in a context that Sunnyvale, CA) software analysis of phosphorimager scans. Description of DNA probes: Probes were used that detect elicits strong C-methylation of intact R-r suggests that the promoter regions and first seven exons of each gene at the ␴ region is needed for induction of C-methylation the R-r complex. The probes used were: pR-nj:1 (Robbins et during paramutation, and that the duplicated gene seg- al. 1991), which detects the promoter and/or first exon of P, 1976 E. L. Walker

Figure 2.—Methylation changes at R-r during paramutation. DNA samples from naive (nonparamutant) homozy- gous R-r (lanes 1 and 2 of each panel) and from first-generation heterozy- gous R-r/R-mb individuals (lanes 3 and 4 of each panel) were digested to com- pletion with the non-methylation-sen- sitive restriction enzyme, HindIII fol- lowed by digestion with the meth- ylation-sensitive restriction enzymes, BstUI (A), HaeII (B), and HpaII (C), which have recognition sites near the ␴ region of R-r. The DNA probe used was ␴1011, an S-specific probe that hy- bridizes to the ␴ region between the S1 and S2 genes and does not recognize R-mb.

q, S1 and S2; ␴1011 (Walker et al. 1995), an S-specific probe of R-r that can be clearly distinguished from R-mb is the that hybridizes to the ␴ region between the S1 and S2 genes; ␴ region. Thus, in order to determine the extent of SAH (Walker et al. 1995), which detects the 5Ј portion of the second intron of P, S1, and S2; and cDNA-B, a probe methylation of R-r, it must be segregated from R-mb by derived from position 812 to 1334 in the Sn cDNA (Consonni sexual crossing of the heterozygote to a tester allele that et al. 1992) that detects r1 exons 4 through 7. To detect specifi- is more readily distinguished from R-r on genomic blots. cally the r-r:N1-3-1 allele, the probe N131 was generated from To do this, the R-r complex was kept heterozygous with primers (oN131-L: 5Ј-CAGGAAACGACTAAATTTGCC-3Ј and the paramutagenic R-mb complex for either two or three oN131-R: 5Ј-GCAACGAAGCATCAGTAT-3Ј) flanking the fil- ler DNA in the r-r:N1-3-1 derivative. The N131 probe was generations, and was then outcrossed via pollen to a generated by amplification from a plasmid clone containing homozygous recessive (r) tester strain. DNA was made the relevant region of r-r:N1-3-1 (Walker et al. 1995). from leaves of the resulting r/R-r″ (second-generation (paramutant), r/R-rٞ (third-generation paramutant plants, and from R-r/R-r (nonparamutant), and r/R-r RESULTS (nonparamutant) individuals. Southern blots were pre- Paramutation results in increased C-methylation of pared and hybridized with probes that allow mapping R-r: In many cases of epigenetic gene silencing, changes of sites within particular genes of the R-r complex. At in the level of C-methylation of the affected gene are least two individuals from each of 10 families (see mate- observed. To determine whether this correlation is ob- rials and methods for derivation of these stocks) were served in paramutation at r1, the methylation status of examined with at least two methylation-sensitive en- the paramutable allele R-r was examined both before zymes known to cut within the genic portions of R-r. and after making it heterozygous with the paramuta- A typical set of blots is shown in Figure 3. In A–C, genic allele R-mb. DNA was extracted from R-r homozy- the probe used hybridizes to the ␴ region of R-r. With gotes and R-r/R-mb heterozygotes, digested with the each C-methylation-sensitive enzyme used, there is an non-methylation-sensitive enzyme, HindIII, and then increase in the level of C-methylation of sites close to with methylation-sensitive restriction enzymes known to the ␴ region in paramutant versus nonparamutant R-r. cut near the ␴ region of R-r. Genomic blots were pre- Thus, the C-methylation that occurred in the heterozy- pared and hybridized with the probe ␴1011 which de- gote was retained following outcrossing. Every r/R-r″ tects the ␴ region of R-r but does not hybridize to R-mb and r/R-rٞ individual tested in all 10 families (not DNA. Representative blots are shown in Figure 2. The shown) exhibited increases in its level of C-methylation level of C-methylation of the paramutable R-r allele, in this region relative to naive (nonparamutant) R-r. measured by resistance to cleavage by methylation-sensi- D–F show the same three blots hybridized with a coding tive restriction enzymes, increases when R-r is heterozy- region probe (cDNA-B) that detects all three of the gous with the paramutagenic R-mb allele. intact components of R-r: P, S1, and S2. No increase in Analysis of the methylation status of R-r in first-gener- the level of methylated sites is evident in the coding ation R-r/R-mb heterozygotes is limited by the ability to regions detected by cDNA-B in paramutant versus non- distinguish between the r1 genes of the R-mb complex paramutant R-r. These results suggest that C-methy- and the r1 genes of the R-r complex. The only region lation changes in paramutation are confined either to R1 Paramutation 1977

Figure 3.—Methylation analysis of paramutant R-r. (A–C) DNA samples were digested to completion with the non-methylation- sensitive restriction enzyme HindIII, followed by digestion with the methylation-sensitive restriction enzymes AvaI (A), PstI (B), and PvuII (C). Lanes 1 and 2 are homozygous R-r; lanes 3 and 4 are r/R-r heterozygotes; lanes 5–8 (or 5–7 in B) are r/R-r ″ heterozygotes; lanes 9 and 10 (or 8 and 9 in B) are r/R-rٞ heterozygotes. The DNA probe used was ␴1011, an S-specific probe that hybridizes to the ␴ region between the S1 and S2 genes. (D–F) The blots shown in A, B, and C were stripped of the original probe and rehybridized with the probe cDNA-B which detects the coding region of each intact gene at R-r (P, S1, and S2). This probe also detects the r tester allele used in these studies; the hybridizing r1 fragments may (E) or may not (D and F) comigrate with fragments from the R-r complex. the 5Ј regions of the genes of the complex and/or are it should be noted that sites within the 5Ј regions of restricted to the S genes. Further mapping experiments these genes were methylated at all times. In order to (see below) also support this idea. detect changes in the P and q genes, those changes ,C-methylation changes map to the 5؅ portions of the would have to involve decreases in C-methylation levels S genes: Because the R-r complex comprises both genes or, in the case of the P gene, the changes would have that are strongly affected by paramutation (the S genes) to occur in the normally unmethylated downstream cod- and a gene which is only weakly affected by paramuta- ing portions. Thus paramutation is correlated with a tion (the P gene) (Brink 1956; Brink and Mikula 1958; striking increase in C-methylation, but only in the 5Ј Brown 1966), it provides an interesting opportunity to regions of the strongly affected S1 and S2 genes. examine whether changes in C-methylation are local- Deletion of ␴ results in loss of paramutation associ- ized to the strongly affected components. Three inde- ated C-methylation changes: The observation that pendently derived r/R-r″ individuals and two indepen- C-methylation changes in paramutation are localized dently derived r/R-rٞ individuals were used for detailed to the area flanking the transposon-derived ␴ region mapping. The maps of each R-r component and the suggests that this region may play a role in inducing methylation status of the C-methylation-sensitive restric- C-methylation changes during paramutation. A sponta- tion sites on these maps are shown in Figure 4. The neous mutant derivative of R-r that has a nearly precise only region of R-r that exhibits changes in the level of deletion of ␴ was used to address the question of C-methylation is a 3.4-kb region flanking ␴ and includ- whether the ␴ region is necessary for C-methylation to ing approximately 1.5 kb of the transcribed portion of take place. This derivative, r-r:N1-3-1 (Kermicle and each gene. No changes in the level of methylation were Axtell 1981) has a 361-bp deletion spanning from the observed in the P or q genes of the R-r complex, although right ␴ end proximally to a position 26 bp from the left 1978 E. L. Walker

Figure 4.—Methylation map of the R-r complex. The methylation status at sites for the methylation-sensitive restriction enzymes AvaI (A), PstI (P), PvuI (P1), PvuII (P2), MluI (M), and NruI (N) were measured from r/R-r″ and r/R-r ٞ plants. The probes used were pR-nj:1 (Robbins et al. 1991), ␴1011 (Walker et al. 1995), SAH (Walker et al. 1995), and cDNA-B which together allow detection of the promoter regions and first seven exons (indicated by black boxes) of each gene at the R-r complex. The accumulated results are represented as restriction maps of each component, P, q, S1, and S2. Circles above the respective restriction sites summarize the methylation profile for each site in paramutant (upper row of circles) and nonparamutant (lower row of circles) R-r. Open circles indicate that fewer than 25% of the sites were methylated. Half-filled circles indicate that 25–50% of the sites were methylated. Closed circles indicate that greater than 50% of the sites were methylated. end of ␴ (Figure 5) (Walker et al. 1995). Thus, r-r:N1- confers no pigmentation to the aleurone presumably 3-1 retains 26 bp of the left end of ␴ but the remainder due to the lack of its promoter, ␴. This allele has been of ␴ has been deleted. The rest of the complex, i.e., the shown to be severely compromised in its ability to ac- P gene, the q gene, and the coding portions of the quire secondary paramutagenicity (Kermicle 1996). It S1 and S2 genes, remains intact (Robbins et al. 1991; is possible to examine whether r-r:N1-3-1 acquires C-meth- Walker et al. 1995). At the breakpoint within r-r:N1-3- ylation following heterozygosity with a paramutagenic 1, an 84-bp stretch of filler DNA has been inserted. This allele, thus testing whether the repeated genic elements filler DNA is derived from adjacent sequences within of R-r (the P, q, S1, and S2 genes) can trigger C-methyla- S1 and S2 (Walker et al. 1995). The r-r:N1-3-1 derivative tion in response to heterozygosity with a paramutagenic allele. The r-r:N1-3-1 derivative was maintained as a heterozy- gote with R-mb for two generations. Even though in- creased C-methylation can be detected (see Figure 1) in first generation heterozygotes, methylation is stronger following several generations of paramutation (E. L. Walker, unpublished results, and see below). Thus, using two generations of heterozygosity gives a better chance of observing weak methylation changes at r-r:N1- 3-1, if such changes occur. DNA was prepared from these second-generation r-r:N1-3-1Ј/R-mb heterozygotes. DNA from a second-generation R-rЈ/R-mb heterozygote was used as a control to ensure that C-methylation of Figure 5.—Structure ofthe r-r:N1-3-1 derivative. Above (and R-rЈ is readily detectable under these heterozygous con- not to scale) are shown the genic components of R-r and its derivative, r-r:N1-3-1. Below is the restriction map of ␴ and its ditions. All DNAs were subjected to Southern analysis flanking DNA in the S1 and S2 genes. Restriction sites shown as described above. The results are shown in Figure 6. are AvaI (A), BstUI (B), PvuII (P), and HpaII (H). The r-r:N1- The relevant portion of S1 and S2 in the control R-rЈ/ 3-1 allele arose spontaneously from hemizygous R-r (Kermicle R-mb heterozygotes is heavily methylated, as shown in Axtell and 1981). This derivative has retained the P, q, S1, Figure 5A. When the same sites were tested in r-r:N1- and S2 genes of the complex, but has suffered a deletion of the ␴ region (white box with arrows representing multiple 3-1Ј/R-mb heterozygotes (Figure 5B), no evidence of subterminal repeat elements of doppia) between the S1 and C-methylation was observed. Thus, removal of the ␴ S2 genes (Robbins et al. 1991). The deletion includes all but region renders the complex ineffective in acquiring the left-most 26 base pairs of ␴ (white box). Between the C-methylation during paramutation. The multiple re- deletion breakpoints, filler DNA (gray box) has been inserted. This filler DNA is derived from nearby S2 sequences (Walker peated structure of R-r, which is maintained in the et al. 1995). Note that the r-r:N1-3-1 allele has gained a BstUI r-r:N1-3-1 derivative, does not, by itself, predispose the site close to the left end of the filler DNA. complex to methylation during paramutation. R1 Paramutation 1979

plex, since the highly paramutable S genes show obvious methylation changes while the much less paramutable P gene was never found to show increased C-methylation under the conditions used in this study. With regard to changes in C-methylation levels, r1 paramutation is distinct from two other maize paramutation systems, b1 and pl1, in which methylation changes are not found following paramutation. The correlation with increased C-methylation is shared with other meiotically stable silencing phenomena, e.g., paramutation of A1 trans- genes (Meyer et al. 1993), meiotically stable silencing associated with the transgenic H locus (Matzke et al. 1994), and with silencing of PAI loci in Arabidopsis (Bender and Fink 1995). Paramutation has been compared to other epigenetic gene silencing phenomena, most notably cases that oc- cur via transcriptional inactivation (Flavell 1994; Jor- gensen 1995; Matzke and Matzke 1995; Meyer and Saedler 1996). These silencing phenomena have been termed “homology-dependent gene silencing” events (Matzke and Matzke 1995) since all are correlated Figure ␴ 6.—C-methylation analysis of the -deletion mutant with the presence of multiple homologous copies of a r-r:N1-3-1. (A and B) As a control for the ability to detect C-methylation in R-mb heterozygotes, DNA from second-gener- gene or promoter. Because both the paramutable R-r ation R-rЈ/R-mb heterozygotes (lanes 1 and 2) and homozy- complex (Robbins et al. 1991; Walker et al. 1995) and gous nonparamutant R-r (lanes 3 and 4), and was digested the paramutagenic R-mb (E. L. Walker, unpublished with HindIII and either BstUI (A) or PvuII (B). Blots were results) and R-st (Eggleston et al. 1995) complexes ␴ Walker prepared and hybridized to the 1011 probe ( et al. contain multiple r1 genes, paramutation at r1 has been 1995), which does not hybridize to genomic DNA from R-mb homozygotes (not shown). (C–F) DNA was prepared from regarded as being consistent with the idea that multiple second-generation r-r:N1-3-1Ј/R-mb heterozygotes (lanes 1–4) gene copies can lead to epigenetic silencing (Assad et and homozygous r-r:N1-3-1 (lane 5), and was digested with al. 1993; Matzke et al. 1996). HindIII and one of the methylation-sensitive restriction en- Recently, an alternative explanation has been sug- zymes, AvaI (C), BstUI (D), HpaII (E), or PvuII (F). Blots were gested: that transposable elements, which make up a prepared and hybridized to the N131 probe which specifically detects the filler DNA of the r-r:N1-3-1 derivative. large part of the moderately repetitive DNA in plant genomes and which are often located in the upstream portions of normal genes, could be responsible for mak- DISCUSSION ing genes nearby susceptible to epigenetic silencing Paramutation of the R-r complex is correlated with events (Martienssen 1996a; Matzke et al. 1996). In increases in its level of C-methylation. The ␴ region of the case of silenced transgenes, the expectation is that R-r was observed to have increased C-methylation while they would have inserted near or within transposable in its first generation of heterozygosity with the paramu- elements, thus coming under their epigenetic influ- tagenic R-mb allele. Thus, methylation of R-r can be ence. Stably active transgenes would have inserted at detected even before the paramutant phenotype of de- positions where there are no transposable elements creased pigmentation in aleurone, which is not observed nearby. This idea has also been applied to naturally without sexual transmission of R-rЈ into a subsequent occurring systems like r1 paramutation. In this case, generation. The methylation changes associated with residual transposon sequences at ␴ might be able to paramutation of R-r are very consistent: in every second- influence the epigenetic state of the R-r complex. and third-generation paramutant plant tested in over Mutant derivatives of R-r exist which have deletions 10 different families, paramutation was always corre- of most or all of ␴ and, usually, portions of the S1 and/ lated with detectable increases in C-methylation levels. or S2 genes. These derivatives can be used to address These changes, however, are not found throughout the the relative contribution of the gene repeats of R-r vs. R-r complex. They occur only in specific portions of the ␴ region of R-r. The r-r:N1-3-1 allele is particularly infor- S1 and S2 genes. C-methylation was found in other parts mative in addressing the role of ␴, since it has lost all of the complex, most notably in the promoter of the P but 26 bp of the left inverted repeat of ␴ (Walker et al. gene, and the q gene fragment, but methylation at P 1995). Using a secondary paramutation test, Kermicle and q was not observed to change following paramuta- (1996) has reported that an r-r:N1-3-1 allele that had tion. Thus the correlation of paramutation and in- been heterozygous for three generations with the strong creased C-methylation exists even within the R-r com- paramutagenic allele, R-st, became very weakly paramu- 1980 E. L. Walker tagenic, thus indicating that paramutation of r-r:N1-3-1 posable elements in maize is well documented (see had occurred, but at greatly reduced efficiency com- Fedoroff 1996 and Martienssen 1996b for reviews). pared to R-r. In the study presented here, r-r:N1-3-1 was Furthermore, the doppia transposon whose remnants used to examine whether C-methylation changes would are found at ␴, is a member of the same superfamily of occur when the ␴ region was absent. No C-methylation transposable elements as Spm, an element with ex- of the S genes in r-r:N1-3-1 was detected in second- tremely well documented epigenetic behavior (Fedor- generation r-r:N1-3-1Ј/R-mb heterozygotes. Second-gen- off 1996). One of the hallmarks of epigenetic silencing eration R-rЈ/R-mb heterozygotes used as a positive con- of Spm is C-methylation of certain regions (Fedoroff trol showed obvious methylation of the same sites that 1996). It is possible that ␴’s role in the C-methylation were unmethylated in r-r:N1-3-1. In comparison to Ker- of the S genes during paramutation reflects a function micle’s secondary paramutation test of r-r:N1-3-1, two of the doppia transposon sequences in ␴. Changes in Spm important differences must be noted. Kermicle’s test methylation can be elicited by an Spm-encoded protein, was carried out using a paramutagenic allele, R-st, that TNPA (Schlappi et al. 1994), that binds to the subtermi- is stronger than the R-mb allele used in the present nal repeat elements of Spm (Gierl et al. 1988; Trent- study. Furthermore, in Kermicle’s experiment, three mann et al. 1993). The Spm subterminal repeats are generations of heterozygosity were used before testing similar in both arrangement and sequence to the subter- for secondary paramutation. It is quite possible that if minal repeat elements from doppia (Fedoroff 1996; C-methylation status of r-r:N1-3-1 were tested under Walker et al. 1995). It is tantalizing to speculate that these conditions, some C-methylation might be found. the doppia subterminal repeats found at ␴ could have a Clearly, though, the level of C-methylation observed in role in making R-r susceptible to paramutation. paramutagenized r-r:N1-3-1 is far smaller (in the present I thank Steve Dellaporta, Alison Delong, Tom Brutnell, study undetectable) than what is routinely observed for Randy Phillis, and Anne Simon for their comments on this manu- R-r. Thus, just as deletion of most of ␴ severely compro- script. This work was supported by the National Science Foundation mises the ability of the complex to acquire secondary (NSF9406483). paramutagenicity, it also severely compromises the abil- ity of the complex to become methylated. Taken together, these results are inconsistent with LITERATURE CITED models in which the highly duplicated structure of the Assad, F., K. L. Tucker and E. R. 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