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Paramutation at the maize pl1 locus is associated with RdDM activity at distal tandem repeats [1]

['Natalie C. Deans', 'Department Of Molecular Genetics', 'The Ohio State University', 'Columbus', 'Ohio', 'United States Of America', 'Centers For Applied Plant Sciences', 'Rna Biology', 'Joy-El R. B. Talbot', 'Department Of Molecular']

Date: 2024-06 

Abstract Exceptions to Mendelian inheritance often highlight novel chromosomal behaviors. The maize Pl1-Rhoades allele conferring plant pigmentation can display inheritance patterns deviating from Mendelian expectations in a behavior known as paramutation. However, the chromosome features mediating such exceptions remain unknown. Here we show that small RNA production reflecting RNA polymerase IV function within a distal downstream set of five tandem repeats is coincident with meiotically-heritable repression of the Pl1-Rhoades transcription unit. A related pl1 haplotype with three, but not one with two, repeat units also displays the trans-homolog silencing typifying paramutations. 4C interactions, CHD3a-dependent small RNA profiles, nuclease sensitivity, and polyadenylated RNA levels highlight a repeat subregion having regulatory potential. Our comparative and mutant analyses show that transcriptional repression of Pl1-Rhoades correlates with 24-nucleotide RNA production and cytosine methylation at this subregion indicating the action of a specific DNA-dependent RNA polymerase complex. These findings support a working model in which pl1 paramutation depends on trans-chromosomal RNA-directed DNA methylation operating at a discrete cis-linked and copy-number-dependent transcriptional regulatory element.

Author summary Although switching genes “on” and “off” in a coordinated fashion is essential to organismal development and homeostasis, some genes ‐ or specific forms thereof ‐ display unusual switching behaviors where the “off” state is sexually transmitted to offspring. Further, only “off” states persist when both “on” and “off” states are combined in heterozygous condition. Examples of this unusual behavior, known as paramutation, exist for certain transgenes and/or endogenous genes in both plants and animals but the chromosome features triggering this switching and the responsible cellular machinery are poorly understood. Here, we used long-read DNA sequencing, comparative genomics, DNA -DNA interaction assays, mutant analyses, and cytosine methylation profiling to identify a switchable regulatory sequence associated with paramutation at the maize purple plant1 (pl1) locus. These key sequences are embedded within a 5-copy tandem repeat array of mostly transposable elements ~14 kilobases distal to the pl1 gene. The “off” state is associated with these sequences producing so-called “small interfering RNAs” (siRNAs) that may direct the recruitment of chromatin-modifying enzymes. Our findings support a molecular model for paramutation and highlight the regulatory importance of repeated sequences found in large eukaryotic genomes.

Citation: Deans NC, Talbot J-ERB, Li M, Sáez-González C, Hövel I, Heavens D, et al. (2024) Paramutation at the maize pl1 locus is associated with RdDM activity at distal tandem repeats. PLoS Genet 20(5): e1011296. https://doi.org/10.1371/journal.pgen.1011296 Editor: Ortrun Mittelsten Scheid, Gregor Mendel Institute of Molecular Plant Biology, AUSTRIA Received: January 9, 2024; Accepted: May 8, 2024; Published: May 30, 2024 Copyright: © 2024 Deans et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: Sequence data from this article can be found in the GenBank, SRA, and GEO data libraries under the following accessions, Pl1-Rhoades haplotype sequence (Genbank OQ405024), 4C libraries (SRA SRR23352217-SRR23352222), Pl-Rh, Pl´, and Pl-Rh / Pl´ cob and seedling sRNA libraries (SRA SRR15400896-SRR15400912), B73 sRNAs (GEO GSE52103), rpd1-1 and rmr1-1 sRNAs (SRA SRR15410418-SRR15410422), and dcl3-2 sRNAs (SRA SRR16938685-SRR16938686). All other relevant data are within the manuscript and its Supporting Information files. For Review: Pl1-Rhoades haplotype sequence (Genbank OQ405024) is provided as a supplemental file S6 rpd1 and rmr1 data: https://dataview.ncbi.nlm.nih.gov/object/PRJNA753531?reviewer=9ddju4gb1e0okftgm0br100tir Pl1-Rh cob and seedling datasets: https://dataview.ncbi.nlm.nih.gov/object/PRJNA753276?reviewer=bbdq0i137ul4fcfqgm92d67t99 4C datasets: https://dataview.ncbi.nlm.nih.gov/object/PRJNA932006?reviewer=p9727ld7ma44k6vs0bemt0m0t9 Funding: Research activities were funded by awards to J.B.H. from the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service (www.usda.gov; 99-35301-7753, 2001-35301-10641, and 2006-35304-17399), the National Science Foundation (www.nsf.gov; MCB-0419909, -0920623, -1715375) and The Ohio State Foundation (www.osu.edu). I.H. was supported by funding of the Research Priority Area Systems Biology of the University of Amsterdam (www.uva.nl). This study and N.C.D. research activity was supported in part by resources and technical expertise from the Georgia Advanced Computing Resource Center (www.gacrc.uga.edu), a partnership between the University of Georgia’s Office of the Vice President for research and Office of the Vice President for Information Technology. Additional support of research activities were provided by the Comprehensive Cancer Center and the National Institutes of Health (www.nih.gov) under grant number P30 CA016058 The views expressed are solely those of the authors and are not endorsed by the sponsors of this work. The sponsors and funders did not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: All rmr materials and their uses are covered by U.S. patent 8134047 assigned to The Regents of the University of California. The authors claim no other known competing interests.

Introduction Patterns of transcriptional regulation governing normal development are consistently replicated from one generation to the next. However, certain trans-homolog interactions known as paramutations facilitate heritable changes in regulation–canonically observed as an active allele being heritably suppressed upon exposure to a repressed allele in trans (reviewed in [1,2]). Susceptible alleles are “paramutable” while repressed derivatives have “paramutagenic” potential. Despite the potential evolutionary significance of such behaviors, the chromosome organization and nuclear feature(s) that define these dynamic properties remain largely unknown. Paramutation-like behaviors have been described for endogenous alleles in several species including maize (Zea mays) [3–7], tomatoes (Solanum lycopersicum) [8], Arabidopsis (Arabidopsis thaliana) [9], mice (Mus musculus) [10], and potentially humans [11]. Specific transgenes displaying paramutation-like behaviors are also documented in petunia [12], tobacco (Nicotiana tobaccum) [13], Arabidopsis [14,15], mice [16,17], flies (Drosophila melanogaster) [18,19], and worms (Caenorhabditis elegans) [20–24]. Many of these pan-eukarya examples implicate roles for small RNA (sRNA)-based chromatin modifications (reviewed in [25]) highlighting an ancient mechanism of genome control having potential for the transgenerational inheritance of novel regulatory programs [26]. In maize, the first paramutation examples were reported for specific red1/colored1 (r1) alleles [3] which encode basic helix-loop-helix (bHLH) transcription factors conferring seed and plant color [27,28] and an allele of the paralogous booster1/colored plant1 (b1) locus [4]. Other alleles exhibiting paramutation are found at the purple plant1 (pl1) [6] and pericarp color1 (p1) [5] loci that encode R2R3 MYB-type transcription factors required for plant and cob pigmentation, respectively [28]. An additional example occurring at the low phytic acid1 (lpa1) locus [7] shows that paramutations in maize are not confined to genes encoding transcriptional regulators of pigment production. Forward genetic screens for mediators of paramutation (mop) of the B1-Intense (B1-I) allele [29,30] and factors required to maintain repression (rmr) of the Pl1-Rhoades allele [31,32] identify both overlapping and locus-specific functions. Several mop and rmr loci encode orthologs of proteins involved in an Arabidopsis RNA-directed DNA methylation (RdDM) pathway. RdDM utilizes two plant-specific DNA-dependent RNA polymerases (RNAPs IV and V) and accessory proteins to guide de novo cytosine methylation. RNAP IV indirectly produces mostly 24 nucleotide (24nt) RNAs that, in complex with Argonaute proteins, help recruit a de novo cytosine methyltransferase to nascent long-non-coding RNAP V transcripts (reviewed in [33]). rmr6/mop3/rpd1 encodes the maize RNAP IV largest subunit, RPD1 [34,35], and rmr7/mop2 encodes one of at least two second largest subunits shared between RNAPs IV and V, RP(D/E)2a [30,36,37]. RPD1 and RP(D/E)2a are orthologous to the Arabidopsis NUCLEAR RNA POLYMERASE D1 (NRPD1) and NRP(D/E)2, respectively. Maize has three genes producing second largest subunits that, according to proteomic data, constitute distinct RNAP IV and RNAP V subtypes [37]. mop1 encodes an ortholog of RNA-DEPENDENT RNA POLYMERASE2 (RDR2) [38] that converts nascent RNAP IV transcripts to a double-stranded substrate for a dicer-like3 (DCL3) endonuclease that generates primarily 24nt RNAs. rmr5 encodes the sole maize DCL3 [39]. The other three RMR proteins identified to date, (RMR1, RMR2, and RMR12/CHD3a), also influence 24nt RNA biogenesis, but neither RMR1 nor RMR12/CHD3a are required to mediate b1 paramutation or to maintain repressed B1-I states ‐ denoted B´ [32,40]. RMR1 is a likely SNF2-type ATPase, potentially orthologous to Arabidopsis CLASSY3 and 4 [32], that co-immunoprecipitates with RPD1 in undifferentiated callus cells [37]. RMR2 is a pioneer protein whose potential Arabidopsis orthologs have no known function [41], and RMR12/CHD3a is a chromodomain helicase DNA-binding3 (CHD3) protein orthologous to the Arabidopsis nucleosome-remodeling protein, PICKLE [40]. Both PICKLE and RMR12/CHD3a influence genome-wide distributions of 24nt RNA production [40,42]. Additional proteins implicated in the repression of B´ include a putative maize ortholog of Arabidopsis ARGONAUTE9 (AGO9) (a member of the AGO4 clade), ZmAGO104, [43] and a novel protein isoform encoded by the unstable factor for orange1 (ufo1) locus [44,45]. A dominant Ufo1-1 allele enhances the expression of both repressed B´ and p1 alleles through an unknown mechanism [44]. One working model for paramutation in maize envisions 24nt RNAs produced from a paramutagenic allele serving as a diffusible substance directing cytosine methylation and/or repressive histone modifications to an active paramutable allele leading to heritable repression. Chromatin modifications, including 5-methylcytosine (5mC), or the 24nt RNAs themselves, may serve as a meiotically heritable mark of paramutagenic alleles [2,25,46]. This model is supported by 1) the associations of MOP and RMR proteins with 24nt RNA biogenesis, 2) transgenic shRNA-mediated induction of b1 paramutation [47], 3) increased 5mC levels at paramutagenic r1 [48], p1 [44,49,50], and b1 alleles [51], and 4) transcriptional repression of paramutagenic B1-I [52], P1-rr [53], and Pl1-Rhoades [54] alleles. However, paramutations at pl1 can still occur with reduced frequencies in rmr1-1/rmr1-2 and rmr2-1 mutants [32,41], paramutation at b1 still occurs in a parent-of-origin specific manner in the rmr7-1 mutant [36], sRNA profiles of active (B-I) and repressed (B´) B1-I states appear similar [47], no 5mC differences have so far been found between active (Pl-Rh) and repressed (Pl´) Pl1-Rhoades states [32,54], and–with the possible exception of RP(D/E)2a –no known MOP or RMR proteins represent RdDM-type components acting downstream of 24nt biogenesis. These findings contrast the expectations of a canonical RdDM-like model. In maize, paramutagenic haplotypes are associated with repetitive sequences. At r1, the repeats include entire r1-coding regions plus flanking intergenic sequences [55–57], while B1-I and P1-rr have intergenic repeats acting as transcriptional enhancers [58,59]. A hepta-repeat of an otherwise unique 853bp sequence found ~100kb 5´ of the B1-I coding sequence (CDS) acts as both an enhancer and an efficient inducer of B1-I paramutation [59,60]. These findings indicate that the b1 hepta-repeat can both promote expression of B-I and confer paramutagenic activity to B´. At both r1 and b1, repeat numbers directly correlate with paramutagenic strength [57,59–61] but the nature of this relationship remains speculative [62]. Although the sequences mediating paramutation at Pl1-Rhoades have remained unknown, a Pl1-Rhoades || pl1-B73 recombinant derivative, pl1-R30, defines a region > 6kb 3´ of the pl1 CDS required for conferring strong pigmentation and for paramutagenicity [63]. pl1-R30 contains the Pl1-Rhoades CDS and a promoter-proximal CACTA-type DNA transposable element (TE) fragment of the doppia subclass [56,63,64] but confers uniformly weak pigmentation similar to that of pl1-B73. 5mC patterns at this doppia fragment are dependent on both RPD1 and RMR1 [32] but appear similar in both Pl-Rh and Pl´ states [54]. This TE fragment likely directs seed-specific expression because 1) a similar doppia fragment appears to serve as a seed-specific promoter in the paramutable R-r:standard haplotype [56]; 2) the non-paramutable Pl1-Blotched (Pl1-Bh) allele that has near-identical sequence to Pl1-Rhoades over at least 3.5kb, including the doppia sequences[65,66], confers blotchy seed pigmentation [65]; and 3) Pl1-Rhoades becomes seed expressed after multi-generational absence of RPD1 [63]. Thus, RNAP IV is responsible for the tissue-restricted expression of Pl1-Rhoades, and other genes [67,68], potentially by repressing regulatory TEs [68,69]. Importantly, the non-paramutable nature of both pl1-R30 and Pl1-Bh indicate the doppia fragment by itself is insufficient for paramutagenic function. Here we document the Pl1-Rhoades haplotype structure and molecular changes occurring within a distal tandem repeat that are associated with paramutation. A 192kb assembly of long-read sequences of bacterial artificial chromosomes (BACs) containing the Pl1-Rhoades CDS revealed a 2092bp sequence present in five tandem direct copies beginning ~14kb 3´ of the pl1 polyadenylation site (PAS). Structure comparisons indicate that three repeat copies are present in another paramutable pl1 haplotype from the CML52 inbred. Circular chromosome conformation capture (4C) results show these repeats are in close proximity to a bait sequence located between the 5´ doppia fragment and Pl1-Rhoades transcription start site (TSS). One or more of these repeats differ in both nuclease sensitivity and polyadenylated RNA levels between active Pl-Rh and repressed Pl´ states. We also show that an otherwise unique portion of the repeat unit produces RNAP IV-dependent 24nt RNAs only in the Pl´ state, identifying a ~200bp segment as having differential RNAP IV recruitment between Pl-Rh and Pl´. We found that this precise positioning of sRNA biogenesis is defined by CHD3a. In contrast, no significant sRNA differences were observed at the doppia fragment or elsewhere in the 192kb haplotype. The differential sRNA abundances also coincide with differing 5mC patterns. Our findings are consistent with a model in which Pl1-Rhoades paramutation involves a maize-specific RNA-directed DNA methylation-like mechanism acting at a copy-number-dependent distal cis-regulatory element.

Discussion Our findings are consistent with the Pl1-Rhoades penta-repeat acting as both a distal transcriptional enhancer and as a feature required for paramutagenic function. The only abundance difference of uniquely mapping sRNAs distinguishing paramutable Pl-Rh and paramutagenic Pl´ across 192kb occurs at one or more USR copies within the penta-repeat. When Pl1-Rhoades is in the highly expressed Pl-Rh state, portions of the USR display differential nuclease sensitivity, increased polyadenylated RNA levels, few 5mCs, and physical association with sequences immediately upstream of the Pl1-Rhoades TSS. From these findings, we conclude that the USR has regulatory significance for the Pl1-Rhoades haplotype. Furthermore, these data indicate that the USR is RNAP II-transcribed in the Pl-Rh state but RNAP IV-transcribed in the Pl´ state. Thus, this small non-coding region within the penta-repeat context appears to serve as a switchable distal element specifying meiotically heritable regulatory states. Although challenges remain in defining features of active plant enhancers, chromatin accessibility, histone acetylation, absence of 5mC, and promoter proximity appear diagnostic [77]. Both the b1 hepta-repeat and proximal P1-rr repeated sequences can promote transcriptional activation in transgenesis-based reporter assays [58,60]. Compared to the repressed B´ state, the b1 hepta-repeat sequences in the active B-I state 1) are more DNase I sensitive, 2) are more represented in a FAIRE-based protein-depleted DNA fraction, 3) are associated with elevated H3 acetylation [51,78], 4) have reduced 5mC levels [51], and 5) physically associate with the b1 CDS at higher frequencies [79]. The hepta-repeat junction sequences are also, like metazoan enhancers (reviewed in [80–82]), bi-directionally RNAP II-transcribed, but this occurs at similar levels in both the B-I and B´ states [47]. Likewise, sRNA levels so far appear similar across the hepta-repeat [38,47] whereas the abundance of both penta-repeat polyadenylated transcripts and RNAP IV-dependent sRNAs distinguish Pl-Rh from Pl´. The P1-rr enhancer is also transcribed at similar rates between allele states, but like Pl1-Rhoades, sRNA levels appear higher in the paramutant state [50]. These distinct characteristics point to variations in the mechanism driving paramutation at different loci; this intriguing finding demands further investigation. The polyadenylated USR-containing RNAs associated with the active Pl-Rh state indicate this region is transcribed, but the origin(s), direction(s), and length(s) of these RNAs remains unknown. Because neither Arabidopsis RNAP IV nor RNAP V produce polyadenylated RNAs [83–87], we infer that these USR-containing transcripts are RNAP II-derived. Whether this transcription reflects a more open chromatin nucleosome-free configuration region, however, remains equivocal. Because the B73 AGPv4 annotated PAS of the adjacent gene immediately downstream of the penta-repeat is less than 100bp from the end of the last repeat, one idea is that pre-termination transcription continues into the repeat array and a downstream PAS is used. Alternatively, the penta-repeat RNAs could initiate within USR sequences or from its resident TE fragments. In either case, a nascent RNA containing USR sequences and/or the adjacent highly repetitive DNA-7_ZM TE could serve as an essential scaffold transcript for the initial recruitment of RNAP V via sRNA-directed argonaute complexes [88]. Hence, a paramutable Pl-Rh state may require such polyadenylated penta-repeat RNAs for paramutation initiation while a paramutagenic Pl´ state likely uses RNAP V-sourced transcripts to maintain sRNA biogenesis. The relationship of sRNAs with endogenous alleles undergoing paramutation has to this point remained unclear. Here we found that the appearance of USR-specific 24nt RNAs in both early seedlings and immature cobs precisely correlates with the reduced plant pigment phenotypes and paramutagenic action diagnostic of Pl´ states [6]. The similar USR sRNA profiles observed between Pl´ and Pl-Rh / Pl´ seedlings indicate that RNAP IV is likely already recruited to the naive Pl-Rh allele state early in development. Our mutant sRNA analyses show that loss of sRNA biogenesis from a discrete USR segment is coincident with increased Pl1-Rhoades transcription (rmr2-1, rpd1-1, and dcl3-3), increased Pl1-Rhoades mRNA-stability (rmr1-1) and, meiotically-heritable reversion of Pl´ to Pl-Rh states (rmr1-1, rmr2-1, rpd1-1 and dcl3-3) [31,32,39,41,74]. Because 24nt USR RNA production depends on RNAP IV and other RMR proteins in an RdDM-like mechanism, it is reasonable to posit that RNAP IV-dependent chromatin changes at the USR impede Pl1-Rhoades transcription. Given that rmr1-1 mutant cobs lose most USR-specific 24mers, this indicates that the RNAP IV subtype occupying the USR in immature cobs is associated with RMR1 and not its CHR167 paralog [37]. The redistribution of sRNAs seen in chd3a mutants argues that CHD3a specifies where the seedling-based RNAP IV complexes function. Since CHD3 proteins are associated with nucleosome remodeling [89], and the Arabidopsis CHD3 PICKLE can move nucleosomes in vitro [90], it is likely that the chd3a-3 mutant penta-repeat sRNA profile reflects changes in nucleosome positioning that impact RNAP IV localization. The appearance of 5mC, coincident with USR sRNA biogenesis, agrees with a model in which CHD3a-positioned nucleosomes direct RNAP IV-dependent 24nt RNA production that, analogous to Arabidopsis RdDM, specifies de novo 5mC patterns. We found a correlation between 5mC appearance at the USR sequences and the Pl´ state. 5mC presence appears generally associated with alleles displaying paramutagenic behaviors, including 1) at the 5´ ends of divergently oriented r1 genes in R-r:standard repressed states [48], 2) within repeats flanking the p1 CDS in paramutagenic p1 haplotypes [5,44,49,58], 3) at the b1 hepta-repeat in the B´ state–with the exception of specific cytosines being more methylated in B-I [51,59,60,78], 4) the repressed phenotype conferred by a paramutagenic lpa1-241 allele is partially compensated by 5-azacytidine treatment [7] implicating 5mC in repression, and 5) in Arabidopsis autotetraploids, a spontaneously silenced epiallele of a partially-repeated hygromycin resistance transgene displays paramutagenic properties coincident with increased 5mC and 24mers primarily across the repeat region [14,91]. Moreover, 6) in tomato, the paramutagenic sulfurea allele also has increased 5mC in the promoter region and part of the 5´ CDS [92]. Changes in 5mC and sRNA production are also common when divergent genomes are combined [9,93,94], but whether these are causative of, or responsive to, changes in gene regulation remains unknown. Although 5mC presence is a unifying hallmark of paramutagenic allele states, 5mC alone is insufficient to predict paramutagenicity. Arabidopsis RdDM facilitates de novo cytosine methylation in CG, CHG, and CHH contexts (reviewed in [33,95]), and once established, these modifications can be maintained in the absence of RdDM by a maintenance methyltransferase (MET1; CG) and a pair of chromomethyltransferases (CMT3; CHG, and CMT2; CHH) [96] that are recruited to methylated H3K9 [97]. Because maize lacks a CMT2 ortholog [96], mCHH presence is assumed to reflect ongoing RdDM [98]. Interestingly, mCHH is specifically found in an enhancer region of the paramutagenic P1-rr´ allele, but not in the stably silenced, non-paramutagenic, P1-prTP allele [44]. Indeed, maize has little genome-wide mCHH (~5%); its presence is more correlated with sRNA production than with mCG or mCHG [94], and loss of MOP1/ZmRDR2, RP(D/E)2a, or RPD1 results in genome-wide reductions [98]. Although most maize mCHH depends on active RdDM, the CMT3 orthologs encoded by zmet2 and zmet5 are required to maintain some mCHH [98]. Since most of our sequenced USR amplicons from Pl´ and Pl-Rh / Pl´ seedlings are devoid of mCHH, we conclude that active RdDM is not occurring in most of the nuclei we sampled. Therefore, the high mCG (88%) and mCHG (55–70%) levels we observed mostly reflect maintenance methylation. While initial recruitment and trans-homolog communication of paramutation may depend on an RdDM-like mechanism primarily occurring in cells not sampled by our analysis, 5mC maintenance pathways are likely required for somatic and intergenerational stability of repressed allele states. While 5mC could be a meiotically heritable feature specifying paramutagenicity, it remains curious that, so far, no downstream (effector) proteins in an RdDM-like pathway have been identified in the mop- and rmr-based genetic screens. In Arabidopsis, sRNAs may direct the action of the SUVH2 and SUVH9 lysine methyltransferases to specify H3K9me2 [99], which can be maintained in a feedforward loop with mCHG through the actions of CMT3 and SUVH4/5/6 [100]. Arabidopsis SAWADEE HOMEODOMAIN HOMOLOGUE1 (SHH1) recruits RNAP IV to H3K9me2 [101]. A related maize ortholog, ZmSHH2a, also binds methylated H3K9 with the highest affinity for H3K9me1 [102]. ZmSHH2a is complexed with RP(D/E)2a and MOP1/ZmRDR2 [37], therefore Wang et al. [102] propose that ZmSHH2a forms a connection between RNAP IV recruitment and H3K9me1. Thus, similar to eukaryotes such as fission yeast that lack any cytosine methylation machinery, H3K9me could represent a heritable mark as well [103]. Indeed, loss of either tomato CMT3 or SUVH4 orthologs lead to reactivation of a paramutagenic sulfurea allele [104]. Additionally, because CHD3 proteins affect the regulation of loci marked by H3K27me3 [105–107], and CHD3a influences penta-repeat 24nt RNA production, it is possible that H3K27 modifications are also involved in maintaining a meiotically heritable epigenetic mark distinguishing repressed Pl1-Rhoades states. Loss of MOP1/ZmRDR2 or RPD1 results in a reduction of both H3K9 and H3K27 dimethylation at the b1 hepta-repeat, suggesting a connection between these marks and b1 paramutation [78]. Indeed, recent work in Drosophila implicates H3K27me3 and inter-chromosomal contacts in facilitating paramutation at the Fab2L transgene [19]. Gametic sRNAs may also contribute to the inheritance of repressed Pl´. In other metazoan examples of paramutation, it is the cytoplasmic transmission of Argonaute-bound sRNAs that confers heritable paramutagenic action (reviewed in [25]). Indeed, Argonaute-bound sRNAs are sufficient to initiate RdDM in Arabidopsis [88] and shRNA constructs can induce the change of B-I to B´ in transgenic maize [47]. In maize, however, paramutagenicity exclusively co-segregates with specific alleles [4,6,58,108]. Additionally, when aneuploid sperm cells are generated from haploid Pl´ gametophytes–as can be accomplished using B-A chromosomes [109]–only sperm cells contributing Pl´ chromosomes transmit paramutagenic function [110]. This result indicates that any sperm-transmitted sRNAs are by themselves insufficient to induce paramutation. We found that, similar to specific r1 and b1 haplotypes, repeat copy numbers correlate with paramutagenic activities [57,59,61]. Importantly, the presence of one or two USRs in non-paramutable pl1 haplotypes indicates that more repeats are required to obtain paramutagenic properties. In r1 paramutation, repeated r1 coding regions and flanking sequences need not be adjacent, or in cis, to promote paramutagenic function [61]. Because the NC350 and CML52 haplotypes share identical 3´ repeats, we expect that either the solo repeat found upstream of Pl1a-CML52 and/or the additional Pl1b-CML52 gene confers its paramutagenic properties. In addition, the pl1-bol3 haplotype containing three pl1 gene copies–none having the doppia fragment [111]–is reportedly paramutagenic [112]. The pl1-bol3 downstream region remains uncharacterized, however, so whether any paramutagenic activity is dependent on pl1 gene copy number or other cis-regulatory features is unclear. Our findings are consistent with the emerging consensus that repeated sequences are essential for paramutagenic function. The distinct sequences and structural arrangements of the B1-I and Pl1-Rh distal repeats may be related to the observed differences in allele stabilities and locus-specific RMR requirements. Neither RMR1 nor RMR12 repress B´ [32,40]. Each individual unit of the b1 hepta-repeat (853bp) consists entirely of unique sequence [59], while the 2092bp penta-repeat unit is mainly consisting of TE fragments with an embedded 390bp unique sequence. Unlike B´, where heritable reversions to a fully expressed B-I state have not been observed [52,113,114], the Pl´ state is less stable and can revert to Pl-Rh when either hemizygous [115], heterozygous with certain recessive alleles [66,115] or in the persistent absence of RMR1 [31,41], RPD1 [74], CHD3a [40], or DCL3 [39]. Whether the size, copy number, location, and/or composition of these b1 and pl1 repeats contribute to the observed differences in allele behaviors and mechanistic requirements remains to be explored. Depending on the phenotypic trait affected, metastable alleles having paramutation-like properties may be desirable or detrimental to breeding efforts. With long-read-based whole genome assemblies now resolving repeated sequences, it should be possible to predict which alleles of genes having agronomic importance might show similar dynamic behaviors. Such efforts will require ATAC-seq profiles and Hi-C interaction maps to identify repeat-associated enhancers, and sRNA distributions to highlight regions of ongoing RdDM. Additionally, RNA-seq profiles of RNAP IV-deficient plants would help validate the regulatory significance of such repeats. Crop improvement programs can now identify and manage these examples of heritable epigenetic variation.

Methods Genetic materials Inbred lines A619, A632, and B73 were sourced from the North Central Regional Plant Introduction Station (USDA-ARS, Ames, IA). The Pl1-Rhoades haplotype introgressed into A619 ([74]; herein designated A619 Pl1-Rhoades; 99.25% A619 for Pl-Rh and 98.5% A619 for Pl´) came from a previously described color-converted W23 stock (herein designated W23 Pl1-Rhoades) [6] developed by Ed Coe, Jr. (USDA-ARS, University of Missouri, Columbia, MO) and maintained in several lines obtained from the Maize Genetics Cooperation Stock Center (MGCSC)(USDA-ARS, University of Illinois, Urbana, IL) and Vicki Chandler (University of Oregon, Eugene, OR). A T6-9 (043–1) [116] interchange chromosome (T Pl1-Rhoades) having linkage between Pl1-Rhoades (6L) and a recessive 9S waxy1 allele (wx1) was introgressed into B73 (herein designated B73 T Pl1-Rhoades; 98.44%) as previously described [66,74] and into A619 (herein designated A619 T Pl1-Rhoades; ~97% A619) and A632 (herein designated A632 T Pl1-Rhoades; 99.25% A632). The BAC library and 4C analysis were derived from Pl-Rh type A619 Pl1-Rhoades plants. qRT-PCR was performed on W23 Pl1-Rhoades, A619 Pl1-Rhoades, and B73 T Pl1-Rhoades individuals. MNase assays were done with A619 Pl1-Rhoades seedlings. 5mC analyses and sRNA libraries were made from isogenic homozygous Pl-Rh, Pl´ and heterozygous (Pl-Rh / Pl´) cobs and seedlings of B73 T Pl1-Rhoades parentage (progenies 161453, 161483, 161482, 170671, 170673, 170674). Additional sRNA libraries were derived from immature cobs of Rpd1/rpd1-1 and rpd1-1/rpd1-1 siblings from progeny 80031 (~25% W23, 25% A632) and Rmr1/rmr1-1 and rmr1-1/rmr1-1 siblings from progeny 80886 in A632 (BC 4 F 3 ). For mutant analyses, all non-mutant siblings were homozygous for Pl1-Rhoades in a Pl´ state while mutants had darky colored anthers consistent with derepression of Pl1-Rhoades. BAC identification and sequencing Snap-frozen developing cobs (progenies 91353 and 91363) were submitted to Amplicon Express (Pullman, WA) to construct a ~3.7X coverage pCC1 BAC-based library and representative Hybond-N+ (Amersham Biosciences) filter arrays. 73,728 clones were screened by formamide-based hybridization to a random-primed 32P radiolabeled 0.9kb HindIII fragment from the pJH1 plasmid [54]. BAC DNA from ten clones was isolated using a Qiagen miniprep kit and evaluated for pl1 sequences using pl1-specific PCR amplification (see S8 Table for primers). Six PCR-positive clones (60_P21, 25_M18, 51_M22, 53_O24, 161_K19, 192_C12) were cultured with Epicentre Biotechnologies (Madison, WI) CopyControl BAC Induction Solution and BAC DNA was isolated with modification to an established protocol [117] including 1μl of 100mg/ml RNaseA in Solution I and adding a final phenol:chloroform extraction followed by chloroform extraction and isopropanol precipitation. BAC ends were Sanger sequenced by the Vincent J. Coates Genome Sequencing Laboratory (UC Berkeley) as per Poulsen and Johnson [118] (see S8 Table for primers) using 2μg of BAC template and 3 pmol of primer, in 20ul Sanger sequencing reactions with the following thermocycle steps: 3 min at 96°C followed by 99 cycles of 10 sec at 96°C, 10 sec at 50°C, 4 min at 60°C. BLASTn aligned the respective end sequences of all six BACs to 6L positions flanking the pl1 gene model. All six BAC clones were later prepared and sequenced on the Pacific Biosciences (PacBio) platform at The Genome Analysis Centre, TGAC, Norwich, UK (Now Earlham Institute). BAC assembly and annotation Raw PacBio reads (see S1 Table for statistics) were filtered and assembled into partial or complete linear contigs at The Genomic Analysis Centre (see S3 File). BAC backbone sequences were identified by BLASTn and removed. The resulting genomic sequences were aligned to each other and the existing Pl1-Rhoades reference using the Multiple Align feature of Geneious (v6.1.8; [119]). Together, these BAC sequences extended the Pl1-Rhoades reference sequence to 192kb, with six independent BACs supporting the sequence over the pl1 coding region and four supporting the sequence over the penta-repeat. One of the BAC sequences (60_P21) was unable to be assembled into a single contig, so only those fragments which overlap the consensus sequence of the other BAC sequences were included. A fragment that only overlapped the consensus by 1kb consisting entirely of Gypsy transposon sequence was also excluded. Gene models were predicted by BLASTing the assembled BAC sequence to cDNA molecules from the B73 AGPv4 genome [70]. Transposons and TE fragments were predicted using CENSOR on April 26, 2022 ([71]; see S4 File). Haplotype comparisons ~70kb extracted from the Pl1-Rhoades BAC contig containing the Pl1-Rhoades allele and surrounding region was aligned to the corresponding region in the B73 AGPv4 genome [70] and the draft CML52 and NC350 genomes [73] with GEvo [120] using the default settings with a minimum high-scoring segment pair size of 400nt. The resulting alignments were manually redrawn using Adobe Illustrator at scale. 4C analysis Tissue was collected from biological triplicate inner husk leaves at silking and from inner stem leaves from A619 Pl1-Rhoades (Pl-Rh) V5 seedlings. Interacting chromatin was isolated from these tissues as described for 3C experiments [79,121,122]. In short, 2-3g tissue was crosslinked in 1X PBS with 2% formaldehyde for 1 hour. Nuclei were resuspended in 1.2X Buffer B (Roche) and treated with 0.2% (w/v) SDS followed by 2% (w/v) Triton X-100 and digested with 400 U DpnII (NEB, 50U/μl) overnight. DpnII was heat-inactivated, and for intra-molecular ligation of crosslinked digested DNA, the sample was incubated in a volume of 7ml with 100U T4 ligase (Promega, 20U/μl) at 16°C for 5 hours, followed by 45 min at RT. Chromatin was de-crosslinked and ligated DNA (3C sample) was recovered by precipitation and the pellet dissolved in 150μl of 10mM Tris-HCl pH 7.5. To generate 4C samples, the 3C samples were digested and ligated essentially as described [123]. The DNA was digested with 50U NlaIII (NEB, 5U/μl), NlaIII was heat-inactivated and the digested DNA was re-ligated overnight in a total volume of 14ml as described above. The ligated DNA was precipitated, dissolved and column purified (QIAquick PCR purification kit- Qiagen). To produce 4C libraries, for each sample, 8 PCR reactions were performed. 100ng of 4C DNA served as template in each inverse PCR reaction. PCR was conducted with the Expand Long Template PCR System (Roche, #11681834001) using primers fused to P5 (forward) and P7 (reverse) illumina sequencing adapters; a forward primer immediately upstream of the Pl1-Rhoades CDS, and a reverse primer at the 5´ end of the doppia transposon fragment upstream of Pl1-Rhoades facing away from the gene (see S8 Table). The program used was 2min 94°C; [30sec 94°C, 30sec 52°C, 2min 72°C] 35x; 10min 72°C, 12°C ∞. Proper 4C amplicons contained: P5 ‐ forward primer (bait sequence) ‐ DpnII ‐ interacting sequence ‐ NlaIII ‐ reverse primer ‐ P7. The 8 PCR reactions per sample were pooled and purified with the Qiaquick PCR purification kit (Qiagen). Library concentrations were measured using the Nanodrop spectrophotometer, mixed in equal concentrations and sequenced on an Illumina 2000 platform by BGI Tech Solutions (Hongkong) Co., producing single-end 49 bp reads. Replicates were processed in different lanes. Reads were first filtered to those containing the DpnII cutsite and the 3 preceding nt of the forward primer to ensure the DpnII site was in the correct context using the BBTools (https://jgi.doe.gov/dataand-tools/bbtools/) function bbduk (options: k = 7 mink = 7 hdist = 1 adapter: CTCGATC). The resulting reads were then trimmed up to the DpnII cutsite using bbduk (options: ktrim = l k = 16 mink = 12 hdist = 2 maxlen = 28, adapter: TACGCCGGCGGAGCTC). Since each read is 49bp, setting the maximum length to 28 ensured that any read where the forward primer could not be recognized due to sequencing errors would not be retained. The NlaIII site and reverse primer, if present, were then trimmed using bbduk (options: ktrim = r k = 12 mink = 7 hdist = 1 minlen = 8, adapter: CATGAGCTATGA). Setting the minimum length to 8 ensured the reads contained an interacting sequence, and eliminated potential primer dimers; reads with longer interacting sequences were not expected to contain the NlaIII site, so presence of this site was not required. Lastly the reads were quality filtered and trimmed using bbduk (options: qtrim = r maq = 20 minlen = 8). After these filtering steps, the third replicate from each tissue had significantly fewer reads than the other two libraries (see S3 Table) and these were excluded from further analyses. The remaining four libraries were aligned to the B73 AGPv4 genome [70] using Bowtie (v0.12.8; options: -v 0—best -m 1 -S) [124]. Those reads which either mapped uniquely or did not map were aligned to a collapsed version of the Pl1-Rhoades BAC-based sequence containing only one copy of the penta-repeat, allowing no mismatches or multiply -mapping reads, using Bowtie (v0.12.8; options: -v 0—best -m 1 -S) [124]. All clean reads (not filtered to the genome) were also aligned to the assembled BAC sequences as above. All alignments were collapsed to their 5´ most coordinate and converted to BED format using awk. The abundance of tags at each position of the BAC sequence was determined using BEDTools genomecov [125]. Only positions with counts were retained, and 2kb on either side of the bait region were excluded to avoid artifacts. The resulting counts were normalized to rpm and ranked by total abundance (see S4 and S5 Tables). Chromatin profiling Mono and dinucleosome-bound DNA was obtained by isolating nuclei from six pooled day-8 A619 Pl1-Rhoades (Pl-Rh) and (Pl´) [74] seedlings in triplicate and fractionating MNase-treated chromatin as described [126]. A range of MNase concentrations was tested to identify a mild treatment which produced a full ladder representing different numbers of nucleosomes bound to DNA following gel electrophoresis. An amount of nuclei yielding ~20μg DNA were incubated for 4 minutes at 37°C with 20U MNase (Thermo Scientific EN0181) in MNase reaction buffer (50mM Tris pH 7.5, 320mM sucrose, 4mM MgCl 2 , 1mM CaCl 2 ) before proteinase K treatment and DNA extraction as described [126]. The bands corresponding to mono and dinucleosomes were extracted from the gel, pooled, and three technical replicate qPCR reactions were performed on three biological replicates. Abundance of each USR amplicon was normalized to abundance of alanine aminotransferase (alt4; Zm00001d014258) (see S8 Table for primers). DNA was amplified with SensiMix SYBR No ROX (Bioline), and data was collected using an Eppendorf Mastercycler EP Gradient S thermocycler. Cycle threshold (Ct) values were calculated using the noiseband option in Eppendorf Mastercycler EP Realplex V2.2 software. qRT-PCR To quantify pl1 mRNA, total RNA was isolated from biological duplicates of Pl-Rh and Pl´ inner seedling and husk leaves from both A619 Pl1-Rhoades and B73 T Pl1-Rhoades lines. cDNA was generated, and qRT-PCR was performed using an Applied Biosystems 7500 Real-Time PCR System on technical duplicates. pl1 mRNAs were normalized to those from actin1 (Zm00001d010159) (see S8 Table). To quantify USR transcripts, total RNA was isolated from male flowers (florets) of triplicate Pl-Rh and Pl´ type W23 Pl1-Rhoades individuals [6] just prior to anthesis. Three florets from a single individual were pooled per sample. RNA was isolated, cDNA was generated, qRT-PCR was performed, and fold expression was calculated as described [40] except the cycle threshold (Ct) values were established using the CalQplex option in Eppendorf Mastercycler EP Realplex V2.2 software. Candidate enhancer transcripts were amplified with P3_F and P3_R primers (see S8 Table) and normalized to gapdh which was amplified using previously published primers ([127]; see S8 Table). Raw Ct values are provided in S5 File. sRNA sequencing Biological triplicate day-8 seedling and 4cm immature cob B73 T Pl1-Rhoades (Pl-Rh), (Pl´) and (Pl-Rh / Pl´) sRNA libraries (SRA accessions SRR15400896-SRR15400912) were made from TRizol-extracted total RNA using the Bioo Scientific (Austin, TX) Nextflex Small RNA-Seq Kit v3 as described [40], and 150bp paired-end sequencing was performed by Novogene Co. Ltd. on an Illumina HiSeq4000 platform. Only two seedling Pl-Rh / Pl´ libraries were successfully sequenced. Between 37M and 198M reads were produced per library (see S6 Table for library statistics). Two B73, two Rpd1 / rpd1-1, one rpd1-1 / rpd1-1, one Rmr1 / rmr1-1, and one rmr1-1 / rmr1-1 4cm immature cob libraries (GEO GSE52103 and SRA SRR15410418-SRR15410422) were prepared as described [41]. Briefly, TRizol-extracted total RNA were separated by PAGE, sRNAs were gel-extracted, and libraries were prepared according to the Illumina sRNA Sequencing Guide. Adapters were trimmed from rmr mutant and heterozygous cob libraries, deduplicated, and quantified using a Perl script. B73 cob libraries were filtered and trimmed using the BBTools (https://jgi.doe.gov/dataand-tools/bbtools/) function bbduk (options: ktrim = r k = 18 mink = 11 hdist = 1 maq = 10 minlen = 18 maxlength = 30); B73 immature cob libraries were not deduplicated. Pl-Rh, Pl´, Pl-Rh / Pl´ and previously published Chd3a / Chd3a and chd3a-3 / chd3a-3 ([40]; GEO GSE158990) raw reads were filtered using bbduk with options (ktrim = r k = 18 mink = 11 hdist = 1 minlen = 26 maxlength = 38). Only mate 1 was used from paired-end libraries. The resulting trimmed reads were converted to .fasta format using seqtk (https://github.com/lh3/seqtk) and deduplicated using the FASTX-Toolkit command fastx_collapser (http://hannonlab.cshl.edu/fastx_toolkit/index.html). The Nextflex Small RNA-Seq Kit v3 adapters have 4N tags flanking the insert. These tags were removed using seqtk (options: trimfq -b 4 -e 4). bbduk was used to filter rpd1, rmr1, and rmr2 immature cob datasets by length (options: minlen = 18 maxlen = 30). rpd1 and rmr1 whole genome mapping statistics (see S7 Table) were obtained as previously described for rmr2 and dcl3 [39,41]. Percent sRNAs of each size class were then normalized by the ratio of the percent 21mers in the mutants to the percent 21mers the corresponding heterozygotes since 21mer levels appear unchanged in these mutants [34,69]. 18-30nt reads from all datasets were aligned to known maize rRNA and tRNA sequences (provided by Dr. Blake Meyers, Donald Danforth Plant Sciences Center) using Bowtie (v0.12.8; options: -v 0—best -m 1 -S) [124]. Those sequences not mapping to tRNA/rRNA were considered “clean” and aligned to the B73 AGPv4 genome [70] using Bowtie (v0.12.8; options: -v 0—best -m 1 -S) [124]. Those reads (excluding the B73 libraries) that either mapped uniquely or did not map were aligned using ShortStack [75] to a modified version of the Pl1-Rhoades haplotype sequence with the five repeats collapsed to a single copy to allow reads which could map to any repeat to map uniquely. ShortStack clusters were called using the six libraries from Pl-Rh and Pl´ cobs since these were the deepest datasets (ShortStack options:—mismatches 0—mmap n—nohp—mincov 152—pad 50), and sRNAs from all other libraries were quantified at these defined loci using the ShortStack option—locifile. Minimum coverage of 152 represents 0.5 rpm for those datasets, and the non-default pad of 50 (minimum distance between independent clusters) was chosen to prevent ShortStack from artificially collapsing independent clusters into a single locus because of the depth of these libraries. Reads mapping uniquely to each nucleotide of the composite unique subregion were collapsed to their 5´ most nucleotide and separated by strand and length using awk, quantified using the samtools function samtools-depth [128], and normalized to rpm. Normalized counts are displayed in 10nt bins (Fig 6H–6M). In parallel, all clean reads from Pl-Rh, Pl´, and Pl-Rh / Pl´ were mapped to the collapsed BAC sequence using Bowtie (v1.3.1; options: -v 0 –best). Representative alignments are shown for each tissue and genotype in S8 Fig. 5mC profiling Genomic DNA was isolated from B73 T Pl1-Rhoades (Pl-Rh), (Pl´) and (Pl-Rh / Pl´) aerial day-8 seedling tissue. ~160ng DNA was mixed with 30ng unmethylated lambda DNA and sheared using a Branson sonicator (two 15 sec pulses at 7watts with 45 sec rest), and unmethylated cytosines were converted to uracils using the NEBNext Enzymatic Methyl-seq Conversion Module (NEB #E7125) followed by PCR using standard Taq DNA polymerase (NEB)(see S8 Table) to amplify fragments within the USR and lambda control. Lambda_F and R were used on the Pl-Rh and Pl´ samples while Lambda_F2 and R2 were used on Pl-Rh / Pl´ sample. The PCR products were then purified using AxyPrep Mag PCR Clean-Up Kit (Axygen MAG-PCR-CL-5). All PCR amplicons ‐ with exception of the lambda amplicon from the Pl-Rh / Pl´ sample ‐ were cloned into pGEM-T easy vectors (Promega A1360) and transformed into competent NEB5-alpha E. coli (NEB #C2987H). Colonies were selected by blue white screening and purified plasmid DNA was Sanger sequenced at The Genomics Shared Resource in the OSU Comprehensive Cancer Center. A 123bp region with full coverage high quality sequence in all amplicons was analyzed by Kismeth [129] for cytosine methylation. For the Pl-Rh / Pl´ sample, Sanger sequencing was performed on the lambda amplicons directly. Complete cytosine conversions were seen for all lambda controls (see S9 Table).

Acknowledgments We thank the Maize Genetics Cooperation Stock Center (MGCSC)(USDA-ARS, University of Illinois, Urbana, IL) and North Central Regional Plant Introduction Station (USDA-ARS, Ames, IA) for contributing valuable genetic materials. We are grateful to Drs. Blake Meyers (Donald Danforth Plant Sciences Center) and Stacey Simon (University of Delaware) for constructing and sequencing the immature cob sRNA libraries from the various rmr mutants and non-mutant siblings. We thank Dr Mario Caccamo (NAIB East Malling Research) for his assistance in sequencing the Pl1-Rhoades BACs (sequencing was delivered via the BBSRC National Capability in Genomics (BB/J010375/1) at the Earlham Institute by members of the Genomics Pipelines Group), Drs. Pawel Krajewski and Dimitrios Zisis (Institute of Plant Genetics, Polish Academy of Sciences) for advice with the 4C data analysis, and Ludek Tikovsky and Harold Lemereis for taking care of the maize plants (Swammerdam Institute for Life sciences, University of Amsterdam). NCBI, Gramene, and MaizeGDB provided critical sequence and data resources. Maize nurseries were supported in part by the University of California College of Natural Resources Oxford Facilities Unit, the Ohio Agricultural Research and Development Centers Waterman Agricultural and Natural Resources Laboratory, and The Ohio State University College of Arts & Sciences Biological Sciences Greenhouse.

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