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Paramutation is an allele-dependent transfer of epigenetic information, which results in the heritable silencing of one allele by another. Paramutation at the b1 locus in maize is mediated by unique tandem repeats that communicate in trans to establish and maintain meiotically heritable transcriptional silencing. The mop1 (mediator of paramutation1) gene is required for paramutation, and mop1 mutations reactivate silenced Mutator elements. Plants carrying mutations in the mop1 gene also stochastically exhibit pleiotropic developmental phenotypes. Here we report the map-based cloning of mop1, an RNA-dependent RNA polymerase gene (RDRP), most similar to the RDRP in plants that is associated with the production of short interfering RNA (siRNA) targeting chromatin. Nuclear run-on assays reveal that the tandem repeats required for b1 paramutation are transcribed from both strands, but siRNAs were not detected. We propose that the mop1 RDRP is required to maintain a threshold level of repeat RNA, which functions in trans to establish and maintain the heritable chromatin states associated with paramutation.

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The maize Myb transcription factor C1 depends on the basic helix-loop-helix (bHLH) proteins R or B for regulatory function, but the closely related Myb protein P does not. We have used the similarity between the Myb domains of C1 and P to identify residues that specify the interaction between the Myb domain of C1 and the N-terminal region of R. Substitution of four predicted solvent-exposed residues in the first helix of the second Myb repeat of P with corresponding residues from C1 is sufficient to confer on P the ability to physically interact with R. However, two additional Myb domain amino acid changes are needed to make the P regulatory activity partially dependent on R in maize cells. Interestingly, when P is altered so that it interacts with R, it can activate the Bz1 promoter, normally regulated by C1 ؉ R but not by P. Together, these findings demonstrate that the change of a few amino acids within highly similar Myb domains can mediate differential interactions with a transcriptional coregulator that plays a central role in the regulatory specificity of C1, and that Myb domains play important roles in combinatorial transcriptional regulation. Combinatorial interactions between transcription factors are of central importance to regulation of gene expression in eukaryotes. These interactions can either modulate transcription factor activity or contribute to the biological specificity of factors with very similar DNA-interaction motifs. Elucidation of the mechanisms by which proteins with very similar DNA-binding domains achieve regulatory specificity remains a fundamental question in biology today.Proteins containing the Myb-homologous DNA-binding domain are widespread in eukaryotes (reviewed in refs. 1 and 2). The vertebrate c-myb gene plays an essential regulatory role in the proliferation and differentiation of hematopoietic cells. Besides c-myb, at least two other myb-related genes (A-myb and B-myb) are present in vertebrates (3). The products of these genes have Myb domains, each consisting of three head-to-tail Myb motifs (R1, R2, and R3). Oncogenic versions of c-myb, such as v-myb, contain only R2 and R3, as do hundreds of plant Myb-domain proteins (4). Myb domains formed by the R2 and R3 Myb motifs bind DNA. Each Myb motif contains three ␣-helices, and the third helix of each Myb motif makes sequencespecific DNA contacts. The second and third helices of each Myb motif form a helix-turn-helix structure when bound to DNA, similar to motifs found in the repressor and in homeo domains (5). In addition to their well-established roles in DNA binding, Myb domains are also emerging as important protein-protein interaction motifs. These Myb domain-mediated proteinprotein interactions play key roles in the biological specificity of the corresponding factors (6-13). However, the mechanisms by which protein-protein interactions contribute to the regulatory specificity of Myb domain proteins are poorly understood.In f lowering plants, several hundred genes containing the conserved Myb DNA-binding domain have b...

Recombination mapping defined a 6-kb region, 100 kb upstream of the transcription start site, that is required for B-I enhancer activity and paramutation-a stable, heritable change in transcription caused by allele interactions in maize (Zea mays). In this region, B-I and B (the only b1 alleles that participate in paramutation) have seven tandem repeats of an 853-bp sequence otherwise unique in the genome; other alleles have one. Examination of recombinant alleles with different numbers of tandem repeats indicates that the repeats are required for both paramutation and enhancer function. The 6-kb region is identical in B-I and B, showing that epigenetic mechanisms mediate the stable silencing associated with paramutation. This is the first endogenous gene for which sequences required for paramutation have been defined and examined for methylation and chromatin structure. The tandem repeat sequences are more methylated in B-I (high expressing) relative to B (low expressing), opposite of the typical correlation. Furthermore, the change in repeat methylation follows establishment of the B epigenetic state. B-I has a more open chromatin structure in the repeats relative to B. The nuclease hypersensitivity differences developmentally precede transcription, suggesting that the repeat chromatin structure could be the heritable imprint distinguishing the two transcription states.

Small RNAs from plants are known to be highly complex and abundant, with this complexity proportional to genome size. Most endogenous siRNAs in Arabidopsis are dependent on RNA-DEPEN-DENT RNA POLYMERASE 2 (RDR2) for their biogenesis. Recent work has demonstrated that the maize MEDIATOR OF PARAMUTATION1 (mop1) gene is a predicted ortholog of RDR2. The mop1 gene is required for establishment of paramutation and maintenance of transcriptional silencing of transposons and transgenes, suggesting the potential involvement of small RNAs. We analyzed small RNAs in wild-type maize and in the isogenic mop1-1 loss-offunction mutant by using Illumina's sequencing-by-synthesis (SBS) technology, which allowed us to characterize the complement of maize small RNAs to considerable depth. Similar to rdr2 in Arabidopsis, in mop1-1, the 24-nucleotide (nt) endogenous heterochromatic short-interfering siRNAs were dramatically reduced, resulting in an enrichment of miRNAs and transacting siRNAs. In contrast to the Arabidopsis rdr2 mutant, the mop1-1 plants retained a highly abundant heterochromatic Ϸ22-nt class of small RNAs, suggesting a second mechanism for heterochromatic siRNA production. The enrichment of miRNAs and loss of 24-nt heterochromatic siRNAs in mop1-1 should be advantageous for miRNA discovery as the maize genome becomes more fully sequenced.miRNA ͉ mop1 ͉ rdr2 ͉ small RNA T he small RNAs found in a typical plant cell include a small number of highly abundant, mainly 21-nt microRNAs (miRNAs) and a large number of small interfering RNAs (mainly 24 nt heterochromatic siRNAs, or simply siRNAs) recognizing many diverse sequences. In addition, several additional subclasses of varying and in some cases overlapping functional importance have been described, including the transacting siRNAs (ta-siRNAs) (1-3), natural antisense siRNAs, a type thus far observed only under stress conditions (4, 5), and natural antisense miRNAs (6).MicroRNAs have a variety of regulatory roles in development and stress responses (for review, see ref. 7). In addition, work in Arabidopsis has led to the hypothesis that transcription of repeats is performed by the plant-specific RNA polymerase IV (pol IV) followed by reverse transcription and cleavage by RDR2 and DCL3, respectively. These repeated sequences include transposons and retrotransposons in plants (8), and this series of events produces a complex set of heterochromatic siRNAs (for review, see ref. 9). The genome size of plants varies substantially among species mainly caused by variation in content of repeated DNA. The complexity of siRNAs is correspondingly greater in rice than in Arabidopsis (10), consistent with larger numbers of repeated sequences in rice.The maize b1 locus is an excellent model for paramutation, a phenomenon in which alleles communicate in trans, resulting in meiotically heritable gene expression changes (11). Molecular work, combined with fine-structure recombination mapping, has demonstrated that this activity is mediated by tandem repeats at b1 that are required to e...

Histone proteins play a central role in chromatin packaging, and modification of histones is associated with chromatin accessibility. SET domain [Su(var)3-9, Enhancer-of-zeste, Trithorax] proteins are one class of proteins that have been implicated in regulating gene expression through histone methylation. The relationships of 22 SET domain proteins from maize (Zea mays) and 32 SET domain proteins from Arabidopsis were evaluated by phylogenetic analysis and domain organization. Our analysis reveals five classes of SET domain proteins in plants that can be further divided into 19 orthology groups. In some cases, such as the Enhancer of zeste-like and trithorax-like proteins, plants and animals contain homologous proteins with a similar organization of domains outside of the SET domain. However, a majority of plant SET domain proteins do not have an animal homolog with similar domain organization, suggesting that plants have unique mechanisms to establish and maintain chromatin states. Although the domains present in plant and animal SET domain proteins often differ, the domains found in the plant proteins have been generally implicated in protein-protein interactions, indicating that most SET domain proteins operate in complexes. Combined analysis of the maize and Arabidopsis SET domain proteins reveals that duplication of SET domain proteins in plants is extensive and has occurred via multiple mechanisms that preceded the divergence of monocots and dicots.

Paramutation is the epigenetic transfer of information from one allele of a gene to another to establish a state of gene expression that is heritable for generations. RNA has recently emerged as a prominent mediator of this remarkable phenomenon in both maize and mice.