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Variation in chromosome number due to polyploidy can seriously compromise meiotic stability. In autopolyploids, the presence of more than two homologous chromosomes may result in complex pairing patterns and subsequent anomalous chromosome segregation. In this context, chromocenter, centromeric, telomeric and ribosomal DNA locus topology and DNA methylation patterns were investigated in the natural autotetraploid, Arabidopsis arenosa. The data show that homologous chromosome recognition and association initiates at telomeric domains in premeiotic interphase, followed by quadrivalent pairing of ribosomal 45S RNA gene loci (known as NORs) at leptotene. On the other hand, centromeric regions at early leptotene show pairwise associations rather than associations in fours. These pairwise associations are maintained throughout prophase I, and therefore likely to be related to the diploid-like behavior of A. arenosa chromosomes at metaphase I, where only bivalents are observed. In anthers, both cells at somatic interphase as well as at premeiotic interphase show 5-methylcytosine (5-mC) dispersed throughout the nucleus, contrasting with a preferential co-localization with chromocenters observed in vegetative nuclei. These results show for the first time that nuclear distribution patterns of 5-mC are simultaneously reshuffled in meiocytes and anther somatic cells. During prophase I, 5-mC is detected in extended chromatin fibers and chromocenters but interestingly is excluded from the NORs what correlates with the pairing pattern.

In genetic hybrids, the silencing of nucleolar rRNA genes inherited from one progenitor is the epigenetic phenomenon known as nucleolar dominance. An RNAi knockdown screen identified the Arabidopsis de novo cytosine methyltransferase, DRM2, and the methylcytosine binding domain proteins, MBD6 and MBD10, as activities required for nucleolar dominance. MBD10 localizes throughout the nucleus, but MBD6 preferentially associates with silenced rRNA genes and does so in a DRM2-dependent manner. DRM2 methylation is thought to be guided by siRNAs whose biogenesis requires RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and DICER-LIKE 3 (DCL3). Consistent with this hypothesis, knockdown of DCL3 or RDR2 disrupts nucleolar dominance. Collectively, these results indicate that in addition to directing the silencing of retrotransposons and noncoding repeats, siRNAs specify de novo cytosine methylation patterns that are recognized by MBD6 and MBD10 in the large-scale silencing of rRNA gene loci.

Nucleolar dominance describes the silencing of one parental set of ribosomal RNA (rRNA) genes in a genetic hybrid, an epigenetic phenomenon that occurs on a scale second only to X-chromosome inactivation in mammals. An RNA interference (RNAi) knockdown screen revealed that the predicted Arabidopsis histone deacetylase, HDA6, is required for rRNA gene silencing in nucleolar dominance. In vivo, derepression of silenced rRNA genes upon knockdown of HDA6 is accompanied by nucleolus organizer region (NOR) decondensation, loss of promoter cytosine methylation, and replacement of histone H3 Lys 9 (H3K9) dimethylation with H3K4 trimethylation, H3K9 acetylation, H3K14 acetylation, and histone H4 tetra-acetylation. Consistent with these in vivo results, purified HDA6 deacetylates lysines modified by histone acetyltransferases whose substrates include H3K14, H4K5, and H4K12. HDA6 localizes, in part, to the nucleolus, supporting a model whereby HDA6 erases histone acetylation as a key step in an epigenetic switch mechanism that silences rRNA genes through concerted histone and DNA modifications.

Allopolyploidy is a significant evolutionary process, resulting in new species with diploid or greater chromosome complements derived from two or more progenitor species. We examined the chromosomal consequences of genomic merger in Arabidopsis suecica, the allotetraploid hybrid of Arabidopsis thaliana and Arabidopsis arenosa. Fluorescence in situ hybridization with centromere, nucleolus organizer region (NOR), and 5S rRNA gene probes reveals the expected numbers of progenitor chromosomes in natural A. suecica, but one pair of A. thaliana NORs and one pair of A. arenosa-derived 5S gene loci are missing. Similarly, in newly formed synthetic A. suecica-like allotetraploids, pairs of A. thaliana NORs are gained de novo, lost, and/or transposed to A. arenosa chromosomes, with genotypic differences apparent between F(3) siblings of the same F(2) parent and between independent lines. Likewise, pairs of A. arenosa 5S genes are lost and novel linkages between 5S loci and NORs arise in synthetic allotetraploids. By contrast, the expected numbers of A. arenosa-derived NORs and A. thaliana-derived 5S loci are found in both natural and synthetic A. suecica. Collectively, these observations suggest that some, but not all, loci are unstable in newly formed A. suecica allotetraploids and can participate in a variety of alternative rearrangements, some of which resemble chromosomal changes found in nature.

In genetic hybrids, nucleolus formation on chromosomes inherited from only one parent is the epigenetic phenomenon, nucleolar dominance. By using Arabidopsis suecica, the allotetraploid hybrid of Arabidopsis thaliana and Arabidopsis arenosa, natural variation in nucleolar dominance was found to occur, providing a unique opportunity to examine homologous nucleolus organizer regions (NORs) in their active and inactive states. In A. suecica strain LC1, NORs derived from A. arenosa are active, whereas A. thaliana-derived NORs are silenced. In A. suecica strain 9502, NORs of both parental species are active. When active, NORs are partially, but not fully, decondensed. Both active and inactive LC1 NORs colocalize with the nucleolus, contradicting the long-standing assumption that rRNA gene transcription drives nucleolus association. Collectively, these observations clarify the relationships among NOR chromatin topology, rRNA gene transcription, and NOR-nucleolus associations. A. suecica strains LC1 and 9502 have each lost one pair of A. thaliana NORs during evolution, and amplified fragment-length polymorphism analysis further indicates that these strains are genetically very similar. These data suggest that nucleolar dominance can result from subtle genetic or epigenetic variation but is not a trait fundamental to a given interspecies hybrid combination.