Gene dosage and stochastic effects determine the severity and direction of uniparental ribosomal RNA gene silencing (nucleolar dominance) in Arabidopsis allopolyploids

Abstract

Nucleolar dominance is an epigenetic phenomenon in which one parental set of ribosomal RNA (rRNA) genes is silenced in an interspecific hybrid. In natural Arabidopsis suecica, an allotetraploid (amphidiploid) hybrid of Arabidopsis thaliana and Cardaminopsis arenosa, the A. thaliana rRNA genes are repressed. Interestingly, A. thaliana rRNA gene silencing is variable in synthetic Arabidopsis suecica F 1 hybrids. Two generations are needed for A. thaliana rRNA genes to be silenced in all lines, revealing a species-biased direction but stochastic onset to nucleolar dominance. Backcrossing synthetic A. suecica to tetraploid A. thaliana yielded progeny with active A. thaliana rRNA genes and, in some cases, silenced C. arenosa rRNA genes, showing that the direction of dominance can be switched. The hypothesis that naturally dominant rRNA genes have a superior binding affinity for a limiting transcription factor is inconsistent with dominance switching. Inactivation of a species-specific transcription factor is argued against by showing that A. thaliana and C. arenosa rRNA genes can be expressed transiently in the other species. Transfected A. thaliana genes are also active in A. suecica protoplasts in which chromosomal A. thaliana genes are repressed. Collectively, these data suggest that nucleolar dominance is a chromosomal phenomenon that results in coordinate or cooperative silencing of rRNA genes.Nucleolar dominance was among the first recognized epigenetic phenomena, discovered in interspecific hybrids in the plant genus Crepis. Navashin noted secondary constrictions at metaphase on D chromosomes inherited from one Crepis species, but not on the D chromosome from the other species (1, 2). Navashin's contemporary, McClintock, showed that these secondary constrictions are sites where nucleoli were organized in the preceding interphase (3). Decades later, nucleolus organizer regions (NORs) were identified as loci where genes encoding the precursor of the 18S, 5.8S, and 25-28S ribosomal RNAs are tandemly arrayed (4-7). Thus, nucleolar dominance was shown ultimately to result from uniparental rRNA gene expression (8).Nucleolar dominance occurs throughout the plant and animal kingdoms (9, 10) via mechanisms that remain unclear. Navashin noted that dominant NOR-bearing chromosomes could be contributed through the pollen or egg (2), ruling out maternal or paternal effects. A dominant D chromosome could also be contributed as part of an incomplete chromosome set. In Drosophila melanogaster ϫ Drosophila simulans hybrids, the D. melanogaster NORs on the X and Y chromosomes are dominant over the single NOR on the D. simulans X (11). In hybrid XO males lacking a D. melanogaster sex chromosome, but containing all autosomes, the D. simulans NOR is active. These results indicate that NOR-bearing chromosomes must be present for nucleolar dominance to occur.

rRNA gene silencing and nucleolar dominance: Insights into a chromosome-scale epigenetic on/off switch

“…Moreover, the demonstration that underdominant rRNA genes are abundantly expressed in Arabidopsis and Brassica upon blocking DNA methylation or histone deacetylation, and dominant genes are also up-regulated, suggests that transcription factors are present in sufficient amounts to support very high levels of transcription provided that the genes are in an accessible chromatin state [39,40,46]. The demonstration that the direction of nucleolar dominance can be reversed in response to changes in ploidy in Arabidopsis thaliana x A. arenosa hybrids bearing 1:3, 2:2 or 3:1 genome dosages of the two progenitors [43] also does not fit with the idea that one parental type of rRNA gene has an inherently higher binding affinty for a limiting transcription factor, in which case that gene type would always recruit transcription factors and be transcribed to some degree. There is no correlation between intergenic spacer length or repeat number in Brassica allotetraploids that display nucleolar dominance [76].…”

“…Foremost of these is the fact that a ten million fold excess of rRNA minigenes is typically injected relative to the number of endogenous rRNA genes, which may artificially create transcription factor deficiencies that endogenous rRNA genes never experience. Analogous tests in plants in which minigenes were transfected into protoplasts in numbers similar to the number of endogenous rRNA genes failed to reveal any differences in the competitive strength of dominant and underdominant genes, nor did competitive in vitro transcription assays [43,86]. Moreover, the demonstration that underdominant rRNA genes are abundantly expressed in Arabidopsis and Brassica upon blocking DNA methylation or histone deacetylation, and dominant genes are also up-regulated, suggests that transcription factors are present in sufficient amounts to support very high levels of transcription provided that the genes are in an accessible chromatin state [39,40,46].…”

“…In A. suecica, the A. thaliana-derived rRNA genes are selectively repressed (underdominant) whereas the A. arenosa-derived genes are active (dominant) [43]. Using ChIP, Lawrence et al [40] found that nucleosomes associated with the promoter regions of underdominant A. thaliana genes are enriched in histone H3 dimethylated on lysine 9 (H3K9me2), a canonical mark of transcriptionally repressed and highly condensed chromatin (heterochromatin) [44].…”

Mechanisms of genomic rearrangements and gene expression changes in plant polyploids

“…(77) However, in Arabidopsis and Brassica allotetraploids, differential rRNA gene expression is not associated with enhancers or availability of species-specific transcription factors. (78,79) Transient and in vitro transcription assays have shown that Arabidopsis and Brassica rRNA promoters can function with the RNA Polymerase I transcription machinery of the other species. (78,80) Silenced rRNA genes in Arabidopsis and Brassica allotetraploids were reactivated by chemical inhibitors for DNA methylation and/or histone deacetylation, suggesting that rRNA genes are silenced by DNA and histone modifications presumably associated with inactive chromatin structure.…”

“…(43,79) For some genes, including rRNA genes, it takes 1-2 generations to establish a differential expression (silencing or activation) pattern. For others, it takes more than two and sometimes five generations to establish expression status.…”