Nuno Neves

A concerted DNA methylation/histone methylation switch regulates rRNA gene dosage control and nucleolar dominance

Eukaryotes regulate the effective dosage of their ribosomal RNA (rRNA) genes, expressing fewer than half of the genes at any one time. Likewise, genetic hybrids displaying nucleolar dominance transcribe rRNA genes inherited from one parent but silence the other parental set. We show that rRNA gene dosage control and nucleolar dominance utilize a common mechanism. Central to the mechanism is an epigenetic switch in which concerted changes in promoter cytosine methylation density and specific histone modifications dictate the on and off states of the rRNA genes. A key component of the off switch is HDT1, a plant-specific histone deacetylase that localizes to the nucleolus and is required for H3 lysine 9 deacetylation and subsequent H3 lysine 9 methylation. Collectively, the data support a model in which cytosine methylation and histone deacetylation are each upstream of one another in a self-reinforcing repression cycle.

Nucleolar dominance in triticales: control by unlinked genes

Hybrid plants and animals often show suppression of activity of ribosomal genes (rDNA) originating from one of the parental or ancestral species. In the wheat × rye amphiploid triticale, containing 28 chromosomes of wheat origin and 14 from rye, rDNA of rye origin (on chromosome 1R) is not normally expressed, while the 1B- and 6B-origin rDNA from wheat shows strong expression. Expression of rDNA can be accurately assessed by the silver staining method, which stains both interphase nucleoli and metaphase rDNA sites that were actively expressed at the previous interphase. We show here that substitution of another rye chromosome, 2R, by a chromosome from hexaploid wheat, 2D(triticale-2D(2R)), prevents suppression of the rye-origin rDNA, and leads to activity of all six major rDNA loci. These results were found in two different triticales and supported by rDNA behaviour in wheat—rye chromosomal addition lines. Models for chromosomal interactions leading to control of rDNA expression are presented.

Ribosomal DNA heterochromatin in plants

The aim of this review is to integrate earlier results and recent findings to present the current state-of-the-art vision concerning the dynamic behavior of the ribosomal DNA (rDNA) fraction in plants. The global organization and behavioral features of rDNA make it a most useful system to analyse the relationship between chromatin topology and gene expression patterns. Correlations between several heterochromatin fractions and rDNA arrays demonstrate the heterochromatic nature of the rDNA and reveal the importance of the genomic environment and of developmental controls in modulating its dynamics.

Development-dependent inheritance of 5-azacytidine-induced epimutations in triticale: analysis of rDNA expression patterns

Genomic imprinting of rye origin rDNA sequences in triticale is modulated by DNA methylation responsible for ontogenic expression patterns of those sequences. Considering the dynamic nature of these phenomena, we evaluated the influence of plant development on the inheritance of modified rye rDNA expression patterns. DNA hypomethylation was induced in triticale by 5-azacytidine (5AC) treatments at distinct developmental stages of M1 plants, and expression patterns were analysed in M2. The activity of rye origin rRNA genes in progeny of untreated and 5AC-treated plants was evaluated by silver staining in meristematic root tip cells and in meiocytes at diplotene. In the progeny of 5AC-treated plants, a significant increase in rye rDNA expression was observed, contrasting with the residual activity in untreated plants. Significant differential effects of 5AC treatments were observed in M2 plants and correlated with the M1 plant developmental stage in which DNA hypomethylation was induced. Hypotheses to explain the origin of those differences are discussed here.

The influence of B chromosomes on rDNA organization in rye interphase nuclei

Patterns of rye rDNA organization in interphase nuclei were studied through the use ofin situ hybridization in spreads of root meristem cells from plants with and without B chromosomes (Bs). In cells from plants without Bs each rDNA locus is organized as a single perinucleolar knob of condensed chromatin with decondensed chromatin inside the nucleolus. In plants with Bs there is a marked modification of the pattern, found in more than 23% of nuclei, which involves several regions of condensed chromatin interspersed with decondensed chromatin inside the nucleolus. This B-induced alteration in rDNA interphase organization suggests a change in expression of the rRNA genes located on the A chromosomes probably related to the reduction in nuclear RNA observed previously in plants with Bs. The influence of the Bs on the expression of A chromosome genes, through rearrangement of interphase chromatin, could provide the basis of an explanation for some of the known phenotypic effects of B chromosomes in rye.

Chromosome and DNA methylation dynamics during meiosis in the autotetraploid Arabidopsis arenosa

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.

Relationships between transcription, silver staining, and chromatin organization of nucleolar organizers in Secale cereale

Summary.The nucleolar organizer regions (NORs) are composed of hundreds of rRNA genes, typically spanning several megabases. Cytologically, NORs include regions that are highly condensed and regions that are decondensed, the latter corresponding to regions at which associated proteins stain intensively with silver (Ag-NORs) and where active rRNA gene transcription is thought to occur. To test the relationship between rRNA gene activity, NOR silver staining, and rDNA (genes coding for rRNA) chromatin condensation, we used the DNA methyl-transferase inhibitor 5-azacytidine to evaluate the correlation between the epigenetic regulation of rRNA genes and NOR silver staining in the plant Secale cereale. Following 5-azacytidine treatment, we observed an increase in rRNA gene transcription as well as a reduction in the number of cells showing a significant difference in the size of the silver-stained domains in the two NORs. These transcriptional changes occurred concomitantly with an increase in nuclear and nucleolar size and were associated with the reallocation of most of the rDNA from perinucleolar heterochromatin into the nucleolus. Collectively, these results suggest that rRNA gene transcription, silver staining, and NOR decondensation are interrelated in S. cereale.

Interplay of ribosomal DNA Loci in nucleolar dominance: dominant NORs are up-regulated by chromatin dynamics in the wheat-rye system

Background Chromatin organizational and topological plasticity, and its functions in gene expression regulation, have been strongly revealed by the analysis of nucleolar dominance in hybrids and polyploids where one parental set of ribosomal RNA (rDNA) genes that are clustered in nucleolar organizing regions (NORs), is rendered silent by epigenetic pathways and heterochromatization. However, information on the behaviour of dominant NORs is very sparse and needed for an integrative knowledge of differential gene transcription levels and chromatin specific domain interactions. Methodology/Principal Findings Using molecular and cytological approaches in a wheat-rye addition line (wheat genome plus the rye nucleolar chromosome pair 1R), we investigated transcriptional activity and chromatin topology of the wheat dominant NORs in a nucleolar dominance situation. Herein we report dominant NORs up-regulation in the addition line through quantitative real-time PCR and silver-staining technique. Accompanying this modification in wheat rDNA trascription level, we also disclose that perinucleolar knobs of ribosomal chromatin are almost transcriptionally silent due to the residual detection of BrUTP incorporation in these domains, contrary to the marked labelling of intranucleolar condensed rDNA. Further, by comparative confocal analysis of nuclei probed to wheat and rye NORs, we found that in the wheat-rye addition line there is a significant decrease in the number of wheat-origin perinucleolar rDNA knobs, corresponding to a diminution of the rDNA heterochromatic fraction of the dominant (wheat) NORs. Conclusions/Significance We demonstrate that inter-specific interactions leading to wheat-origin NOR dominance results not only on the silencing of rye origin NOR loci, but dominant NORs are also modified in their transcriptional activity and interphase organization. The results show a cross-talk between wheat and rye NORs, mediated by ribosomal chromatin dynamics, revealing a conceptual shift from differential amphiplasty to ‘mutual amphiplasty’ in the nucleolar dominance process.

Reprogramming of rye rDNA in triticale during microsporogenesis

To test the hypothesis that interspecific genomic and chromosome interactions leading to nucleolar dominance could be reprogrammed in meiosis, we compared the expression of distinct nucleolar organizing region (NOR) loci in hexaploid triticale root tip meristematic cells, pollen mother cells and young pollen grains. Interphase and metaphase cells were silver stained to quantify nucleoli and active NOR loci respectively. A marked difference in the ribosomal RNA gene activity of each locus was observed when different types of cells were compared: in somatic and pollen mother cells, rRNA gene activity was mainly restricted to major wheat NORs (1B and 6B) with only a small contribution from rye NORs (1R). In contrast, in young pollen grains, all NORs present, including the 1R NORs, were consistently active. The expression of all NORs just after meiosis is considered to be a consequence of meiotic reprogramming of rye origin rDNA. Gene reprogramming mediated by the resetting of methylation patterns established early in embryogenesis is suggested to be responsible for the differential expression of the NORs of rye origin in distinct developmental stages of triticale.

Genomic interactions: gene expression, DNA methylation and nuclear architecture

New varieties of plants are obtained by breeders producing new combinations of characters. With this goal, some strategies aim to introduce alien chromatin from related species into cultivated crops. Genetic engineering and hybridization techniques permit assembly in a single organism of the advantages of different organisms; however, in many cases the regular expression of the inserted genes cannot be ensured. Thus, integrated in a context of gene, chromosome and genomic manipulation, further studies of genomic interactions are vital for fundamental studies of gene control and for plant breeding.