Mutational Analysis of the Structure and Localization of the Nucleolus in the Yeast Saccharomyces cerevisiae

The nucleolus in Saccharomyces cerevisiae is a crescent-shaped structure that makes extensive contact with the nuclear envelope. In different chromosomal rDNA deletion mutants that we have analyzed, the nucleolus is not organized into a crescent structure, as determined by immunofluorescence microscopy, fluorescence in situ hybridization, and electron microscopy. A strain carrying a plasmid with a single rDNA repeat transcribed by RNA polymerase I (Pol I) contained a fragmented nucleolus distributed throughout the nucleus, primarily localized at the nuclear periphery. A strain carrying a plasmid with the 35S rRNA coding region fused to the GAL7 promoter and transcribed by Pol II contained a rounded nucleolus that often lacked extensive contact with the nuclear envelope. Ultrastructurally distinct domains were observed within the round nucleolus. A similar rounded nucleolar morphology was also observed in strains carrying the Pol I plasmid in combination with mutations that affect Pol I function. In a Pol I–defective mutant strain that carried copies of the GAL7-35S rDNA fusion gene integrated into the chromosomal rDNA locus, the nucleolus exhibited a round morphology, but was more closely associated with the nuclear envelope in the form of a bulge. Thus, both the organization of the rDNA genes and the type of polymerase involved in rDNA expression strongly influence the organization and localization of the nucleolus.     

Nucleolar rDNA folds into condensed foci with a specific combination of epigenetic marks

Arabidopsis thaliana 45S ribosomal genes (rDNA) are located in tandem arrays called nucleolus organizing regions on the termini of chromosomes 2 and 4 (NOR2 and NOR4) and encode rRNA, a crucial structural element of the ribosome. The current model of rDNA organization suggests that inactive rRNA genes accumulate in the condensed chromocenters in the nucleus and at the nucleolar periphery, while the nucleolus delineates active genes. We challenge the perspective that all intranucleolar rDNA is active by showing that a subset of nucleolar rDNA assembles into condensed foci marked by H3.1 and H3.3 histones that also contain the repressive H3K9me2 histone mark. By using plant lines containing a low number of rDNA copies, we further found that the condensed foci relate to the folding of rDNA, which appears to be a common mechanism of rDNA regulation inside the nucleolus. The H3K9me2 histone mark found in condensed foci represents a typical modification of bulk inactive rDNA, as we show by genome-wide approaches, similar to the H2A.W histone variant. The euchromatin histone marks H3K27me3 and H3K4me3, in contrast, do not colocalize with nucleolar foci and their overall levels in the nucleolus are very low. We further demonstrate that the rDNA promoter is an important regulatory region of the rDNA, where the distribution of histone variants and histone modifications are modulated in response to rDNA activity.     

Phenotypic reversion in fas mutants of Arabidopsis thaliana by reintroduction of FAS genes: variable recovery of telomeres with major spatial rearrangements and transcriptional reprogramming of 45S rDNA genes

Arabidopsis thaliana mutants dysfunctional in the evolutionarily conserved protein complex chromatin assembly factor‐1 (CAF‐1), which deposits the canonical histone H3 variant H3.1 during DNA synthesis‐dependent chromatin assembly, display complex phenotypic changes including meristem and growth alterations, sensitivity to DNA‐damaging agents, and reduced fertility. We reported previously that mutants in the FAS1 subunit of CAF‐1 progressively lose telomere and 45S rDNA repeats. Here we show that multiple aspects of the fas phenotype are recovered immediately on expression of a reintroduced FAS1 allele, and are clearly independent of the recovery of rDNA copy‐numbers and telomeres. In reverted lines, 45S rDNA genes are recovered to diverse levels with a strikingly different representation of their variants, and the typical association of nucleolar organizing region 4 with the nucleolus is perturbed. One of 45S rDNA variants (VAR1), which is silenced in wild‐type (WT) plants without mutation history (Col‐0 WT), dominates the expression pattern, whereas VAR2 is dominant in Col‐0 WT plants. We propose an explanation for the variability of telomere and 45S rDNA repeats associated with CAF‐1 function, suggesting that the differences in nuclear partitioning and expression of the rDNA variants in fas mutants and their revertants provide a useful experimental system to study genetic and epigenetic factors in gene dosage compensation.