Postembryonic establishment of megabase-scale gene silencing in nucleolar dominance

Nucleolar dominance is an epigenetic phenomenon in plant and animal genetic hybrids that describes the expression of 45S ribosomal RNA genes (rRNA genes) inherited from only one progenitor due to the silencing of the other progenitor's rRNA genes. rRNA genes are tandemly arrayed at nucleolus organizer regions (NORs) that span millions of basepairs, thus gene silencing in nucleolar dominance occurs on a scale second only to X-chromosome inactivation in female mammals. In Arabidopsis suecica, the allotetraploid hybrid of A. thaliana and A. arenosa, the A. thaliana -derived rRNA genes are subjected to nucleolar dominance and are silenced via repressive chromatin modifications. However, the developmental stage at which nucleolar dominance is established in A. suecica is currently unknown. We show that nucleolar dominance is not apparent in seedling cotyledons formed during embryogenesis but becomes progressively established during early postembryonic development in tissues derived from both the shoot and root apical meristems. The progressive silencing of A. thaliana rRNA genes correlates with the transition of A. thaliana NORs from a decondensed euchromatic state associated with histone H3 that is trimethylated on lysine 4 (H3K4me3) to a highly condensed heterochromatic state in which the NORs are associated with H3K9me2 and 5-methylcytosine-enriched chromocenters. In RNAi-lines in which the histone deacetylases HDA6 and HDT1 are knocked down, the developmentally regulated condensation and inactivation of A. thaliana NORs is disrupted. Collectively, these data demonstrate that HDA6 and HDT1 function in the postembryonic establishment of nucleolar dominance, a process which recurs in each generation.     

Evidence for nucleolus organizer regions as the units of regulation in nucleolar dominance in Arabidopsis thaliana interecotype hybrids

Nucleolar dominance describes the silencing of one parent's ribosomal RNA (rRNA) genes in a genetic hybrid. In Arabidopsis thaliana, rRNA genes are clustered in two nucleolus organizer regions, NOR2 and NOR4. In F(8) recombinant inbreds (RI) of the A. thaliana ecotypes Ler and Cvi, lines that display strong nucleolar dominance inherited a specific combination of NORs, Cvi NOR4 and Ler NOR2. These lines express almost all rRNA from Cvi NOR4. The reciprocal NOR genotype, Ler NOR4/Cvi NOR2, allowed for expression of rRNA genes from both NORs. Collectively, these data reveal that neither Cvi rRNA genes nor NOR4 are always dominant. Furthermore, strong nucleolar dominance does not occur in every RI line inheriting Cvi NOR4 and Ler NOR2, indicating stochastic effects or the involvement of other genes segregating in the RI mapping population. A partial explanation is provided by an unlinked locus, identified by QTL analysis, that displays an epistatic interaction with the NORs and affects the relative expression of NOR4 vs. NOR2. Collectively, the data indicate that nucleolar dominance is a complex trait in which NORs, rather than individual rRNA genes, are the likely units of regulation.     

Locus-specific ribosomal RNA gene silencing in nucleolar dominance

The silencing of one parental set of rRNA genes in a genetic hybrid is an epigenetic phenomenon known as nucleolar dominance. We showed previously that silencing is restricted to the nucleolus organizer regions (NORs), the loci where rRNA genes are tandemly arrayed, and does not spread to or from neighboring protein-coding genes. One hypothesis is that nucleolar dominance is the net result of hundreds of silencing events acting one rRNA gene at a time. A prediction of this hypothesis is that rRNA gene silencing should occur independent of chromosomal location. An alternative hypothesis is that the regulatory unit in nucleolar dominance is the NOR, rather than each individual rRNA gene, in which case NOR localization may be essential for rRNA gene silencing. To test these alternative hypotheses, we examined the fates of rRNA transgenes integrated at ectopic locations. The transgenes were accurately transcribed in all independent transgenic Arabidopsis thaliana lines tested, indicating that NOR localization is not required for rRNA gene expression. Upon crossing the transgenic A. thaliana lines as ovule parents with A. lyrata to form F1 hybrids, a new system for the study of nucleolar dominance, the endogenous rRNA genes located within the A. thaliana NORs are silenced. However, rRNA transgenes escaped silencing in multiple independent hybrids. Collectively, our data suggest that rRNA gene activation can occur in a gene-autonomous fashion, independent of chromosomal location, whereas rRNA gene silencing in nucleolar dominance is locus-dependent.     

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.     

Cloning and restriction analysis of ribosomal RNA genes from Mycobacterium smegmatis

A genomic library of Mycobacterium smegmatis DNA was constructed in phage EMBL3. A clone (gamma HB85) containing rRNA genes was isolated using as probes, fragments of E. coli rRNA cistron B. This cloned DNA fragment was mapped by restriction analysis and was shown to contain one complete set of rRNA genes (rRNA A). The physical mapping of the second set of rRNA genes of M. smegmatis (rRNA B) was done by restriction analysis of total chromosomal DNA. The two sets of rRNA genes showed highly conserved restriction sites within the respective sets but not in the flanking regions. The two rRNA sets of genes are organised like in the other eubacteria in the order 16S-23S-5S.     

Molecular cytogenetics and allotetraploidy in the red vizcacha rat, Tympanoctomys barrerae (Rodentia, Octodontidae)

The theoretical impossibility of polyploidy in mammals was overturned by the discovery of tetraploidy in the red vizcacha rat, Tympanoctomys barrerae (2n = 102). As a consequence of genome duplication, remarkably increased cell dimensions are observed in the spermatozoa and in different somatic cell lines of this species. Locus duplication had been previously demonstrated by in situ PCR and Southern blot analysis of single-copy genes. Here, we corroborate duplication of loci in multiple-copy (major rDNAs) and single-copy (Hoxc8) genes by fluorescence in situ hybridization. We also demonstrate that nucleolar dominance, a large-scale epigenetic silencing phenomenon characteristic of allopolyploids, explains the presence of only one Ag-NOR chromosome pair in T. barrerae. Nucleolar dominance, together with the chromosomal heteromorphism detected in the G-banding pattern and synaptonemal complexes of the species' diploid-like meiosis, consistently indicates allotetraploidy. Allotetraploidization can coherently explain the peculiarities of gene silencing, cell dimensions, and karyotypic features of T. barrerae that remain unexplained by assuming diploidy and a large genome size attained by the dispersion of repetitive sequences.     

Conserved boxes C and D are essential nucleolar localization elements of U14 and U8 snoRNAs.

Sequences necessary for nucleolar targeting were identified in Box C/D small nucleolar RNAs (snoRNAs) by fluorescence microscopy. Nucleolar preparations were examined after injecting fluorescein-labelled wild-type and mutated U14 or U8 snoRNA into Xenopus oocyte nuclei. Regions in U14 snoRNA that are complementary to 18S rRNA and necessary for rRNA processing and methylation are not required for nucleolar localization. Truncated U14 molecules containing Boxes C and D with or without the terminal stem localized efficiently. Nucleolar localization was abolished upon mutating just one or two nucleotides within Boxes C and D. Moreover, the spatial position of Boxes C or D in the molecule is essential. Mutations in Box C/D of U8 snoRNA also impaired nucleolar localization, suggesting the general importance of Boxes C and D as nucleolar localization sequences for Box C/D snoRNAs. U14 snoRNA is shown to be required for 18S rRNA production in vertebrates.     

Fluctuations in ribosomal RNA gene content and nucleolar activity in the cambial region of Abies balsamea (Pinaceae) shoots during reactivation

Tissue was collected from the vascular cambial region of 1-year-old balsam fir shoots over an 11-week period during which cambial reactivation occurred. The amount of rDNA (ribosomal RNA genes) relative to total genomic DNA was determined by quantitative slot blots for three trees, one of which showed a 3-week delay in reactivation. In addition, nucleolar activity was estimated by measuring nucleolar volume, number, and staining intensity. Relative rRNA gene content increased transiently prior to the onset of cambial cell periclinal division. Nucleolar volume also increased transiently, but 1–2 weeks prior to the maximal relative rDNA value. The increases in relative rDNA and nucleolar activity were delayed in the tree in which reactivation was late. We interpret these changes as reflecting the amplification and loss of genes encoding rRNA to facilitate cambial cell reactivation.     

Ribosomal RNA gene number and sequence divergence in the diploid-tetraploid species pair of north American hylid tree frogs

Hyla chrysoscelis (2n = 24) and H. versicolor (2n = 48) are a diploid-tetraploid species pair of tree frogs. Hybridization saturation of isolated ¹²⁵I-labeled ribosomal RNAs (rRNAs) with filter-immobilized DNA shows that there are twice as many rRNA genes in the tetraploid as in the diploid. For the diploid, saturation occurs at 0.037%, from which it is calculated that there are about 618 copies of the (18 S + 28 S) rRNA genes per haploid genome. Analysis of the extent of hybridization and also the thermal stability of homologous and heterologous hybrids shows that considerably more base substitutions have occurred in the tetraploid rDNA genes than in the diploid since their divergence. This is interpreted to reflect either a relaxation of the gene regulatory correction mechanism hypothesized to be responsible for the maintenance of identical tandem rRNA genes in the tetraploid or a release of one gene set from the normal selective constraints.This research supported by NSF Grants CDP-8002341 and PRM-8106947 to J.C.V. and by research grants from Miami University to L.A.T., D.T.C., R.J.D., and S.W.K.     

Construction of the physical map of the chloroplast DNA of Phaseolus vulgaris and localization of ribosomal and transfer RNA genes

Construction of a physical map of the chloroplast DNA from Phaseolus vulgaris showed that this circular molecule is segmentally organized into four regions. Unlike other chloroplast DNAs which have analogous organization, two single-copy regions that separate two inverted repeats have been demonstrated to exist in both relative orientations, giving rise to two populations of DNA molecules.Hybridization studies using individual rRNA and tRNA species revealed the location of a set of rRNA genes and at least seven tRNA genes in each inverted repeat region, a minimum of 17 tRNA genes in the large single-copy region and one tRNA gene in the small single-copy region. The tRNA genes code for 24 tRNA species corresponding to 16 amino acids. Comparison of this gene map with those of other chloroplast DNAs suggests that DNA sequence rearrangements, involving some tRNA genes, have occurred.