111 research outputs found
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
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 alsomodified 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 βmutual amphiplastyβ in the nucleolar dominance process.This work was supported by the Fundação para a CiΓͺncia e Tecnologia (projects POCI/BIA-BDE/57575/2004 to M.S. and POCI/BIA-BCM/59389/2004 to N.N.
Trait evaluation and trial cultivation of Dongfang No. 2, the hybrid of a male gametophyte clone of Laminaria longissima (Laminariales, Phaeophyta) and a female one of L. japonica
Direct cultivation of the first filial generation of gametophyte clones from different Laminaria species is a highly effective way of utilizing kelp heterozygous vigor (heterosis). A male gametophyte clone of L. longissima Miyabe and a female one of L. japonica Areschoug were hybridized, generating Dongfang No. 2 hybrid kelp. This hybrid kelp was used directly in trial cultivation, and its agronomical traits were evaluated. L. longissima and L. japonica are obviously different and complement each other in their morphological characteristics and ecological performances. The hybrid of their gametophyte clones, Dongfang No. 2, showed 56.8% heterozygous vigor in yield. It also showed increased yields of 41.0 and 76.4% compared to the widely used commercial kelps Variety 1 and Variety 2, respectively. In large-scale cultivation trials at different locations and in different years, Dongfang No. 2 attained significantly higher yields than Varieties 1 and 2, increasing yield by 26.4% on average over Variety 1 and by 65.0% over the other. Dongfang No. 2 has a robust holdfast and a wide, long and deep-brown uniform blade, which shows a distinct middle groove. In addition to yield, Dongfang No. 2 also demonstrates obvious heterozygous vigor in other agronomic traits. It is resistant to strong irradiance, as the two commercial varieties are, has an appropriate vegetative maturation time, and adapts well to a range of different culture conditions. The parentage analysis using AFLP of total DNA and SNP of the ITS region of ribosomal RNA transcription unit showed that Dongfang No. 2 is the real hybrid of L. japonica and L. longissima
Metal A and Metal B Sites of Nuclear RNA Polymerases Pol IV and Pol V Are Required for siRNA-Dependent DNA Methylation and Gene Silencing
Plants are unique among eukaryotes in having five multi-subunit nuclear RNA polymerases: the ubiquitous RNA polymerases I, II and III plus two plant-specific activities, nuclear RNA polymerases IV and V (previously known as Polymerases IVa and IVb). Pol IV and Pol V are not required for viability but play non-redundant roles in small interfering RNA (siRNA)-mediated pathways, including a pathway that silences retrotransposons and endogenous repeats via siRNA-directed DNA methylation. RNA polymerase activity has not been demonstrated for Polymerases IV or V in vitro, making it unclear whether they are catalytically active enzymes. Their largest and second-largest subunit sequences have diverged considerably from Pol I, II and III in the vicinity of the catalytic center, yet retain the invariant Metal A and Metal B amino acid motifs that bind magnesium ions essential for RNA polymerization. By using site-directed mutagenesis in conjunction with in vivo functional assays, we show that the Metal A and Metal B motifs of Polymerases IV and V are essential for siRNA production, siRNA-directed DNA methylation, retrotransposon silencing, and the punctate nuclear localization patterns typical of both polymerases. Collectively, these data show that the minimal core sequences of polymerase active sites, the Metal A and B sites, are essential for Pol IV and Pol V biological functions, implying that both are catalytically active
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 progenitorβs 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, theA. thaliana βderived 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 generatio
Diversity of Pol IV Function Is Defined by Mutations at the Maize rmr7 Locus
Mutations affecting the heritable maintenance of epigenetic states in maize identify multiple small RNA biogenesis factors including NRPD1, the largest subunit of the presumed maize Pol IV holoenzyme. Here we show that mutations defining the required to maintain repression7 locus identify a second RNA polymerase subunit related to Arabidopsis NRPD2a, the sole second largest subunit shared between Arabidopsis Pol IV and Pol V. A phylogenetic analysis shows that, in contrast to representative eudicots, grasses have retained duplicate loci capable of producing functional NRPD2-like proteins, which is indicative of increased RNA polymerase diversity in grasses relative to eudicots. Together with comparisons of rmr7 mutant plant phenotypes and their effects on the maintenance of epigenetic states with parallel analyses of NRPD1 defects, our results imply that maize utilizes multiple functional NRPD2-like proteins. Despite the observation that RMR7/NRPD2, like NRPD1, is required for the accumulation of most siRNAs, our data indicate that different Pol IV isoforms play distinct roles in the maintenance of meiotically-heritable epigenetic information in the grasses
A Position Effect on the Heritability of Epigenetic Silencing
In animals and yeast, position effects have been well documented. In animals, the best example of this process is Position Effect Variegation (PEV) in Drosophila melanogaster. In PEV, when genes are moved into close proximity to constitutive heterochromatin, their expression can become unstable, resulting in variegated patches of gene expression. This process is regulated by a variety of proteins implicated in both chromatin remodeling and RNAi-based silencing. A similar phenomenon is observed when transgenes are inserted into heterochromatic regions in fission yeast. In contrast, there are few examples of position effects in plants, and there are no documented examples in either plants or animals for positions that are associated with the reversal of previously established silenced states. MuDR transposons in maize can be heritably silenced by a naturally occurring rearranged version of MuDR. This element, Muk, produces a long hairpin RNA molecule that can trigger DNA methylation and heritable silencing of one or many MuDR elements. In most cases, MuDR elements remain inactive even after Muk segregates away. Thus, Muk-induced silencing involves a directed and heritable change in gene activity in the absence of changes in DNA sequence. Using classical genetic analysis, we have identified an exceptional position at which MuDR element silencing is unstable. Muk effectively silences the MuDR element at this position. However, after Muk is segregated away, element activity is restored. This restoration is accompanied by a reversal of DNA methylation. To our knowledge, this is the first documented example of a position effect that is associated with the reversal of epigenetic silencing. This observation suggests that there are cis-acting sequences that alter the propensity of an epigenetically silenced gene to remain inactive. This raises the interesting possibility that an important feature of local chromatin environments may be the capacity to erase previously established epigenetic marks
A comparative approach for the investigation of biological information processing: An examination of the structure and function of computer hard drives and DNA
The Pattern of R2 Retrotransposon Activity in Natural Populations of Drosophila simulans Reflects the Dynamic Nature of the rDNA Locus
The pattern and frequency of insertions that enable transposable elements to remain active in a population are poorly understood. The retrotransposable element R2 exclusively inserts into the 28S rRNA genes where it establishes long-term, stable relationships with its animal hosts. Previous studies with laboratory stocks of Drosophila simulans have suggested that control over R2 retrotransposition resides within the rDNA loci. In this report, we sampled 180 rDNA loci of animals collected from two natural populations of D. simulans. The two populations were found to have similar patterns of R2 activity. About half of the rDNA loci supported no or very low levels of R2 transcripts with no evidence of R2 retrotransposition. The remaining half of the rDNA loci had levels of R2 transcripts that varied in a continuous manner over almost a 100-fold range and did support new retrotransposition events. Structural analysis of the rDNA loci in 18 lines that spanned the range of R2 transcript levels in these populations revealed that R2 number and rDNA locus size varied 2-fold; however, R2 activity was not readily correlated with either of these parameters. Instead R2 activity was best correlated with the distribution of elements within the rDNA locus. Loci with no activity had larger contiguous blocks of rDNA units free of R2-insertions. These data suggest a model in which frequent recombination within the rDNA locus continually redistributes R2-inserted units resulting in changing levels of R2 activity within individual loci and persistent R2 activity within the population
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