Histone methyltransferases regulating rRNA gene dose and dosage control in Arabidopsis

Department of Biology, Indiana University, Bloomington, Indiana 47405, USA.
Genes & development (Impact Factor: 10.8). 05/2012; 26(9):945-57. DOI: 10.1101/gad.182865.111
Source: PubMed


Eukaryotes have hundreds of nearly identical 45S ribosomal RNA (rRNA) genes, each encoding the 18S, 5.8S, and 25S catalytic rRNAs. Because cellular demands for ribosomes and protein synthesis vary during development, the number of active rRNA genes is subject to dosage control. In genetic hybrids, one manifestation of dosage control is nucleolar dominance, an epigenetic phenomenon in which the rRNA genes of one progenitor are repressed. For instance, in Arabidopsis suecica, the allotetraploid hybrid of Arabidopsis thaliana and Arabidopsis arenosa, the A. thaliana-derived rRNA genes are selectively silenced. An analogous phenomenon occurs in nonhybrid A. thaliana, in which specific classes of rRNA gene variants are inactivated. An RNA-mediated knockdown screen identified SUVR4 {SUPPRESSOR OF VARIEGATION 3-9 [SU(VAR)3-9]-RELATED 4} as a histone H3 Lys 9 (H3K9) methyltransferase required for nucleolar dominance in A. suecica. H3K9 methyltransferases are also required for variant-specific silencing in A. thaliana, but SUVH5 [SU(VAR)3-9 HOMOLOG 5] and SUVH6, rather than SUVR4, are the key activities in this genomic context. Mutations disrupting the H3K27 methyltransferases ATXR5 or ATXR6 affect which rRNA gene variants are expressed or silenced, and in atxr5 atxr6 double mutants, dominance relationships among variants are reversed relative to wild type. Interestingly, these changes in gene expression are accompanied by changes in the relative abundance of the rRNA gene variants at the DNA level, including overreplication of the normally silenced class and decreased abundance of the normally dominant class. Collectively, our results indicate that histone methylation can affect both the doses of different variants and their differential silencing through the choice mechanisms that achieve dosage control.

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Available from: Frédéric Pontvianne, Oct 04, 2015
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    • "Wild-type nontransgenic plants are shown as controls. HDA6 (Aufsatz et al., 2002; Pontvianne et al., 2012). "
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    ABSTRACT: In order to investigate the role of chromatin regulators in patterning gene expression we employed a novel epigenetically controlled and highly tissue-specific GFP reporter line in Arabidopsis (Arabidopsis thaliana). Using a combination of forward and reverse genetic approaches on this line we show here that distinct epigenetic regulators are involved in silencing the transgene in different tissues. The forward genetic screen led to the identification of a novel HDA6 mutant allele (epic1, hda6-8). This allele differs from the previously reported alleles, as it did not affect DNA methylation and only had a very modest effect on the release of transposable elements and other heterochromatic transcripts. Overall our data shows that HDA6 has at least two clearly separable activities in different genomic regions. In addition, we present an unexpected role for HDA6 in the control of CG methylation. Copyright © 2015, Plant Physiology.
    Plant physiology 04/2015; 168(4). DOI:10.1104/pp.15.00177 · 6.84 Impact Factor
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    • "The mechanism of maintenance of DNA methylation in the symmetric CHG context is unique to plants, and differs significantly from maintenance of CG methylation (Cokus et al., 2008). In A. thaliana, the majority of CHG context methylation depends on the plant-specific DMTase CHRO- MOMETHYLASE 3 (CMT3) and the H3K9-specific histone methyltransferases SUPPRESSOR OF VARIEGATION 39 HOMOLOG 4–6 (SUVH4–6) (Jackson et al., 2002; Malagnac et al., 2002; Ebbs and Bender, 2006; Pontvianne et al., 2012). Experiments showing in vitro affinity of the chromodomain of CMT3 for K9-methylated H3-derived peptides and a decrease of CHG methylation in mutants deficient in dimethylation at lysine 9 of histone H3 (H3K9me2) suggest that H3K9 methylation serves as a label for CMT3 target sites (Lindroth et al., 2004), which is also consistent with results from genome-wide superimposition of CMT3 binding sites and H3K9me2-marked nucleosomes (Du et al., 2012). "
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    ABSTRACT: In plants, 24-nucleotide short interfering RNAs serve as a signal to direct cytosine methylation at homologous DNA regions in the nucleus. If the targeted DNA has promoter function, this RNA-directed DNA methylation can result in transcriptional gene silencing. In a genetic screen for factors involved in RNA-directed transcriptional silencing of a ProNOS-NPTII reporter transgene in Arabidopsis thaliana, we captured alleles of DOMAINS REARRANGED METHYLTRANSFERASE 2, the gene encoding the DNA methyltransferase mainly responsible for de novo DNA methylation in the context of RNA-directed DNA methylation. Interestingly, reporter gene ProNOS methylation was not erased completely in these mutants, but persisted in symmetric CG context, indicating that RNA-directed DNA methylation had been consolidated by DNA methylation maintenance. Taking advantage of the segregation of the transgenes giving rise to ProNOS short interfering RNAs and carrying the ProNOS-NPTII reporter in our experimental system, we found that ProNOS DNA methylation maintenance became first evident after two generations of ongoing RNA-directed DNA methylation, and then increased in its extent with further generations. As ProNOS DNA methylation already had reached its final level in the first generation of RNA-directed DNA methylation, our findings suggest that setting up DNA methylation at a particular region can be divided into distinct stages. An initial phase of efficient, but still fully reversible de novo DNA methylation and transcriptional gene silencing is followed by the transition to efficient maintenance of cytosine methylation in symmetric sequence context accompanied by persistence of gene silencing.This article is protected by copyright. All rights reserved.
    The Plant Journal 07/2014; 80(2). DOI:10.1111/tpj.12630 · 5.97 Impact Factor
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    • "The presence of more than one promoter in animal and plant IGS is well documented [8], [13], [43], [80], [85], [86], and their functional significance has been related to their transcriptional activity [46], [80], [85], [86]. Although long IGSs seems to have higher transcription rates that can favor these variants instead of smaller ones [8], [59], in Arabidopsis only the short rDNA variants are mainly expressed in adult leaves [87]. Moreover, since transcripts from the spacer promoter have been implicated in the silencing of rRNA genes in humans and Arabidopsis [88]–[90] perhaps our smaller variants lacking the spacer promoter are preferentially transcribed rather than the longer ones. "
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    ABSTRACT: The 35S ribosomal DNA (rDNA) units, repeated in tandem at one or more chromosomal loci, are separated by an intergenic spacer (IGS) containing functional elements involved in the regulation of transcription of downstream rRNA genes. In the present work, we have compared the IGS molecular organizations in two divergent species of Fagaceae, Fagus sylvatica and Quercus suber, aiming to comprehend the evolution of the IGS sequences within the family. Self- and cross-hybridization FISH was done on representative species of the Fagaceae. The IGS length variability and the methylation level of 18 and 25S rRNA genes were assessed in representatives of three genera of this family: Fagus, Quercus and Castanea. The intergenic spacers in Beech and Cork Oak showed similar overall organizations comprising putative functional elements needed for rRNA gene activity and containing a non-transcribed spacer (NTS), a promoter region, and a 5'-external transcribed spacer. In the NTS: the sub-repeats structure in Beech is more organized than in Cork Oak, sharing some short motifs which results in the lowest sequence similarity of the entire IGS; the AT-rich region differed in both spacers by a GC-rich block inserted in Cork Oak. The 5'-ETS is the region with the higher similarity, having nonetheless different lengths. FISH with the NTS-5'-ETS revealed fainter signals in cross-hybridization in agreement with the divergence between genera. The diversity of IGS lengths revealed variants from ∼2 kb in Fagus, and Quercus up to 5.3 kb in Castanea, and a lack of correlation between the number of variants and the number of rDNA loci in several species. Methylation of 25S Bam HI site was confirmed in all species and detected for the first time in the 18S of Q. suber and Q. faginea. These results provide important clues for the evolutionary trends of the rDNA 25S-18S IGS in the Fagaceae family.
    PLoS ONE 06/2014; 9(6):e98678. DOI:10.1371/journal.pone.0098678 · 3.23 Impact Factor
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