Mechanisms of HDA6-mediated rRNA gene silencing: suppression of intergenic Pol II transcription and differential effects on maintenance versus siRNA-directed cytosine methylation

Abstract

The Arabidopsis histone deacetylase HDA6 is required to silence transgenes, transposons, and ribosomal RNA (rRNA) genes subjected to nucleolar dominance in genetic hybrids. In nonhybrid Arabidopsis thaliana, we show that a class of 45S rRNA gene variants that is normally inactivated during development fails to be silenced in hda6 mutants. In these mutants, symmetric cytosine methylation at CG and CHG motifs is reduced, and spurious RNA polymerase II (Pol II) transcription occurs throughout the intergenic spacers. The resulting sense and antisense spacer transcripts facilitate a massive overproduction of siRNAs that, in turn, direct de novo cytosine methylation of corresponding gene sequences. However, the resulting de novo DNA methylation fails to suppress Pol I or Pol II transcription in the absence of HDA6 activity; instead, euchromatic histone modifications typical of active genes accumulate. Collectively, the data reveal a futile cycle of unregulated transcription, siRNA production, and siRNA-directed DNA methylation in the absence of HDA6-mediated histone deacetylation. We propose that spurious Pol II transcription throughout the intergenic spacers in hda6 mutants, combined with losses of histone deacetylase activity and/or maintenance DNA methylation, eliminates repressive chromatin modifications needed for developmental rRNA gene dosage control.[Keywords: Chromatin; histone deacetylation; DNA methylation; epigenetics; RNA polymerase I; ribosomal RNA genes] Supplemental material is available at http://www.genesdev.org.

HISTONE DEACETYLASE6 Interacts with FLOWERING LOCUS D and Regulates Flowering in Arabidopsis

“…Genome-wide gene expression analysis using the Affymetrix GeneChip indicates that down-regulation of HDA6 affected genes involved in a variety of biological processes. In addition to genes involved in flowering, genes involved in gene silencing and stress responses were also affected in hda6 mutants, supporting multiple functions of HDA6 (Aufsatz et al, 2007;Wu et al, 2008;Chen et al, 2010;Earley et al, 2010). We found that the expression of two genes encoding AGO proteins, AGO5 and AGO3, was up-regulated in hda6 mutants.…”

“…By using microarray analysis, two additional MADS box genes, AGL37 and AT5G59930, were also identified to be up-regulated and hyperacetylated in axe1-5 plants. Since HDA6 is a trichostain A-sensitive histone deacetylase capable of removing acetyl groups from multiple Lys residues of histones (Earley et al, 2006(Earley et al, , 2010, HDA6 may repress FLC as well as other MADS box genes through deacetylating their chromatin. ChIP analysis indicated that HDA6 bound directly to the promoters of FLC, MAF4, and AT5G59930, suggesting that they may be the direct targets of HDA6.…”

“…The AGO proteins are involved in RNA-silencing processes that also involve 21-to 25-nucleotide short RNAs cleaved from double-stranded RNAs by the RNaseIII enzyme Dicer (Baumberger and Baulcombe, 2005). It is suggested that HDA6 plays an important role in RNAimediated heterochromatin formation (Aufsatz et al, 2007;Earley et al, 2010). Up-regulation of AGO5 and AGO3 in hda6 mutants suggests that HDA6 may be involved in RNAi-mediated gene silencing by regulating the expression of AGO-like genes.…”

Subnuclear partitioning of rRNA genes between the nucleolus and nucleoplasm reflects alternative epiallelic states

“…HISTONE DEACETYLASE 6 (HDA6) is required for uniparental rRNA gene silencing in hybrid Arabidopsis suecica and for developmentally regulated silencing of variant 1 rRNA genes in nonhybrid A. thaliana (Earley et al 2006(Earley et al , 2010. HDA6 localizes throughout nuclei, including the nucleolus (Fig.…”

“…All subtypes are expressed immediately following germination, but by ;10 d of seedling growth, the variant 1 subtype (Fig. 1A), accounting for ;50% of all rRNA genes, is silenced via epigenetic mechanisms that include changes in DNA methylation and histone modification (Earley et al 2006(Earley et al , 2010Pontvianne et al 2010Pontvianne et al , 2012. Chromatin modifications mediate rRNA gene dosage control in yeast, mouse, and human cells as well (Sandmeier et al 2002;McStay and Grummt 2008;Grummt and Langst 2013).…”