Arabidopsis histone deacetylase HDA6 is required for maintenance of transcriptional gene silencing and determines nuclear organization of rDNA repeats

Abstract

Histone acetylation and deacetylation are connected with transcriptional activation and silencing in many eukaryotic organisms. Gene families for enzymes that accomplish these modifications show a surprising multiplicity in sequence and expression levels, suggesting a high specificity for different targets. We show that mutations in Arabidopsis (Arabidopsis thaliana) HDA6, a putative class I histone deacetylase gene, result in loss of transcriptional silencing from several repetitive transgenic and endogenous templates. Surprisingly, total levels of histone H4 acetylation are only slightly affected, whereas significant hyperacetylation is restricted to the nucleolus organizer regions that contain the rDNA repeats. This switch coincides with an increase of histone 3 methylation at Lys residue 4, a modified DNA methylation pattern, and a concomitant decondensation of the chromatin. These results indicate that HDA6 might play a role in regulating activity of rRNA genes, and this control might be functionally linked to silencing of other repetitive templates and to its previously assigned role in RNA-directed DNA methylation.

Figure 1.

sil1 Has a Mutation in the AtHDA6 Gene. The sil1 mutation maps between markers CER456030 and CER455379 at the bottom of chromosome 5. Sequencing of the HDA6 gene in the sil1 mutant reveals a point mutation, 46 bases after the ATG initiation codon, leading to the replacement of Gly₁₆ by Arg. The axe1-5 mutant has a base substitution at position 1635 downstream of the ATG at the third exon-intron junction. Alignment of AtHDA6 with Arabidopsis (At) and human (Hs) RPD3-like HDACs and yeast (Sc) RPD3 reveals a conservation of Gly₁₆ in plant and human RPD3-like HDACs.

Figure 2.

The 35S:GUS Transgene at the L5 Locus Is Methylated and Transcriptionally Silenced. The transcriptionally active transgenic line Hc1 (1) and the silenced line L5 (2) were characterized by a combination of DNA gel blot (A), RNA gel blot (B), and nuclear run-on analysis (C). The presence of high molecular weight fragments observed after digestion by the methylation-sensitive restriction enzymes HpaII (H) and MspI (M) and hybridization with radiolabeled 35S and GUS probes indicate that the entire insert in line L5 is strongly methylated (A). Hybridization of 10 μg of total RNA with a probe corresponding to the GUS coding region (top panel) or a 25S rDNA probe reveals the absence of GUS cytoplasmic transcript in line L5 (B). Run-on experiments using labeled RNA extracted from leaf nuclei of adult plants for hybridization of dot blots demonstrate the lack of nascent GUS transcript in line L5. Dots contain 2 μg DNA each of the 25S rDNA-containing plasmid (25S), single-stranded pBluescript KS+ (plasmid), and GUS-containing plasmids (GUS−, sense single-stranded; GUS+, antisense single-stranded; GUS, double-stranded) (C).

Figure 3.

sil1 and axe1-5 Alleles Release Silencing of an Endogenous TSI Sequence. (A) RNA gel blot analysis using the TSI pA2 fragment as probe reveals TSI transcripts in the two HDA6 mutant alleles axe1-5 and sil1. Lanes 1, 2, and 5 show silencing of the endogenous TSI repeats in the transgenic background of the axe1-5 mutants (DR5) and the Ler ecotype, whereas lanes 3, 4, 6, and 7 show reactivation of TSI in the two HDA6 mutant alleles and the mom1-1 mutant, respectively. Predominantly, two transcripts are expressed—a longer, polyadenylated one (Steimer et al., 2000) as well as a shorter transcript. nts, nucleotides. (B) The blot was reprobed with RAN (small GTP binding protein) (Haizel et al., 1997) as a loading reference. Total RNA (20 μg per lane) was extracted from rosette leaves of adult plants.

Figure 4.

rDNA Repeats Are Hyperacetylated in Nuclei of HDA6 Mutants. (A) to (D) Distribution of histone H4 acetylation revealed by DAPI staining of DNA (blue, left panel) and immunodetection with an antibody specific for tetra-acetylated H4 (green, middle panel) in nuclei of control lines DR5 (A) and Ler (C) and in axe1-5 (B) and sil1 (D) mutant nuclei. Right panels show merged images. For each nucleus, two layers were selected from deconvoluted image stacks, arrows mark the nucleolus. (E) and (F) FISH using rDNA repeats (red, left panel) after immunostaining with α-H4ac antibodies (green, middle panels) shows that the rDNA loci indeed are devoid of H4ac staining in the wild type (E) but become highly enriched with H4ac in mutant nuclei (F).

Figure 5.

Changes in Levels of H4ac and H3K4met Are Limited to Specific Loci in HDA6 Mutants. (A) Protein gel blot analysis detecting H4ac (top panel) and H3K4met (middle panel) using α-H4ac and α-H3K4met antibodies, respectively, on protein extracts from wild-type (DR5) and axe1-5 mutant plants. Bottom panel, Coomassie staining shows equal protein loading. (B) ChIP performed in the control line DR5 and the mutant allele axe1-5 reveals an increase in H4ac and H3K4met at rDNA repeats. The Actin2/7 gene is equally present in mutant and control precipitates. If the antibodies are omitted during the procedure (mock), neither target is amplified, whereas the equal strength of bands after PCR with the input fraction indicates equal amounts of chromatin before immunoprecipitation. (C) and (D) Distribution of histone H3 methylated at Lys 4 revealed by DAPI staining (blue, left panel) and immunodetection with an antibody specific for H3K4met (green, middle panel) in nuclei of control lines (top row) DR5 (C), Ler (D), and mutants (bottom row) axe1-5 (C) and sil1 (D). Right panels show merged images.

Figure 6.

rDNA Expression Is Not Increased in HDA6 Mutant Plants. Total RNA from control lines DR5 and Ler and mutants axe1-5 and sil1 was subjected to S1 nuclease protection using probes specific for the 5′ end of pre-rRNA transcripts and compared with total RNA amounts, as seen from ethidium bromide staining (A). The signals obtained for rRNA (1) of DR5 and axe1-5 were normalized against signals obtained with probes specific for protein-coding genes ubiquitin (2) or actin (3) (B). All lanes are from the same exposure of the same autoradiogram.

Figure 7.

rDNA Loci, but Not Chromocenters in General, Are Decondensed in HDA6 Mutant Nuclei. Interphase nuclear spreads of control lines DR5 and Ler and mutants axe1-5 and sil1 stained with DAPI (black and white in left panel, blue in merged images in the right panel) and FISH with biotin-labeled probes for rDNA repeats (A) and centromeric (180 bp) repeats (B). Arrows in the black and white images point to decondensed rDNA repeats in mutant nuclei in (A) and (B).

Figure 8.

DNA Methylation Patterns at rDNA Repeats Are Affected in HDA6 Mutants. Genomic DNA samples from wild-type Columbia (1) and Ler (4), the transgenic line DR5 (2), the HDA6 mutant alleles axe1-5 (3) and sil1 (5), and from the DNA methylation mutant ddm1-5 (6) (Jeddeloh et al., 1999) were analyzed. The DNA was digested with methylation-sensitive restriction enzymes CfoI, HpaII, MaeII, and AvaII ([A] and [B]), subjected to DNA gel blot analysis, and probed with rDNA (Vongs et al., 1993). A CfoI digest hybridized with a FWA probe (Saze et al., 2003) is shown in (C).