AtHDA6 functions as an H3K18ac eraser to maintain pericentromeric CHG methylation in Arabidopsis thaliana

Abstract

Pericentromeric DNA, consisting of high-copy-number tandem repeats and transposable elements, is normally silenced through DNA methylation and histone modifications to maintain chromosomal integrity and stability. Although histone deacetylase 6 (HDA6) has been known to participate in pericentromeric silencing, the mechanism is still yet unclear. Here, using whole genome bisulfite sequencing (WGBS) and chromatin immunoprecipitation-sequencing (ChIP-Seq), we mapped the genome-wide patterns of differential DNA methylation and histone H3 lysine 18 acetylation (H3K18ac) in wild-type and hda6 mutant strains. Results show pericentromeric CHG hypomethylation in hda6 mutants was mediated by DNA demethylases, not by DNA methyltransferases as previously thought. DNA demethylases can recognize H3K18ac mark and then be recruited to the chromatin. Using biochemical assays, we found that HDA6 could function as an 'eraser' enzyme for H3K18ac mark to prevent DNA demethylation. Oxford Nanopore Technology Direct RNA Sequencing (ONT DRS) also revealed that hda6 mutants with H3K18ac accumulation and CHG hypomethylation were shown to have transcriptionally active pericentromeric DNA.

Figure 1.

Schematic representation of chromatin dynamics mediated by DNA methylation and histone modifications. (A) Euchromatin, with H3K9ac and H3K18ac histone marks and minimal levels of methylated DNA after catalysis by DNA demethylases (in green above right-pointing arrow). (B) Euchromatin, with relatively high levels of methylated DNA (shown as light and dark grey –CH3 groups) catalyzed by DNA methyltransferases (in green below left-pointing arrow), or transitioned from facultative heterochromatin after catalysis by IDM1 and HAG1. (C) Facultative heterochromatin, transitioned from highly methylated euchromatin after catalysis by HDA6, or transitioned from constitutive heterochromatin after IBM1 catalysis. (D) Constitutive heterochromatin, transitioned from facultative heterochromatin after catalysis by SUVH4/5/6.

Figure 2.

Genome-wide distributions of DNA methylation levels in Arabidopsis ecotype C24, ros1-1, ros1-1&hda6-9, and ros1-1&hda6-10. (A) An IGV plot showing the distributions of differentially methylated loci (DMLs) in C24/ros1-1, ros1-1&hda6-9/ros1-1, and ros1-1&hda6-10/ros1-1 in the CG, CHG and CHH contexts along the five chromosomes, with the corresponding genic and transposable element (TE) regions at the bottom in grey. Red: hypermethylated DMLs; blue: hypomethylated DMLs. (B) Venn diagrams showing the overlapping hypo-DMRs (differentially methylated regions) among the C24/ros1-1, ros1-1&hda6-9/ros1-1 and ros1-1&hda6-10/ros1-1 comparisons in the CG, CHG and CHH contexts. (C) Heat maps of DMR clusters sorted according to their relative DNA methylation levels across the four genotypes. Each genotype is presented with two replicates (R1 and R2). (D) Proportions of the DMRs being located in each of the four genomic regions (gene, intergenic, promoter and transposon) within each cluster from (C) in the CG, CHG and CHH contexts respectively.

Figure 3.

Genome-wide distributions of DMRs and differential H3K18ac accumulation regions at the CHG context. (A) A Circos plot showing the distributions of DMRs in the CHG context between C24/ros1-1 (a), ros1-1&hda6-9/ros1-1 (b), and ros1-1&hda6-10/ros1-1 (c), and the differential H3K18ac accumulation between C24/ros1-1 (d), ros1-1&hda6-9/ros1-1 (e), and ros1-1&hda6-10/ros1-1 (f), with the corresponding gene densities (g) and transposable element (TE) densities (h). The dark red bars on the chromosomes indicate centromeric regions. (B) Upper panel: numbers of DMRs in C24/ros1-1, ros1-1&hda6-9/ros1-1 and ros1-1&hda6-10/ros1-1; lower panel: numbers of differential H3K18ac peaks in C24/ros1-1, ros1-1&hda6-9/ros1-1 and ros1-1&hda6-10/ros1-1.

Figure 4.

HDA6 can effectively deacetylate H3K9ac and H3K18ac in vitro. The hydrolysis of acetylated peptides by FLAG-tagged HDA6 was analyzed by LC–MS. Black traces show the total ion intensity for all ion species with m/z ranging from 300 to 2000; red traces show the ion intensity for the mass of unmodified (deacetylated) peptides; and blue traces show the ion intensity for the mass of acetylated peptides. TIC: total ion count.

Figure 5.

DNA methylation, histone H3K18ac accumulation and gene expression patterns of clustered DMRs in the CG (A), CHG (B) and CHH contexts (C). DMRs were clustered according to their relative DNA methylation levels across the four genotypes (C24, ros1-1, ros1-1&hda6-9 and ros1-1&hda6-10). Each genotype is represented by two biological replicates (R1 and R2).

Figure 6.

HDA6 increased DNA methylation, suppressed histone H3K18ac accumulation and reduced transcript abundance in the centromeric regions in the CHG context. (A) Numbers of DMLs or DMRs in the CHG context along all five chromosomes. Horizontal grey bars at the bottom indicate centromeric regions. Turquoise lines represent the distribution of hypomethylated loci; red lines represent the hypermethylated loci; vertical grey bars represent the hyper-/hypo- DMRs. (B) Representative sections from each chromosomal centromeric region showing the DNA methylation levels, H3K18ac accumulation and transcript abundance in the CHG context in each genotype.