Arabidopsis HDA6 Regulates Locus-Directed Heterochromatin Silencing in Cooperation with MET1

Abstract

Heterochromatin silencing is pivotal for genome stability in eukaryotes. In Arabidopsis, a plant-specific mechanism called RNA–directed DNA methylation (RdDM) is involved in heterochromatin silencing. Histone deacetylase HDA6 has been identified as a component of such machineries; however, its endogenous targets and the silencing mechanisms have not been analyzed globally. In this study, we investigated the silencing mechanism mediated by HDA6. Genome-wide transcript profiling revealed that the loci silenced by HDA6 carried sequences corresponding to the RDR2-dependent 24-nt siRNAs, however their transcript levels were mostly unaffected in the rdr2 mutant. Strikingly, we observed significant overlap of genes silenced by HDA6 to those by the CG DNA methyltransferase MET1. Furthermore, regardless of dependence on RdDM pathway, HDA6 deficiency resulted in loss of heterochromatic epigenetic marks and aberrant enrichment for euchromatic marks at HDA6 direct targets, along with ectopic expression of these loci. Acetylation levels increased significantly in the hda6 mutant at all of the lysine residues in the H3 and H4 N-tails, except H4K16. Interestingly, we observed two different CG methylation statuses in the hda6 mutant. CG methylation was sustained in the hda6 mutant at some HDA6 target loci that were surrounded by flanking DNA–methylated regions. In contrast, complete loss of CG methylation occurred in the hda6 mutant at the HDA6 target loci that were isolated from flanking DNA methylation. Regardless of CG methylation status, CHG and CHH methylation were lost and transcriptional derepression occurred in the hda6 mutant. Furthermore, we show that HDA6 binds only to its target loci, not the flanking methylated DNA, indicating the profound target specificity of HDA6. We propose that HDA6 regulates locus-directed heterochromatin silencing in cooperation with MET1, possibly recruiting MET1 to specific loci, thus forming the foundation of silent chromatin structure for subsequent non-CG methylation.

Histone Deacetylase Complex1 Expression Level Titrates Plant Growth and Abscisic Acid Sensitivity in Arabidopsis

“…Before proceeding to ChIPqPCR, DNA samples were amplified using GenomePlex Complete Whole Genome Amplification (WGA2; Sigma-Aldrich) following the manufacturer's protocol. As a quality control for successful ChIP, existence or absence of sequences previously found to be associated (positive control) or not (negative control) with H3K9K14Ac (Zhou et al, 2010;To et al, 2011) in the ChIP samples was confirmed by PCR. Supplemental Table 1 online lists primer pairs and positions of the amplified regions for Actin2 (positive control), AT1G37110 (negative control), and the tested genes.…”

“…Within this context, deacetylation of Lys residues in histones 3 and 4, catalyzed by histone deacetylases (HDACs), establishes a repressive mark (Kouzarides, 2007;Roudier et al, 2009). In plants, histone deacetylation supports fundamental life functions, including maintenance of genome stability (Probst et al, 2004;To et al, 2011;Liu et al, 2012), determination of cell-type specificity (Xu et al, 2005;Hollender and Liu, 2008), and transition between developmental stages (Tanaka et al, 2008;Yu et al, 2011). Gene repression through histone deacetylation is also an important part of the hormonal signaling pathways that orchestrate plant responses to biotic or abiotic stress factors in the environment (Zhou et al, 2005;.…”

“…The Class1-REDUCED POTASSIUM DEPENDENCY3 (Rpd3)-type HISTONE DEACETYLASE6 (HDA6; often acting redundantly with another enzyme of the same family, HDA19) has emerged as a major player in many of these functions (Kim et al, 2012). For example, HDA6 silences transposons and repetitive elements in a process that is at least partially coupled to DNA methylation (Probst et al, 2004;To et al, 2011;Liu et al, 2012). HDA6 and HDA19 redundantly silence embryonic genes after germination, and in mature plants, HDA6 induces flowering by repressing the flowering inhibitor FLOWERING LOCUS C (FLC; Tanaka et al, 2008;Yu et al, 2011).…”

Ordered changes in histone modifications at the core of the Arabidopsis circadian clock

“…Among others, processes such as flowering time, transposon repression, light signaling, and abiotic stress responses are modulated by posttranslational modifications of histones on specific residues (e.g., refs. [8][9][10][11][12]. The genomic distribution and combinatorial association of different histone marks in Arabidopsis appear to define various chromatin states that can be correlated with transcriptionally active or inactive genes (13).…”

HISTONE DEACETYLASE6 Controls Gene Expression Patterning and DNA Methylation-Independent Euchromatic Silencing

“…A clear difference between hda6-8 and hda6-7 was observed when we assessed transcription of the HDA6 targets AT5G41660 and AT3G4470 ( Fig. 4A; To et al, 2011). In contrast to hda6-7, hda6-8 showed no release of transcriptional suppression of these two targets.…”