Plant Genomic Network Research Team, RIKEN Plant Science Center, Yokohama, Kanagawa, Japan.
PLoS Genet. 2011 Apr;7(4):e1002055. doi: 10.1371/journal.pgen.1002055. Epub 2011 Apr 28.
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.
异染色质沉默对于真核生物的基因组稳定性至关重要。在拟南芥中，一种称为 RNA 指导的 DNA 甲基化 (RdDM) 的植物特异性机制参与异染色质沉默。组蛋白去乙酰化酶 HDA6 已被鉴定为这种机制的组成部分；然而，其内源性靶标和沉默机制尚未进行全面分析。在这项研究中，我们研究了由 HDA6 介导的沉默机制。全基因组转录谱分析表明，HDA6 沉默的基因座携带与 RDR2 依赖性 24-nt siRNA 对应的序列，但在 rdr2 突变体中其转录水平大多不受影响。引人注目的是，我们观察到 HDA6 沉默的基因与 CG DNA 甲基转移酶 MET1 沉默的基因有显著重叠。此外，无论是否依赖 RdDM 途径，HDA6 缺陷都会导致异染色质表观遗传标记丢失和 HDA6 直接靶标上常染色质标记异常富集，以及这些基因座的异位表达。在 hda6 突变体中，除了 H4K16 外，H3 和 H4 N 末端所有赖氨酸残基的乙酰化水平都显著增加。有趣的是，我们在 hda6 突变体中观察到两种不同的 CG 甲基化状态。在 hda6 突变体中，一些被侧翼 DNA 甲基化区域包围的 HDA6 靶标基因中的 CG 甲基化仍然存在。相比之下，在与侧翼 DNA 甲基化分离的 HDA6 靶标基因中，hda6 突变体中完全失去了 CG 甲基化。无论 CG 甲基化状态如何，CHG 和 CHH 甲基化丢失，转录去抑制发生在 hda6 突变体中。此外，我们表明 HDA6 仅与靶基因座结合，而不与侧翼甲基化 DNA 结合，表明 HDA6 具有深刻的靶基因座特异性。我们提出 HDA6 与 MET1 合作调节基因座导向的异染色质沉默，可能将 MET1 招募到特定基因座，从而为随后的非 CG 甲基化形成沉默染色质结构奠定基础。
