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

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

Arabidopsis Homologs of Retinoblastoma-Associated Protein 46/48 Associate with a Histone Deacetylase to Act Redundantly in Chromatin Silencing

RNA molecules such as small-interfering RNAs (siRNAs) and antisense RNAs (asRNAs) trigger chromatin silencing of target loci. In the model plant Arabidopsis, RNA–triggered chromatin silencing involves repressive histone modifications such as histone deacetylation, histone H3 lysine-9 methylation, and H3 lysine-27 monomethylation. Here, we report that two Arabidopsis homologs of the human histone-binding proteins Retinoblastoma-Associated Protein 46/48 (RbAp46/48), known as MSI4 (or FVE) and MSI5, function in partial redundancy in chromatin silencing of various loci targeted by siRNAs or asRNAs. We show that MSI5 acts in partial redundancy with FVE to silence FLOWERING LOCUS C (FLC), which is a crucial floral repressor subject to asRNA–mediated silencing, FLC homologs, and other loci including transposable and repetitive elements which are targets of siRNA–directed DNA Methylation (RdDM). Both FVE and MSI5 associate with HISTONE DEACETYLASE 6 (HDA6) to form complexes and directly interact with the target loci, leading to histone deacetylation and transcriptional silencing. In addition, these two genes function in de novo CHH (H = A, T, or C) methylation and maintenance of symmetric cytosine methylation (mainly CHG methylation) at endogenous RdDM target loci, and they are also required for establishment of cytosine methylation in the previously unmethylated sequences directed by the RdDM pathway. This reveals an important functional divergence of the plant RbAp46/48 relatives from animal counterparts

Evolutionary origins of Brassicaceae specific genes in Arabidopsis thaliana

<p>Abstract</p> <p>Background</p> <p>All sequenced genomes contain a proportion of lineage-specific genes, which exhibit no sequence similarity to any genes outside the lineage. Despite their prevalence, the origins and functions of most lineage-specific genes remain largely unknown. As more genomes are sequenced opportunities for understanding evolutionary origins and functions of lineage-specific genes are increasing.</p> <p>Results</p> <p>This study provides a comprehensive analysis of the origins of lineage-specific genes (LSGs) in <it>Arabidopsis thaliana </it>that are restricted to the Brassicaceae family. In this study, lineage-specific genes within the nuclear (1761 genes) and mitochondrial (28 genes) genomes are identified. The evolutionary origins of two thirds of the lineage-specific genes within the <it>Arabidopsis thaliana </it>genome are also identified. Almost a quarter of lineage-specific genes originate from non-lineage-specific paralogs, while the origins of ~10% of lineage-specific genes are partly derived from DNA exapted from transposable elements (twice the proportion observed for non-lineage-specific genes). Lineage-specific genes are also enriched in genes that have overlapping CDS, which is consistent with such novel genes arising from overprinting. Over half of the subset of the 958 lineage-specific genes found only in <it>Arabidopsis thaliana </it>have alignments to intergenic regions in <it>Arabidopsis lyrata</it>, consistent with either <it>de novo </it>origination or differential gene loss and retention, with both evolutionary scenarios explaining the lineage-specific status of these genes. A smaller number of lineage-specific genes with an incomplete open reading frame across different <it>Arabidopsis thaliana </it>accessions are further identified as accession-specific genes, most likely of recent origin in <it>Arabidopsis thaliana</it>. Putative <it>de novo </it>origination for two of the <it>Arabidopsis thaliana</it>-only genes is identified via additional sequencing across accessions of <it>Arabidopsis thaliana </it>and closely related sister species lineages. We demonstrate that lineage-specific genes have high tissue specificity and low expression levels across multiple tissues and developmental stages. Finally, stress responsiveness is identified as a distinct feature of Brassicaceae-specific genes; where these LSGs are enriched for genes responsive to a wide range of abiotic stresses.</p> <p>Conclusion</p> <p>Improving our understanding of the origins of lineage-specific genes is key to gaining insights regarding how novel genes can arise and acquire functionality in different lineages. This study comprehensively identifies all of the Brassicaceae-specific genes in <it>Arabidopsis thaliana </it>and identifies how the majority of such lineage-specific genes have arisen. The analysis allows the relative importance (and prevalence) of different evolutionary routes to the genesis of novel ORFs within lineages to be assessed. Insights regarding the functional roles of lineage-specific genes are further advanced through identification of enrichment for stress responsiveness in lineage-specific genes, highlighting their likely importance for environmental adaptation strategies.</p

Stress-Induced Activation of Heterochromatic Transcription

Constitutive heterochromatin comprising the centromeric and telomeric parts of chromosomes includes DNA marked by high levels of methylation associated with histones modified by repressive marks. These epigenetic modifications silence transcription and ensure stable inheritance of this inert state. Although environmental cues can alter epigenetic marks and lead to modulation of the transcription of genes located in euchromatic parts of the chromosomes, there is no evidence that external stimuli can globally destabilize silencing of constitutive heterochromatin. We have found that heterochromatin-associated silencing in Arabidopsis plants subjected to a particular temperature regime is released in a genome-wide manner. This occurs without alteration of repressive epigenetic modifications and does not involve common epigenetic mechanisms. Such induced release of silencing is mostly transient, and rapid restoration of the silent state occurs without the involvement of factors known to be required for silencing initiation. Thus, our results reveal new regulatory aspects of transcriptional repression in constitutive heterochromatin and open up possibilities to identify the molecular mechanisms involved

DETORQUEO, QUIRKY, and ZERZAUST Represent Novel Components Involved in Organ Development Mediated by the Receptor-Like Kinase STRUBBELIG in Arabidopsis thaliana

Intercellular signaling plays an important role in controlling cellular behavior in apical meristems and developing organs in plants. One prominent example in Arabidopsis is the regulation of floral organ shape, ovule integument morphogenesis, the cell division plane, and root hair patterning by the leucine-rich repeat receptor-like kinase STRUBBELIG (SUB). Interestingly, kinase activity of SUB is not essential for its in vivo function, indicating that SUB may be an atypical or inactive receptor-like kinase. Since little is known about signaling by atypical receptor-like kinases, we used forward genetics to identify genes that potentially function in SUB-dependent processes and found recessive mutations in three genes that result in a sub-like phenotype. Plants with a defect in DETORQEO (DOQ), QUIRKY (QKY), and ZERZAUST (ZET) show corresponding defects in outer integument development, floral organ shape, and stem twisting. The mutants also show sub-like cellular defects in the floral meristem and in root hair patterning. Thus, SUB, DOQ, QKY, and ZET define the STRUBBELIG-LIKE MUTANT (SLM) class of genes. Molecular cloning of QKY identified a putative transmembrane protein carrying four C2 domains, suggesting that QKY may function in membrane trafficking in a Ca2+-dependent fashion. Morphological analysis of single and all pair-wise double-mutant combinations indicated that SLM genes have overlapping, but also distinct, functions in plant organogenesis. This notion was supported by a systematic comparison of whole-genome transcript profiles during floral development, which molecularly defined common and distinct sets of affected processes in slm mutants. Further analysis indicated that many SLM-responsive genes have functions in cell wall biology, hormone signaling, and various stress responses. Taken together, our data suggest that DOQ, QKY, and ZET contribute to SUB-dependent organogenesis and shed light on the mechanisms, which are dependent on signaling through the atypical receptor-like kinase SUB