ARTADE2DB: Improved Statistical Inferences for Arabidopsis Gene Functions and Structure Predictions by Dynamic Structure-Based Dynamic Expression (DSDE) Analyses

Recent advances in technologies for observing high-resolution genomic activities, such as whole-genome tiling arrays and high-throughput sequencers, provide detailed information for understanding genome functions. However, the functions of 50% of known Arabidopsis thaliana genes remain unknown or are annotated only on the basis of static analyses such as protein motifs or similarities. In this paper, we describe dynamic structure-based dynamic expression (DSDE) analysis, which sequentially predicts both structural and functional features of transcripts. We show that DSDE analysis inferred gene functions 12% more precisely than static structure-based dynamic expression (SSDE) analysis or conventional co-expression analysis based on previously determined gene structures of A. thaliana. This result suggests that more precise structural information than the fixed conventional annotated structures is crucial for co-expression analysis in systems biology of transcriptional regulation and dynamics. Our DSDE method, ARabidopsis Tiling-Array-based Detection of Exons version 2 and over-representation analysis (ARTADE2-ORA), precisely predicts each gene structure by combining two statistical analyses: a probe-wise co-expression analysis of multiple transcriptome measurements and a Markov model analysis of genome sequences. ARTADE2-ORA successfully identified the true functions of about 90% of functionally annotated genes, inferred the functions of 98% of functionally unknown genes and predicted 1,489 new gene structures and functions. We developed a database ARTADE2DB that integrates not only the information predicted by ARTADE2-ORA but also annotations and other functional information, such as phenotypes and literature citations, and is expected to contribute to the study of the functional genomics of A. thaliana. URL: http://artade.org

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

Sm-like protein-mediated RNA metabolism is required for heat stress tolerance in Arabidopsis.

Sm-like proteins play multiple functions in RNA metabolism, which is essential for biological processes such as stress responses in eukaryotes. The Arabidopsis thaliana sad1 mutant has a mutation of sm-like protein 5 (LSM5) and shows impaired drought and salt stress tolerances. The lsm5/sad1 mutant also showed hypersensitivity to heat stress. GFP-fused LSM5/SAD1 was localized in the nucleus under optimal growth conditions. After heat stress treatment, GFP-fused LSM5/SAD1 fluorescence was also observed as small cytoplasmic dots, in addition to nuclear localization. Whole genome transcriptome analysis revealed that many genes in Arabidopsis were drastically changed in response to heat stress. More heat-responsive genes were highly expressed in lsm5/sad1 mutant at both 2 h and 6 h after heat stress treatment. Additionally, intron-retained and capped transcripts accumulated in the lsm5/sad1 mutant after heat stress treatment. In this study, we also identified non-Arabidopsis Genome Initiative (AGI) transcripts that were expressed from unannotated regions. Most of these transcripts were antisense transcripts, and many capped non-AGI transcripts accumulated in the lsm5/sad1 mutant during heat stress treatment. These results indicated that LSM5/SAD1 functions to degrade aberrant transcripts through appropriate mRNA splicing and decapping, and precise RNA metabolic machinery is required for heat stress tolerance

Genome-wide analysis of endogenous abscisic acid-mediated transcription in dry and imbibed seeds of Arabidopsis using tiling arrays

SUMMARY The phytohormone abscisic acid (ABA) plays important roles in the induction and maintenance of seed dormancy. Although application of exogenous ABA inhibits germination, the effects of exogenous ABA on ABA-mediated gene transcription differ from those of endogenous ABA. To understand how endogenous ABA regulates the transcriptomes in seeds, we performed comprehensive expression analyses using wholegenome Affymetrix tiling arrays in two ABA metabolism mutants -an ABA-deficient mutant (aba2) and an ABA over-accumulation mutant (cyp707a1a2a3 triple mutant). Hierarchical clustering and principal components analyses showed that differences in endogenous ABA levels do not influence global expression of stored mRNA in dry seeds. However, the transcriptome after seed imbibition was related to endogenous ABA levels in both types of mutant. Endogenous ABA-regulated genes expressed in imbibed seeds included those encoding key ABA signaling factors and gibberellin-related components. In addition, cohorts of ABA-upregulated genes partially resembled those of dormant genes, whereas ABA-downregulated genes were partially overlapped with after-ripening-regulated genes. Bioinformatic analyses revealed that 6105 novel genes [non-Aradopsis Genome Initiative (AGI) transcriptional units (TUs)] were expressed from unannotated regions. Interestingly, approximately 97% of non-AGI TUs possibly encoded hypothetical non-protein-coding RNAs, including a large number of antisense RNAs. In dry and imbibed seeds, global expression profiles of non-AGI TUs were similar to those of AGI genes. For both non-AGI TUs and AGI code genes, we identified those that were regulated differently in embryo and endosperm tissues. Our results suggest that transcription in Arabidopsis seeds is more complex and dynamic than previously thought

A Stress-Activated Transposon in Arabidopsis Induces Transgenerational Abscisic Acid Insensitivity

Transposable elements (TEs), or transposons, play an important role in adaptation. TE insertion can affect host gene function and provides a mechanism for rapid increases in genetic diversity, particularly because many TEs respond to environmental stress. In the current study, we show that the transposition of a heat-activated retrotransposon, ONSEN, generated a mutation in an abscisic acid (ABA) responsive gene, resulting in an ABA-insensitive phenotype in Arabidopsis, suggesting stress tolerance. Our results provide direct evidence that a transposon activated by environmental stress could alter the genome in a potentially positive manner. Furthermore, the ABA-insensitive phenotype was inherited when the transcription was disrupted by an ONSEN insertion, whereas ABA sensitivity was recovered when the effects of ONSEN were masked by IBM2. These results suggest that epigenetic mechanisms in host plants typically buffered the effect of a new insertion, but could selectively "turn on" TEs when stressed

Transduction of RNA-directed DNA methylation signals to repressive histone marks in Arabidopsis thaliana

RNA-directed modification of histones is essential for the maintenance of heterochromatin in higher eukaryotes. In plants, cytosine methylation is an additional factor regulating inactive chromatin, but the mechanisms regulating the coexistence of cytosine methylation and repressive histone modification remain obscure. In this study, we analysed the mechanism of gene silencing mediated by MORPHEUS' MOLECULE1 (MOM1) of Arabidopsis thaliana. Transcript profiling revealed that the majority of up-regulated loci in mom1 carry sequences related to transposons and homologous to the 24-nt siRNAs accumulated in wild-type plants that are the hallmarks of RNA-directed DNA methylation (RdDM). Analysis of a single-copy gene, SUPPRESSOR OF drm1 drm2 cmt3 (SDC), revealed that mom1 activates SDC with concomitant reduction of di-methylated histone H3 lysine 9 (H3K9me2) at the tandem repeats in the promoter region without changes in siRNA accumulation and cytosine methylation. The reduction of H3K9me2 is not observed in regions flanking the tandem repeats. The results suggest that MOM1 transduces RdDM signals to repressive histone modification in the core region of RdDM