Waraporn Tongprasit

Analysis of Transcription Factor HY5 Genomic Binding Sites Revealed Its Hierarchical Role in Light Regulation of Development

The transcription factor LONG HYPOCOTYL5 (HY5) acts downstream of multiple families of the photoreceptors and promotes photomorphogenesis. Although it is well accepted that HY5 acts to regulate target gene expression, in vivo binding of HY5 to any of its target gene promoters has yet to be demonstrated. Here, we used a chromatin immunoprecipitation procedure to verify suspected in vivo HY5 binding sites. We demonstrated that in vivo association of HY5 with promoter targets is not altered under distinct light qualities or during light-to-dark transition. Coupled with DNA chip hybridization using a high-density 60-nucleotide oligomer microarray that contains one probe for every 500 nucleotides over the entire Arabidopsis thaliana genome, we mapped genome-wide in vivo HY5 binding sites. This analysis showed that HY5 binds preferentially to promoter regions in vivo and revealed >3000 chromosomal sites as putative HY5 binding targets. HY5 binding targets tend to be enriched in the early light-responsive genes and transcription factor genes. Our data thus support a model in which HY5 is a high hierarchical regulator of the transcriptional cascades for photomorphogenesis.

High-Resolution Mapping of Epigenetic Modifications of the Rice Genome Uncovers Interplay between DNA Methylation, Histone Methylation, and Gene Expression

We present high-resolution maps of DNA methylation and H3K4 di- and trimethylation of two entire chromosomes and two fully sequenced centromeres in rice (Oryza sativa) shoots and cultured cells. This analysis reveals combinatorial interactions between these epigenetic modifications and chromatin structure and gene expression. Cytologically densely stained heterochromatin had less H3K4me2 and H3K4me3 and more methylated DNA than the less densely stained euchromatin, whereas centromeres had a unique epigenetic composition. Most transposable elements had highly methylated DNA but no H3K4 methylation, whereas more than half of protein-coding genes had both methylated DNA and di- and/or trimethylated H3K4. Methylation of DNA but not H3K4 was correlated with suppressed transcription. By contrast, when both DNA and H3K4 were methylated, transcription was only slightly reduced. Transcriptional activity was positively correlated with the ratio of H3K4me3/H3K4me2: genes with predominantly H3K4me3 were actively transcribed, whereas genes with predominantly H3K4me2 were transcribed at moderate levels. More protein-coding genes contained all three modifications, and more transposons contained DNA methylation in shoots than cultured cells. Differential epigenetic modifications correlated to tissue-specific expression between shoots and cultured cells. Collectively, this study provides insights into the rice epigenomes and their effect on gene expression and plant development.

Genome-wide transcription analyses in rice using tiling microarrays.

Sequencing and computational annotation revealed several features, including high gene numbers, unusual composition of the predicted genes and a large number of genes lacking homology to known genes, that distinguish the rice (Oryza sativa) genome from that of other fully sequenced model species. We report here a full-genome transcription analysis of the indica rice subspecies using high-density oligonucleotide tiling microarrays. Our results provided expression data support for the existence of 35,970 (81.9%) annotated gene models and identified 5,464 unique transcribed intergenic regions that share similar compositional properties with the annotated exons and have significant homology to other plant proteins. Elucidating and mapping of all transcribed regions revealed an association between global transcription and cytological chromosome features, and an overall similarity of transcriptional activity between duplicated segments of the genome. Collectively, our results provide the first whole-genome transcription map useful for further understanding the rice genome.

A pilot study of transcription unit analysis in rice using oligonucleotide tiling-path microarray.

As the international efforts to sequence the rice genome are completed, an immediate challenge and opportunity is to comprehensively and accurately define all transcription units in the rice genome. Here we describe a strategy of using high-density oligonucleotide tiling-path microarrays to map transcription of the japonica rice genome. In a pilot experiment to test this approach, one array representing the reverse strand of the last 11.2 Mb sequence of chromosome 10 was analyzed in detail based on a mathematical model developed in this study. Analysis of the array data detected 77% of the reference gene models in a mixture of four RNA populations. Moreover, significant transcriptional activities were found in many of the previously annotated intergenic regions. These preliminary results demonstrate the utility of genome tiling microarrays in evaluating annotated rice gene models and in identifying novel transcription units that will facilitate rice genome annotation.

Transcriptional analysis of highly syntenic regions between Medicago truncatula and Glycine max using tiling microarrays

BackgroundLegumes are the third largest family of flowering plants and are unique among crop species in their ability to fix atmospheric nitrogen. As a result of recent genome sequencing efforts, legumes are now one of a few plant families with extensive genomic and transcriptomic data available in multiple species. The unprecedented complexity and impending completeness of these data create opportunities for new approaches to discovery.ResultsWe report here a transcriptional analysis in six different organ types of syntenic regions totaling approximately 1 Mb between the legume plants barrel medic (Medicago truncatula) and soybean (Glycine max) using oligonucleotide tiling microarrays. This analysis detected transcription of over 80% of the predicted genes in both species. We also identified 499 and 660 transcriptionally active regions from barrel medic and soybean, respectively, over half of which locate outside of the predicted exons. We used the tiling array data to detect differential gene expression in the six examined organ types and found several genes that are preferentially expressed in the nodule. Further investigation revealed that some collinear genes exhibit different expression patterns between the two species.ConclusionThese results demonstrate the utility of genome tiling microarrays in generating transcriptomic data to complement computational annotation of the newly available legume genome sequences. The tiling microarray data was further used to quantify gene expression levels in multiple organ types of two related legume species. Further development of this method should provide a new approach to comparative genomics aimed at elucidating genome organization and transcriptional regulation.

In-Depth Query of Large Genomes Using Tiling Arrays

Identification of the transcribed regions in the newly sequenced genomes is one of the major challenges of postgenomic biology. Among different alternatives for empirical transcriptome mapping, whole-genome tiling array experiment emerged as the most comprehensive and unbiased approach. This relatively new method uses high-density oligonucleotide arrays with probes chosen uniformly from both strands of the entire genomes including all genic and intergenic regions. By hybridizing the arrays with tissue specific or pooled RNA samples, a genome-wide picture of transcription can be derived. This chapter discusses computational tools and techniques necessary to successfully conduct genome tiling array experiments.

Analysis of oligo hybridization properties by high-resolution tiling microarrays in rice

Rice genome sequencing and computational annotation provide a static map for understanding this model of Gramineae species. With the development of in situ oligonucleotide synthesis technology, tiling-path microarrays have become a dynamic and efficient way for monitoring large-scale transcriptional activities and detecting novel transcribed elements missed by software. Unlike conventional cDNA or oligonucleotide arrays, tiling-path platforms employ the full extent of oligos covering given genomic regions, and thus offer excellent experimental conditions in which to assay the properties of oligos in terms of their specificity and efficiency of hybridization to their corresponding targets. Here, we report a tiling-path microarray analysis of a 1-Mb region (10 to 11 Mb) in japonica rice chromosome 10, which was tiled by a 36-mer oligo set at a resolution of 5 bp. Our analysis focused on three major factors of oligo hybridization properties, including GC content, melting temperature (Tm), and the repetitiveness of oligo sequences.