Unknown,Transcriptomics,Genomics,Proteomics

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Genome-wide mapping of the HY5-mediated gene networks in Arabidopsis that involve both transcriptional and post-transcriptional regulations: ChIP-Chip

ABSTRACT: LONG HYPOCOTYL 5 (HY5) is a basic leucine zipper transcription factor (TF) that functions downstream of multiple families of photoreceptors. Mutations in the HY5 gene cause a myriad of aberrant phenotypes in Arabidopsis, including elongated hypocotyl, reduced accumulation of pigments, halted chloroplast development in greening hypocotyls, altered root morphology and defective hormonal and stimulus responses. HY5 thus acts as an integrater that links various gene networks to coordinate plant development. Here we report an effort to experimentally map the HY5-mediated gene networks in Arabidopsis by integrating genomic loci occupied by HY5 and HY5-dependent gene expression profiles. Our results indicate HY5 binds to over 9,000 genes, which detectably impact the expression of over 1,100 genes, either positively or negatively. Further, HY5 indirectly regulates many other genes through sub-networks mediated by other regulators. In particular, we show that HY5 regulates eight microRNA (miRNA) genes, which in turn control transcript abundance of specific target genes. Over-expressing the HY5-targeted miR408 resulted in phenotypes that are opposite to hy5 mutants. Together our results revealed both the transcriptional and post-transcriptional components of the HY5-mediated gene networks responsible for the phenotypic complexity in plants. Chromatin isolation was performed with 4-day-old whole seedlings grown under wild type light (WL) or 8hr darkness treatment according to Bowler et al. (2004). The resuspended chromatin pellet was sonicated at 4M-BM-0C with a Diagenode Bioruptor set at high intensity for 10 min (30 s on, 30 s off intervals). The DNA was sheared to an average size of approximately 500 bp. Chromatin was immunoprecipitated, washed, reverse cross-linked, amplified, and hybridized according to the Affymetrix Chromatin Immunoprecipitation Assay Protocol Rev.3. A polyclonal HY5 antibody purified from anti-HY5 rabbit IgG using Sepharose 4B beads coupled with purified recombinant HY5 protein was used. M-bM-^@M-^XFlow-throughM-bM-^@M-^Y IgG devoid of the HY5 antibody was used as a negative control. An aliquot of untreated sonicated chromatin was reverse cross-linked and used as a total input DNA control for ChIP-PCR experiments. Four biological replicates for each experimental condition and two biological replicates for each control condition were hybridized to tiling arrays, resulting in a total of 12 chips.

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Hang He

PROVIDER: E-GEOD-24973 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:LONG HYPOCOTYL 5 (HY5) is a basic leucine zipper transcription factor (TF) that functions downstream of multiple families of photoreceptors. Mutations in the HY5 gene cause a myriad of aberrant phenotypes in Arabidopsis, including elongated hypocotyl, reduced accumulation of pigments, halted chloroplast development in greening hypocotyls, altered root morphology and defective hormonal and stimulus responses. HY5 thus acts as an integrater that links various gene networks to coordinate plant development. Here we report an effort to experimentally map the HY5-mediated gene networks in Arabidopsis by integrating genomic loci occupied by HY5 and HY5-dependent gene expression profiles. Our results indicate HY5 binds to over 9,000 genes, which detectably impact the expression of over 1,100 genes, either positively or negatively. Further, HY5 indirectly regulates many other genes through sub-networks mediated by other regulators. In particular, we show that HY5 regulates eight microRNA (miRNA) genes, which in turn control transcript abundance of specific target genes. Over-expressing the HY5-targeted miR408 resulted in phenotypes that are opposite to hy5 mutants. Together our results revealed both the transcriptional and post-transcriptional components of the HY5-mediated gene networks responsible for the phenotypic complexity in plants. DNase I-treated total RNA was prepared from wild type and hy5-215 seedlings grown under wild type light (WL). The mRNA fraction was extracted from total RNA using Dynabeads oligo(dT)25 (Invitrogen) following the manufacturerM-bM-^@M-^Ys directions. First- and second-strand cDNA were generated usingSuperScript II and random hexamer primers. Double-stranded cDNA was fragmented by nebulization and used for mRNA library construction following the manufacturerM-bM-^@M-^Ys protocol (Illumina). Bowtie (Langmead et al., 2009) was used to map the sequence reads to the Arabidopsis genome and only uniquely mapped reads were used in subsequent analyses.

Project description:Although landscapes of several epigenetic marks are now available for Arabidopsis and rice, such profiles remain static and do not provide information about dynamic changes of plant epigenomes in response to developmental or environmental cues. Here we analyzed the effects of light regulation on four epigenetic histone modifications (H3K9ac, H3K9me3, H3K27ac, H3K27me3). Our genome-wide profiling of H3K9ac and H3K27ac revealed that these modifications are gene-specific. In contrast, we found that H3K9me3 and H3K27me3 target genes, but also intergenic-regions and transposable elements. Specific light conditions affected the number of epigenetically modified regions, as well as the overall correlation strength between the presence of specific epigenetic modifications and transcription. Futhermore, we observed that acetylation is an important contributor to light-regulated genome expression not only through its activating action on HY5 and HYH but also on their downstream targets. We found that dynamic acetylation changes in response to light have a major role in the active regulation of photosynthetic gene expression, while H3K27ac and H3K27me3 are major contributors to light regulation of the gibberellin metabolism. We also identified distinct epigenetic patterns in the epigenome of the photomorphogenic mutant cop1-4, suggesting that COP1 might have a role in the establishment of specific epigenetic modifications. Thus, this work provides a dynamic portrait of the variations in histone modifications in response to the plantM-bM-^@M-^Ys changing light environment and strengthens the concept that epigenetic modifications represent another layer of control for light-regulated genes involved in photomorphogenesis. Arabidopsis ecotype Columbia (Col-0) was the genetic background used for this study. Seeds were stratified for two days at 4M-BM-0C, then germinated and grown for five days at 22M-BM-0C in continuous darkness (D). Six hours before harvest, a number of plates were transferred to continuous white light (D to L). The cop1-4 mutant was also used in this study. Seedlings from the different conditions and genotypes were harvested at the same time of day. Chromatin was immunoprecipitated, washed, reverse crosslinked, amplified and hybridized according to AffymetrixM-bM-^@M-^Y Chromatin Immunoprecipitation Assay Protocol Rev.3. Three M-NM-<l of the following antibodies were used: anti-H3K9ac (Upstate 06-942), anti-H3K9me3 (Upstate 07-442), anti-H3K27ac (Upstate 07-360), anti-H3K27me3 (Upstate 07-449), and anti-H3 (Upstate 07-690). Four biological replicates were hybridized to tiling arrays.

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Genome-wide mapping of the HY5-mediated gene networks in Arabidopsis that involve both transcriptional and post-transcriptional regulations: ChIP-Chip

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