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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.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE24973 | GEO | 2010/12/31

SECONDARY ACCESSION(S): PRJNA133459

REPOSITORIES: GEO

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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: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.

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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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