Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Genome-wide analysis of SPL7 regulated genes in Arabidopsis

ABSTRACT: To study the genes regulated by transcription factor SQUAMOSA PROMOTER BINDING PROTEIN-LIKE7 (SPL7) in Arabidopsis thaliana, we conducted chromatin immunoprecipitation-based sequencing (ChIP-seq) in 35S:FALG-SPL7 transgenic plant and spl7 mutant, and genome-wide sequencing-based transcript profiling of Arabidopsis thaliana wild-type plants and of spl7 mutant under MS medium and MS supplement with 5µM CuSO4. These experiments led to the identification of genes that are direct target of SPL7 and genes differentially expressed in an SPL7-dependent or Cu-dependent manner. This study provides a framework for the identification of SPL7 regulated genes towards characterization of SPL7 in copper homeostasis. ChIP-Seq: Chromatin isolation was performed with 7-day-old whole seedlings of spl7 mutant (negative control) and 35S:FALG-SPL7 transgene (sample) grown on standard MS medium under continuous white light according to Bowler et al. (2004). The resuspended chromatin pellet was sonicated at 4°C 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 with FLAG antibody (Sigma) according to the Affymetrix Chromatin Immunoprecipitation Assay Protocol Rev.3. Reverse cross-linked DNA was purified and used for sequencing libraries construction following the manufacture’s protocol (Illumina). RNA-Seq: DNase I-treated total RNA was prepared from 7-day-old whole seedlings of wild type (Col-0) and spl7 mutant grown on standard MS medium and MS supplement with 5µM CuSO4 under continuous white light (WL). First- and second-strand cDNA were generated using SuperScript II and random hexamer primers. Double-stranded cDNA was fragmented by nebulization and used for mRNA library construction following the manufacturer’s protocol (Illumina). Tophat was used to map the sequence reads to the Arabidopsis genome and only uniquely mapped reads were used in subsequent analyses. mRNA profiles of 7-day-old whole seedlings of wild type (Col-0) or spl7 mutant grown on standard MS medium and MS supplement with 5µM CuSO4 under continuous white light (WL) were generated by deep sequencing using illumine HiSeq2000.

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: xin zhao

PROVIDER: E-GEOD-45213 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

MicroRNA408 is critical for the HY5-SPL7 gene network that mediates the coordinated response to light and copper.

Zhang Huiyong H Zhao Xin X Li Jigang J Cai Huaqing H Deng Xing Wang XW Li Lei L

The Plant cell 20141216 12

Light and copper are important environmental determinants of plant growth and development. Despite the wealth of knowledge on both light and copper signaling, the molecular mechanisms that integrate the two pathways remain poorly understood. Here, we use Arabidopsis thaliana to demonstrate an interaction between SQUAMOSA PROMOTER BINDING PROTEIN-LIKE7 (SPL7) and ELONGATED HYPOCOTYL5 (HY5), which mediate copper and light signaling, respectively. Through whole-genome chromatin immunoprecipitation ...[more]

PMID: 25516599

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Project description:FAR-RED ELONGATED HYPOCOTYL 3 (FHY3) and its homolog FAR-RED IMPAIRED RESPONSE 1 (FAR1) are two transposase-derived transcription factors initially identified as the key components in phytochrome A signaling and recently shown to function in the circadian clock. However, whether FHY3 and FAR1 are involved in other processes of plant development remains largely unknown. Here, we explored chromatin immunoprecipitation-based sequencing (ChIP-seq) analysis to identify 1745 and 1171 FHY3 direct binding target genes in darkness and far-red light conditions, respectively in the Arabidopsis thaliana genome. This analysis revealed that FHY3 preferentially binds to the gene promoters through the previously identified typical FHY3/FAR1 binding motif. Interestingly, FHY3 also binds to two novel motifs in the 178-bp repeats of the Arabidopsis centromere regions in vivo. Comparison between the ChIP-seq and microarray data indicates that FHY3 regulates the expression of 196 and 85 genes in dark and far-red respectively by directly binding to their promoters. FHY3 also co-regulates a number of common target genes with PHYTOCHROME INTERACTING FACTOR 3-LIKE 5 (PIL5) and ELONGATED HYPOCOTYL 5 (HY5). Moreover, our genome-wide identification of FHY3 direct target genes ultimately led to the discovery and validation of a new role of FHY3 in controlling chloroplast development, by directly activating the expression of ACCUMULATION AND REPLICATION OF CHLOROPLASTS5 (ARC5), a key gene regulating chloroplast constriction and division. Taken together, our data suggest that FHY3 is involved in regulating multiple facets of plant development, thus providing new insights into the functions of this type of transposase-derived transcription factors. To determine the global in vivo binding sites of FHY3, we performed ChIP-seq analysis using 35S:3FLAG-FHY3-3HA fhy3-4 transgenic lines in which the 3FLAG-FHY3-3HA fusion proteins could largely rescue the long hypocotyl phenotype of the fhy3-4 mutants The seedlings were grown in D or continuous FR light conditions for 4 days, and then chromatin fragments were prepared using the commercially monoclonal anti-FLAG antibodies. We then generated two DNA libraries, one for D and one for FR -grown samples, which were then subjected to ultra-high throughput Solexa (Illumina) sequencing

Dataset Information

Genome-wide analysis of SPL7 regulated genes in Arabidopsis

Publications

MicroRNA408 is critical for the HY5-SPL7 gene network that mediates the coordinated response to light and copper.

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