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Genome-wide Identification of SHR Direct Targets Using a Custom Agilent Promoter Array

ABSTRACT: SHR is a key regulator of stem cell renewal and radial patterning in the Arabidopsis root. In previous studies we showed that the SHR is a transcriptional regulator and it regulates gene transcription directly. To fully understand the SHR developmental pathway, we aim to identify its direct target at the genome scale using the ChIP-on-chip method. To this end, we have designed a promoter microarray for Arabidopsis, which contains probes that tile the intergenic regions as well as the first intron and the 3' UTR for all annotated genes including miRNA genes. Chromatin immunoprecipitation (ChIP) was performed using an anti-GFP antibody on the root of a transgenic line expressing under the SHR promoter a functional fusion protein between SHR and GFP. After labeling with Cy5 and Cy3 respectively, DNA recoverd from the ChIP and mock experiments was hybridized to the same microarray. The array was scanned using an Agilent scanner and the signal intensity for each channel was retrieved and normalized by the Agilent feature extraction software.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE21338 | GEO | 2010/07/19

SECONDARY ACCESSION(S): PRJNA126153

REPOSITORIES: GEO

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Genome-wide Identification of SHR Direct Targets Using a Custom Agilent Promoter Array

Project description:SHR is a key regulator of stem cell renewal and radial patterning in the Arabidopsis root. In previous studies we showed that the SHR is a transcriptional regulator and it regulates gene transcription directly. To fully understand the SHR developmental pathway, we aim to identify its direct target at the genome scale using the ChIP-on-chip method. To this end, we have designed a promoter microarray for Arabidopsis, which contains probes that tile the intergenic regions as well as the first intron and the 3' UTR for all annotated genes including miRNA genes. Chromatin immunoprecipitation (ChIP) was performed using an anti-GFP antibody on the root of a transgenic line expressing under the SHR promoter a functional fusion protein between SHR and GFP. After labeling with Cy5 and Cy3 respectively, DNA recoverd from the ChIP and mock experiments was hybridized to the same microarray. The array was scanned using an Agilent scanner and the signal intensity for each channel was retrieved and normalized by the Agilent feature extraction software. Two bilogical replicate ChIP-on-chip experiments were performed; Each array is hybridized with both ChIP and control DNA samples, which are labeled with Cy5 and Cy3 dye respectively;

2010-07-18 | E-GEOD-21338 | biostudies-arrayexpress

The SHR/SCR pathway directly activates genes involved in asymmetric cell division in the Arabidopsis root

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The SHR/SCR pathway directly activates genes involved in asymmetric cell division in the Arabidopsis root

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Project description:Transcriptional profiling in the shoot after SHR induction by DEX. We used Affymetrix ATH1 microarrays to identify new target genes of SHR and the effect of SHR on growth and development of the Arabidopsis shoot system by global transcriptome analysis.

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Genome-wide identification of SCARECROW (SCR) direct targets using a custom Agilent promoter array

Project description:ChIP-chip analysis was used to identify direct targets of the transcription factor SCR SCARECROW (SCR) is a key regulatory of radial patterning and stem cell renewal in the primary root. We have shown in previous studies that SCR is a direct transcriptional regulator in Arabidopsis thaliana. With the aim of understanding the molecular pathway acting downstream of SCR in primary root development, we identified SCR direct targets at the genome-wide level in primary roots.To do this we designed a promoter microarray (chip) for Arabidopsis containing probes tiling the intergenic regions, first intron, and 3’UTR of all annotated genes. We performed Chromatin Immunoprecipitation (ChIP) using an antibody to Green Fluorescent Protein (GFP) on roots of transgenic plants expressing a functional fusion protein of SCR and GFP that is driven by the SCR promoter. Our control was performing ChIP in parallel using the same anti-GFP antibody on roots of Columbia-O wild-type plants. DNA recovered from the SCR and wild-type ChIP experiments with Cy5 and Cy3 dye, respectively, and then hybridized to the same microarray. The array was scanned using an Agilent scanner and the signal intensity for each channel was retrieved and normalized by Agilent feature extraction software. Two biological replicates of this ChIP-chip experiment were performed.

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Project description:Hydrogen peroxide (H2O2) is a potent signaling molecule influencing various aspects of plant growth and development. Its limited lifetime and specific production sites in the plant cell necessitate the existence of specialized mechanisms that relay H2O2-encoded information. To discover such mechanisms, we focused on peroxisomal H2O2 production triggered by enhanced photorespiration in Arabidopsis mutants lacking catalase activity (cat2-2), and looked for second-site mutations that attenuate the negative effects (Fv'/Fm' decline and lesion formation) of H2O2 build up. A mutation residing in the GRAS family transcriptional regulator SHORT-ROOT (SHR) was found to underlie the increased performance of cat2-2 knock-outs under photorespiratory stress. In contrast to shr, introduction of the scr mutation in cat2-2 background did not improve the photorespiratory performance of plants lacking peroxisomal catalase. The absence of SHR negatively affected the activity of the photorespiratory enzymes glycolate oxidase and catalase, which was accompanied with elevated glycolate content and inability to accumulate glycine under conditions promoting photorespiration. The transcriptome signature of cat2-2 shr-6 double mutants exposed to photorespiratory stress lacked jasmonate-dependent signaling components, otherwise induced in cat2-2. The photorespiratory phenotype of cat2-2 was found to be modulated by exogenous sugars both in the presence and absence of shr. Taken together, these findings highlight a crucial role for SHR in H2O2 signal transduction and stress tolerance.

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Genome-wide analysis of SHORTROOT targets in the Arabidopsis root stele

Project description:Asymmetric cell division (ACD) and positional signals play critical roles in the tissue patterning during multicellular organ development. In the Arabidopsis root meristem, two major phloem cell types arise via ACDs of distinct origins: one for companion cells and the other for proto- and metaphloem sieve elements. The molecular mechanisms underlying each of these processes have been reported, however, how these two are coordinated has remained elusive. Here, we report that SHORTROOT (SHR) is the key coordinator of two ACD processes for phloem development. SHR moving into the endodermis regulates ACD for companion cells by turning on microRNA165/6. ACD that generates phloem sieve elements is mediated by SHR moving into phloem precursors. To find the downstream targets of SHR in the stele, pCRE1::erGFP was introduced into wild type and shr-2 backgrounds to express GFP in the stele cells in the root meristem. We then collected GFP-expressing stele cells from the wild type, shr-2 and pCRE1::SHRΔNLELDV:nlsGFP; shr-2 through Fluorescence Activated Cell Sorter (FACS). RNAs were extracted from the sorted cells of each line and processed for generating labeled probes to be hybridized onto GeneChip Arabidopsis Tiling 1.0R Array (Affymetrix). We then examined the influence of SHR on phloem enriched genes in the tiling array data. In this analysis, we found 224 genes that are down-regulated in shr-2 in comparison to the wild type and then restore expression in pCRE1::SHRΔNLELDV:nlsGFP; shr-2.

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