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

ABSTRACT: 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.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE30104 | GEO | 2011/11/23

SECONDARY ACCESSION(S): PRJNA155185

REPOSITORIES: GEO

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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 3M-bM-^@M-^YUTR 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. Two biological replicates of the ChIP-chip experiment were performed using samples isolated from primary roots. Each Agilent array was hybridized with both SCR ChIP and wild-type control samples differentially labeled with Cy5 and Cy3 dye, respectively.

2011-11-23 | E-GEOD-30104 | biostudies-arrayexpress

Cell-type, developmental stage, and whole root responses to low pH and sulfur deficiency

Project description:This SuperSeries is composed of the following subset Series: GSE30091: Expression analysis of the effect of protoplasting and sorting in roots exposed to low pH GSE30095: Expression analysis of root cell types after treatment with low pH GSE30096: Expression analysis of developmental stages of Arabidopsis roots exposed to low pH GSE30097: Time-course expression analysis of the low pH (pH 4.6) response in Arabidopsis whole roots GSE30098: Expression analysis time-course of Arabidopsis roots to sulfur deficiency GSE30099: Expression analysis of root cell types after treatment with sulfur deficient media GSE30100: Expression analysis of developmental stages of Arabidopsis roots exposed to sulfur deficient media GSE30104: Genome-wide identification of SCARECROW (SCR) direct targets using a custom Agilent promoter array Refer to individual Series

2011-11-23 | E-GEOD-30166 | biostudies-arrayexpress

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

2010-07-19 | GSE15876 | GEO

Cell signalling by microRNA165/6 directs gene dose dependent root cell fate

Project description:A key question in developmental biology is how cells exchange positional information for proper patterning during organ development. In plant roots the radial tissue organization is highly conserved with a central vascular cylinder in which two water conducting cell types, protoxylem and metaxylem, are patterned centripetally. We show that this patterning occurs through crosstalk between the vascular cylinder and the surrounding endodermis mediated by cell-to-cell movement of a transcription factor in one direction and microRNAs in the other. SHORT ROOT, produced in the vascular cylinder, moves into the endodermis to activate SCARECROW. Together these transcription factors activate MIR165a and 166b. Endodermally produced miR165/6 then acts to degrade its target mRNAs encoding class III homeodomain-leucine zipper transcription factors in the endodermis and stele periphery. The resulting differential distribution of target mRNA in the vascular cylinder determines xylem cell types in a dosage dependent manner. To investigate the global regulation of SHR and SCR in the root, direct and indirect target genes of SHR and SCR were cross-compared with selected cell type specific expression profiles in the Arabidopsis root published in Birnbaum et al. (2003), Nawy et al. (2005), Lee et al. (2006), and Brady et al. (2007) [pubmed IDs: 14671301, 15937229, 16581911, 17975066]. Furthermore, the mature endodermis specific transcript profiles were generated using a GFP marker line, E30, and used together with published profiles in this comparison.

2010-04-27 | E-GEOD-16469 | biostudies-arrayexpress

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

Project description:Asymmetric division of cortex/endodermal initials (CEI) in the Arabidopsis root generates two layers of ground tissue and is controlled by a finely orchestrated interplay between the transcription factors, SHORT ROOT (SHR) and SCARECROW (SCR). To understand the dynamics of the SHR/SCR regulatory network we performed microarray time course experiments using inducible versions of SHR and SCR and examined their transcriptional effects specifically in the ground tissue. Keywords: SHR and SCR time course and CEI cell-type analyses We performed a time course analysis (TC data set) of the ground tissue (sorted J0571 enancher trap) after transfer either SHR or SCR inducible plants to Dex containing plates 0, 1, 3, 6 and 12 hours. We sorted for CEI cells using the pCYCD6::GFP marker line. Seedlings were grown for 5 days. SHR and SCR inducible plants were transfer to 1microM Dex MS plates. J0571 and CYCD6 sorted fluorecent cells were harvested for RNA extraction.

2010-07-19 | E-GEOD-15876 | biostudies-arrayexpress

SHORT-ROOT and SCARECROW regulate leaf growth in Arabidopsis by stimulating S-phase progression of the cell cycle

Project description:SHORT-ROOT (SHR) and SCARECROW (SCR) are required for stem cell maintenance in the Arabidopsis thaliana root meristem, ensuring its indeterminate growth. Mutation of SHR and SCR genes results in disorganization of the quiescent center and loss of stem cell activity, resulting in the cessation of root growth. This manuscript reports on the role of SHR and SCR in the development of leaves, which, in contrast to the root, have a determinate growth pattern and lack a persistent stem-cell niche. Our results demonstrate that inhibition of leaf growth in shr and scr mutants is not a secondary effect of the compromised root development, but is caused by a direct effect on cell division in the leaves: a reduced cell division rate and early exit of proliferation phase. Consistent with the observed cell division phenotype, the expression of SHR and SCR genes in leaves is closely associated with cell division activity in most cell types. The increased cell cycle duration is due to a prolonged S-phase duration, which is mediated by up-regulation of cell cycle inhibitors known to restrain the activity of the transcription factor, E2Fa. Therefore, we conclude that, in contrast to their specific role in cortex/endodermis differentiation and stem cell maintenance in the root, SHR and SCR primarily function as general regulators of cell proliferation in leaves.

2010-08-26 | GSE21629 | GEO

SHORT-ROOT and SCARECROW regulate leaf growth in Arabidopsis by stimulating S-phase progression of the cell cycle

2010-08-26 | E-GEOD-21629 | biostudies-arrayexpress

Arabidopsis thaliana

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

| PRJNA155185 | ENA

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.

2010-07-19 | GSE21338 | GEO

New insights on the role of HCN in root hair elongation through single cell proteomics.

Project description:In order to identify the possible protein targets of cyanide in the regulation of root hair, we have carried out a single cell proteomic approach to characterize the protein atlas in wild type root hair cells compared to the cas-c1 mutant cells. Root hair specific cells were obtained from isolated protoplast from root tissues of Arabidopsis wt-pCOBL9:GFP and cas-c1-pCOBL9:GFP lines. To analyze the effect of the CAS-C1 mutation, we isolated 1 x 106 root hair cells by Fluorescence-activated cell sorting (FACS) from three independent replicate of wt-pCOBL9:GFP and cas-c1-pCOBL9:GFP roots. The extracted proteins from each sample were trypsin-digested and the digested peptides were analyzed by liquid chromatography-high resolution mass spectrometry (LC-MS/MS) for protein identification. In wild type samples, we have identified 3829 unique proteins at a false discovery rate below 1% (FDR<1%), that represent almost 10% of the total Arabidopsis proteome. In protein extract of the cas-c1-pCOBL9:GFP roots samples, we have identified 3972 proteins of which 3515 were commons in both cell types, 310 were only identified y wild type and 457 only in cas-c1 mutant (Fig. 5).

2024-01-26 | PXD028991 | Pride

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