Unknown,Transcriptomics,Genomics,Proteomics

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Identification of BABY BOOM downstream targets

ABSTRACT: A glucocorticoid-regulated BBM protein (35S:BBM-GR) was used in combination with microarray analysis to identify genes directly activated by BBM. We employed the system described by (Lloyd et al., 1994) in which dexamethasone (DEX) and cycloheximide (CHX) are applied together to respectively, induce nuclear localization of the BBM-GR protein and prevent translation of the primary targets mRNAs. In this way it is possible to identify direct targets of a transcriptional activator by comparing gene expression profiles between DEX+CHX-treated transgenic and wild-type tissues. The ability of the 35S:BBM GR construct to induce somatic embryogenesis in Arabidopsis seedlings was determined by phenotypic observation of 35S:BBM GR seeds germinated and grown in the presence of 10 ÂµM dexamethasone (DEX). As in 35S:BBM seedlings, we observed somatic embryo formation on the cotyledons, first leaves and shoot meristem of DEX-treated 35S:BBM GR seedlings. We identified a set of 20 genes (including BBM itself) and our analysis indicates that BBM directly activates a signaling pathway comprising transcription factors and other signaling molecules, but which does not initially include genes known to induce somatic embryogenesis, such as LEC1, LEC2 or WUS. The functions of the BBM target genes are unknown, however a number of them have recently been identified in microarray screens for meristem-expressed genes. The identification of BBM-interacting partners and downstream targets provides new tools for unraveling pathways related to plant cell growth and organogenesis. Keywords: transcriptional activation To identify candidate BBM targets, we treated 4 day old 35S:BBM-GR seedlings for 8 hours with 10 ÂµM DEX in the presence of 10 ÂµM CHX and compared the mRNA population from these seedlings using Arabidopsis Operon microarrays (http://www.ag.arizona.edu/microarray/) to that of 4 day old wild-type seedlings treated in the same way. The seed batches used in this analysis showed 100% EFS when grown continuously on media with 10 ÂµM DEX. Four day old seedlings were chosen as the experimental material because they are highly responsive to BBM GR activation and provide sufficient RNA for the microarray experiments. The biological reproducibility of the data was monitored using two independent single locus 35S:BBM GR lines. The technical reproducibility of the data was monitored using two independently DEX + CHX-treated samples from each transgenic line. Dye effects were monitored in a dye-swap experiment using one of the four RNA samples from the two biological replicates

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Hiroyuki Fukuoka

PROVIDER: E-GEOD-3399 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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BABY BOOM target genes provide diverse entry points into cell proliferation and cell growth pathways.

Passarinho Paul P Ketelaar Tijs T Xing Meiqing M van Arkel Jeroen J Maliepaard Chris C Hendriks Mieke Weemen MW Joosen Ronny R Lammers Michiel M Herdies Lydia L den Boer Bart B van der Geest Lonneke L Boutilier Kim K

Plant molecular biology 20080729 3

Ectopic expression of the Brassica napus BABY BOOM (BBM) AP2/ERF transcription factor is sufficient to induce spontaneous cell proliferation leading primarily to somatic embryogenesis, but also to organogenesis and callus formation. We used DNA microarray analysis in combination with a post-translationally regulated BBM:GR protein and cycloheximide to identify target genes that are directly activated by BBM expression in Arabidopsis seedlings. We show that BBM activated the expression of a large ...[more]

PMID: 18663586

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Project description:Cell-cell communication is critical for stem cell maintenance. Shoot apical meristem (SAM) located at the shoot tip harbors stem cells within the central zone (CZ). Their progeny differentiate in the adjacent peripheral zone (PZ). WUSCHEL (WUS) is a homeodomain transcription factor produced in a few cells of the organizing center (OC), located beneath the CZ. It has been shown to specify stem cell fate and also activate CLAVATA3 (CLV3) expression in cells of the CZ. CLV3 is a secreted peptide that activates a membrane bound receptor kinase-CLAVATA1 to restrict WUS transcription to the OC. It has been hypothesized that WUS activates CLV3 expression and stem cell fate in adjacent cells of the CZ by activating a non-cell autonomous signal. Contrary to this hypothesis, here we show that the WUS protein after being synthesized in cells of the OC, migrates into the superficial cell layers of the CZ where it activates CLV3 transcription by binding to its promoter elements. WUS also migrates laterally into the PZ to repress the expression of differentiation promoting transcription factors by binding to their regulatory regions. Migration of a stem cell inducing transcription factor into adjacent cells to activate a negative regulator, whereby restricting its own accumulation is unique to plant stem cell niches. While stem cell promoting transcription factor directly repressing differentiation promoting transcription factors to prevent premature differentiation of stem cell progenitors is conserved among diverse stem cell niches. A dexamethasone inducible version of WUSCHEL was used to identify its direct tragets. To analyze WUS-regulated gene expression programs at a higher spatial resolution, we introduced a dexamethasone (Dex)-inducible form of WUS consisting of the WUS protein-coding region fused to sequences coding for the ligand-binding domain of the rat glucocorticoid receptor (GR), expressed under a ubiquitous promoter (35S::WUS-GR) into apetala1-1;cauliflower1-1 (ap1-1;cal1-1) double mutant background. 35S::WUS-GR; ap1-1;cal1-1 SAMs were either treated with 10μM Dex or mock solution for four hours and RNA samples were hybridized to Arabidopsis ATH1 gene Chip (Affymmetrix). To identify putative genes that are directly regulated by WUS, in independent experiments, SAMs were simultaneously treated with 10μM Dex and 10μM Cycloheximide (Cyc), protein synthesis inhibitor and Cyc alone for four hours. A comparison of Dex-treated samples with mock identified 641 genes as differentially expressed [DEGs] (≥/≤2 fold; p < 0.01) and comparison of Dex+Cyc with Cyc identified 457 genes as DEGs (≥/≤2 fold; p < 0.01).

Dataset Information

Identification of BABY BOOM downstream targets

Publications

BABY BOOM target genes provide diverse entry points into cell proliferation and cell growth pathways.

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