Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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BABY BOOM (BBM) ChIP-seq in Arabidopsis somatic embryo tissue

ABSTRACT: After fertilization, a plant's life relies on progression through embryogenesis and maintenance of the stem cell niches from which all post-embryonic organs arise. BABY BOOM (BBM) and other members of the AINTEGUMENTA-LIKE (AIL)/PLETHORA (PLT) clade of the AP2-type transcription factor family play important roles controlling these processes in Arabidopsis thaliana (Arabidopsis). Development of the plt2/bbm double mutant is blocked at during early embryogenesis (Galinha et al., 2007), and combinations of bbm with plt1 and plt3 lead to short roots as a result of meristem differentiation. In contrast, overexpression of BBM in Arabidopsis seedlings induces the formation of somatic embryos on cotyledons and leaves (Boutilier, 2002), showing that BBM is a key regulator of cell identity and proliferation. Although the functions of BBM and other AIL genes have been well described, the molecular mode of action of these transcription factors has not been well examined (reviewed in Horstman et al., 2013). Our previous study provided the first insight into BBM molecular function by identifying BBM targets through a microarray-based approach (Passarinho, 2008), but only a few BBM targets have been functionally characterized. To obtain a better understanding of BBM function, we identified direct BBM targets using a chromatin immunoprecipitation (ChIP) combined with massively-parallel DNA sequencing (ChIP-seq) approach. Somatic embryo tissue was used for the ChIP-seq experiments with the native BBM promoter (pBBM::BBM-YFP), with a line expressing nuclear-localized GFP from the same BBM promoter (pBBM::NLS-GFP) as a negative control. Whole, embryogenic seedlings of the 35S::BBM-GFP line were used for the 35S::BBM-GFP ChIP-seq experiments, with 35S::BBM embryogenic seedlings serving as a negative control. Both experiments were performed once, making a total of 4 samples.

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Kim Boutilier

PROVIDER: E-GEOD-52400 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description: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

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BABY BOOM (BBM) ChIP-seq in Arabidopsis somatic embryo tissue

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