Project description:Here we report proteins identified after conducting Tandem Affinity Purification (TAP) of the TOPLESS (TPL) corepressor from Arabidopsis. We generated transgenic plants harboring TPL fused to the GS-TAG, "Boosting tandem affinity purification of plant protein complexes" (Van Leene et al., 2008) [1]. Four independent biological replicates of a selected TPL-GS-TAG line were grown simultaneously, crosslinked with formaldehyde, and proteins were isolated from whole plant tissue via TAP. Purified proteins were treated with trypsin, and the peptides were analyzed via mass spectrometry. Datasets are hosted in the MassIVE public repository (reference number: MSV000082477, https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=f16255fb7080426a9fe1926b4d3d5862). The data in this article has not been published elsewhere and is original to this work.
Project description:To identify the direct target genes of PPD2, tandem chromatin affinity purification (TChAP, Verkest et al. (2014)), a variant of chromatin immunoprecipitation (ChIP) in which tandem affinity tags are used instead of epitope tags, followed by sequencing (TChAP-Seq) was performed. An Arabidopsis cell suspension culture overexpressing an HBH-tagged PPD2 was used for the purification of the chromatin bound by PPD2.
Project description:The Arabidopsis VEL proteins VIN3 and VRN5 are accessory proteins of the Polycomb Repressive Complex 2 (PRC2). Their function is most well-understood in the epigenetic PRC2-mediated silencing of the floral repressor gene Flowering Locus C (FLC) during prolonged cold (vernalization). To identify protein interactors of VIN3 and VRN5, also dependent on their C-terminal VEL domains, we used stable Arabidopsis transgenic lines expressing VIN3-GFP WT, VIN3-GFP deltaVEL, VRN5-SYFP2 WT and VRN5-SYFP2 deltaVEL under endogenous promoters to perform native co-immunoprecipitation assays in seedlings vernalized for six weeks at 5 °C. Non-transgenic Col-FRI was used as a negative control.
Project description:The Arabidopsis VEL proteins VIN3 and VRN5 are accessory proteins of the Polycomb Repressive Complex 2 (PRC2). Their function is most well-understood in the epigenetic PRC2-mediated silencing of the floral repressor gene Flowering Locus C (FLC) during prolonged cold (vernalization). To identify protein interactors of VIN3 and VIN3 chimeric mutant protein carrying VRN5VEL instead of VIN3VEL, we used stable Arabidopsis transgenic lines expressing VIN3-GFP WT and VIN3-GFP VRN5VEL under endogenous promoters to perform native co-immunoprecipitation assays in seedlings vernalized for six weeks at 5 °C. Non-transgenic Col-FRI was used as a negative control.
Project description:To identify the genomic binding sites of FRS12, Tandem Chromatin Affinity Purification â Seq (TChAP-Seq) was performed on 7-d-old Pro35S:FRS12-HBH and Pro35S:NLS-GFP-HBH expressing Arabidopsis thaliana PSB-D cells. Cultures were transferred to long days (16:8) conditions two weeks before harvesting at night time (NT) at zeitgeber time (ZT) 20 (time of lights on usually defines zeitgeber time zero). Chromatin was isolated from formaldehyde-treated cell cultures following two affinity purification steps; first by IMAC using a Ni-NTA Superflow resin (Qiagen), then by a Biotin binding step using a Streptavidin Sepharose resin (GE Healthcare). Finally, protein-DNA bound fragments were decrosslinked, deproteinized and purified using QIAquick PCR Purification Kit (Qiagen). Pro35S:FRS12-HBH samples were carried in two replicates whereas background Pro35S:NLS-GFP-HBH sample was prepared in a single replicate. The TChAP DNA samples were processed by first preparing a TruSeq ChIPseq library (Illumina) and then sequenced using Illumina HiSeq 2000 at 50bp single read at an average depth of 15 million reads.
Project description:The POLYCOMB proteins are required for maintenance of silent chromatin states mediated by H3K27 trimethylation in animals, but POLYCOMB homologues are not found in plant genomes. Using DamID-chip, we found that the Arabidopsis chromodomain-containing protein LHP1 localizes to chromatin associated with H3K27me3 genome-wide. Furthermore, the LHP1 chromodomain binds H3K27me3 with high affinity. These results suggest that LHP1 shares similar functions with POLYCOMB. Keywords: DamID-Chip
Project description:Numerous gene expression datasets from diverse tissue samples from the plant variety Arabidopsis thaliana have been already deposited in the public domain. There have been several attempts to do large scale meta-analyses of all of these datasets. Most of these analyses summarize pairwise gene expression relationships using correlation, or identify differentially expressed genes in two conditions. We propose here a new large scale meta-analysis of all of the publicly available Arabidopsis datasets to identify Boolean logical relationships between genes. Boolean logic is a branch of mathematics that deals with two possible values. In the context of gene expression datasets we use qualitative high and low expression values. A strong logical relationship between genes emerges if at least one of the quadrants is sparsely populated. We put together a web resource where gene expression relationships can be explored online which helps visualize the logical relationships between genes. We believe that this website will be useful in identifying important genes in different biological context. The web link is http://hegemon.ucsd.edu/plant/.