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:This dataset provides a proteomic resource of non-specific protein interactions in Leishmania infantum identified by affinity purification coupled to mass spectrometry (AP-MS/MS). Protein extracts from promastigotes expressing Cas9/T7 RNA polymerase were subjected to immunoprecipitation using HA, myc, and His affinity systems, followed by LC–MS/MS analysis. Raw spectral data were processed using MaxQuant against the L. infantum reference proteome from UniProtKB. The dataset includes raw mass spectrometry files, peptide and protein identification tables, and label-free quantification (LFQ) data, all publicly available via the ProteomeXchange Consortium under the identifier PXD067464. After data filtering, a total of 566 unique proteins were identified across all conditions, including 60 proteins consistently detected in all three affinity systems, representing putative non-specific binders. Among these, metabolism-related proteins (30%) and ribosomal components (28%) were the most abundant functional classes. This dataset provides a valuable reference for background protein binding in AP-MS/MS experiments in Leishmania , supporting improved interpretation of interactome studies and benchmarking of affinity purification strategies.