ABSTRACT: Whi3 associated mRNAs were identified by immunoprecipitation of TAP-tagged Whi3 followed by microarray analysis RNA IP of Whi3-TAP and Whi3-dRRM-TAP with total RNA as reference. Each sample has 2 replicates
Project description:Identification of RNAs bound to Whi3 and Whi4. Mock experiment and several other proteins (Dpm1 and Sfb3) were used as controls. TAP versions of the proteins were used for affinity purification. RNAs retained in the columns were identified by microarray hybridizations.
Project description:To establish the molecular basis for CEP215 function in centrosome-spindle pole attachment, we employed an unbiased proteomic approach to isolat e and identify CEP215 interactors. To this end, affinity purification tags ( Gs TAP containing protein G and streptavidin-binding protein) were inserted in-frame into both alleles of the CEP215 gene (CEP215-TAP cell line) in the chicken B cell line, DT40. Following affinity purification, protein complexes were analysed by mass spectr ometry
Project description:The goal of this experiment was to identify transcripts associated with the S. cerevisiae Upf1 protein. Experiment Overall Design: The RNA population from four independent pairs of Upf1p-TAP and mock affinity selections were analyzed by using Affymetrix Yeast Genome S98 Arrays.
Project description:RPF-1 binds promoters of genes modulated by its induction in HEK/RPF-1 transfectant Chromatin IP (ChIP) combined to chromatin array analysis (ChIP-chip) shows that RPF-1 transcription factor bind to promoter elements of genes repressed or activated by RPF-1 expression Comparison of cells induced for RPF-1 expression (Tet-) over uninduced control cells (Tet+)
Project description:Notch signalling plays crucial roles in mediating cell fate choices in all metazoans largely by specifying the transcriptional output of one cell in response to a neighbouring cell. The DNA-binding protein RBPJ is the principle effector of this pathway in mammals and together with the transcription factor moiety of Notch (NICD) it regulates the expression of target genes. The prevalent view presumes that RBPJ statically occupies consensus binding sites while exchanging repressors for activators in response to NICD. We present the first specific RBPJ chromatin immunoprecipitation and high-throughput sequencing study in mammalian cells. To dissect the mode of transcriptional regulation by RBPJ and identify its direct targets, whole genome binding profiles were generated for RBPJ, its coactivator p300, NICD and the histone H3 modifications H3K4me3, H3K4me1 and H3K27ac in myogenic cells under active or inhibitory Notch signalling conditions. Our results demonstrate dynamic binding of RBPJ in response to Notch activation at essentially all sites co-occupied by NICD. Additionally, we identify a distinct set of sites where RBPJ recruits neither NICD nor p300, and binds DNA statically, irrespective of Notch activity. These findings significantly modify our views on how RBPJ and Notch signalling mediate their activities and consequently impact on cell fate decisions. ChIP (chromatin immunoprecipitation) is followed by deep sequencing to generate genome-wide patterns of RBP-J binding in mouse C2C12 cells under various conditions. Cells were either Notch activated by exposure to immobilized ligand or by overexpression of NICDGFP, or Notch inhibited by treatment with DAPT. Notch activation and inhibition treatments were applied for 6h and 24h. In addition to RBP-J, p300 and NICDGFP were profiled by ChIP-Seq and gene expression was assessed by RNA-Seq.
Project description:To discover new miRNA targets, we generated a C. elegans transgenic line expressing a functional N-terminally Tandem Affinity Purification (TAP) tagged ALG-1 protein (C. elegans strain WS4303). We crossed the TAP::ALG-1 transgene into the mir-58(n4640) mutant background to generate the strain WS5041. For simplicity, we will hereafter term the TAP::ALG-1 transgenic animals as wild typeand the transgenic WS5041 animals as mir-58. We compared the mRNA population that coimmunopurified with TAP::ALG-1 from synchronized L4 stage wild-type animals with that from synchronized L4 stage mir-58 mutant animals by one-color Affymetrix gene arrays. miR-58 target mRNAs should be specifically underrepresented in the latter samples. Strains WS4303 (wt) and WS5041 (mir-58) were used for TAP::ALG-1 IPs. All experiments were conducted in three independent replicates. For each replicate, WS4303 and WS5041 were grown in parallel. 150 ng of TAP::ALG-1 associated RNA isolated from synchronized late L4 animals were sent to the GeneCore facilty in Heidelberg, Germany (http://www.genecore.embl.de/index.cfm), and the microarray data were generated according to their standard protocol (Weinmann et al. 2009).
Project description:Expression data from Kc167 cells under normal conditions. Used to assess expression levels of genes with ORC bound at promoter. Control experiments in support of our previous modencode submssion GSE17282. Kc167 cells were harvested from log phase gorwth. RNA extraction and hybridization on Affymetrix microarrays. Chromatin immunoprecipitation with IgG against TAP-ORC2 or control cells.
Project description:During development, specialized cell lineages are generated through the establishment of cell type-specific transcriptional patterns and epigenetic programs. However, the precise mechanisms and regulators that maintain these specialized cell states remain largely elusive. To identify molecules that safeguard somatic cell identity, we performed two comprehensive RNAi screens targeting known and predicted chromatin regulators during transcription factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPSCs). Remarkably, subunits of the chromatin assembly factor-1 (CAF-1) complex emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation, DNA methylation and heterochromatin maintenance. Suppression of CAF-1 increased reprogramming efficiencies by several orders of magnitude and generated iPSCs two to three times faster compared to controls without affecting cell proliferation. We demonstrate that suppression of CAF-1 leads to a more accessible chromatin structure specifically at enhancer elements early during reprogramming. These changes were accompanied by increased binding of the reprogramming factor Sox2 to ESC-specific regulatory elements and earlier activation of pluripotency-associated genes. Notably, suppression of CAF-1 also enhanced iPSC formation from blood progenitors as well as the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 as an unanticipated regulator of somatic cell identity and provide a potential strategy to modulate cellular plasticity in a regenerative setting. Keywords: Genome binding/occupancy profiling by high throughput sequencing Chromatin accessibility and Sox2 bindings in CAF-1 knockdown and Renilla control during early OKSM reprogramming by high throughput sequencing
Project description:TAP-GluN1 (840 kDa and 1.5 MDa), PSD95-TAP (1.5 MDa) and WT (control; 1.5 MDa) native complexes from Grin1^TAP/TAP, Dlg4^TAP/TAP, and WT mouse forebrain (cortex and hippocampus), respectively. Purified samples were seperated by blue native PAGE. Bands were excised, digested with trypsin. Peptides were identified by LC-MS/MS.
Project description:This study involves the role of yeast mRNA decay factors in transcription. The experiment included here are the ChIP-exo results of three decay factors: Xrn1, Dcp2 & Lsm1. Four experiments were made: Xrn1, Dcp2, Lsm1 and control (no-TAP tag), in two replicates.