Project description:Analysis of specific genes regulated by IWR1 and Y27632 (Y27), respectivly. Total RNA obtained from No Treatment (NT) control EpiSCs and EpiSCs treated by 2 µM IWR1 or 0.5 µM Y27.
Project description:Analysis of the genome-wide transcrptional effects caused by the deletion of the IWR1 gene by comparing the transcriptional profile of a iwr1 mutant strain with the isogenic wild type strain when cells were exponentially grown in YPD medium.
Project description:We identified in a forward genetic screen a novel factor required for RNA-directed DNA methylation (IWR1-like transcription factor). The aim of the study was to identify natural targets of this factor and to compare it to targets of drd1 another major key factor required for the RNA silencing process in plants. We performed transcriptome analysis to identify targets of this factor. For this we used whole plants at seedling stage, isolated total RNA and then used the standard Affymetrix pipeline/chemistry. The mutated transcription factor, dms4-1 was compared to its respective wildtype (EDT, Col-O background). For microarray analysis THREE biological replicates were run.<br><br>For transcriptome comparison we analyzed another factor required for the process RNA-directed DNA methylation in plants, drd1 also identified in a forward genetic screen and previously characterized in depth (Kanno et al. Current Biology, 14, 801-805, (2004), Huettel et al. EMBO J. 25, 2828-2836 (2006)). For transcriptome analysis two alleles of drd1 (drd1-1 and drd1-6) with two biological replicates each was compared to the respective wildtype DT, which served as starting material in this forward genetic screen (DT, Col-0 background). For drd1 mutants and DT wildtype analysis 10 day old seedlings were used as starting matieral while for dms4-1 and EDT 3 week old seedlings were analyzed.
Project description:Analysis of the genome-wide transcrptional effects caused by the deletion of the IWR1 gene by comparing the transcriptional profile of a iwr1 mutant strain with the isogenic wild type strain when cells were exponentially grown in YPD medium. Three independent cultures for the wt and the iwr1 mutant were used for the whole genome transcription analysis and they were grown in YPD at the early exponential phase. Total RNA was isolated and cDNA synthesis and labeling, filter hybridization and quantification/normalization of hybridization signals were performed as described in Garcia-Martinez, J., Aranda, A. and Perez-Ortin, J.E. (2004) Mol Cell 15(2),303-313.
Project description:The folding of the optic cup during embryonic development is a highly mechanic process. In our laboratory we want to demonstrate how the Retinal Pigmented Epithelium (RPE) cells are essential for the correct morphogenesis of the eye. Our experimental workflow has shown that the hippo and B-catenin pathways are involved in the acquisition of the particular geometry of the RPE cells and that this is what drives the proper folding of the optic cup. The aim of this RNA-seq is to analyse how transcription is affected in the zebra fish embryos after disturbing mechanosensitive pathways that alter RPE cells morphology and, therefore, optic cup folding. Dasatinib (150uM), IWR1 (25uM) and Rockout (100uM) are inhibitory drugs of the hippo pathway, Wnt pathway and tension respectively. Yoda1 (10uM) y an agonist of the mechanosensitive Piezo1 calcium channel, and CaliculynA (0.1uM) increase tension and was used as a positive control. WT zebra fish embryos were collected in 1 cell stage and incubated at 28ºC until 7hpf. Embryos were dechorionated with a pronase solution. Then, 50 embryos were added to each plate with E3 medium and the proper concentration of the corresponding drug. Embryos were incubated at 25ºC until the next morning. When the optic cups of the control condition (DMSO) were folded, heads of 10 embryos of each drug treatment were cut and added to a RNAse free Eppendorf and snap-freeze in liquid Nitrogen. We collected 3 biological replicates for each drug treatment. RNA extraction was done following the manufacturer instructions of the RNeasy Mini Kit from QIAGEN, Cat no. 74104. Extracted RNA eluted in 15uL Nuclease-free H2O was sent for library preparation and sequencing to Novogene. This enterprise creates RNA libraries through polyA capture and reverse transcription into cDNA, followed by quality assessment. 12G of sequencing was performed in 150 bp paired-end sequencing using Illumina PE150 technology.
Project description:Saccharomyces cerevisiae strains carrying mutations of the essential Mediator subunit Med11 as well as strains lacking the non-essential Mediator subunits Med2 and Med20 were compared to the corresponding wild-type strains.
Project description:A 2 x 2 factorial design was used to elucidate the genome-wide transcriptional responses of old and young cells to the medium pH control. ?HO strain (BY4742 background) was cultivated batch-wise in SDC media and in fully controlled fermenters. pH was maintained at 4.5 via automatic NaOH addition for pH controlled case while this control was turned off for the unbuffered experiments. Samples of the young and old cells, collected at the 3rd and 7th day of the experiment respectively, were transferred to stimulating media containing fresh SDC medium prior to sample collection for transcriptional profiling.
Project description:Using chromatin-immunoprecipitation followed by next-generation sequencing, we studied the effect of WNT pathway inhibition of the binding of SMAD1 in mouse mesodermal cells from Embryoid Bodies (EB) cultures. The WNT inhibitor IWR1 (or the vehicle control DMSO) were added in day 3 EB cultures and cells were collected 24h later for ChIP assay.
Project description:Full title: Deep sequencing of small RNAs in the trasngenic wild type plant (contains trigger and silencer transgenes) and the IWR1-type transcription factor mutant, dms4 RNA-directed DNA methylation (RdDM) in plants requires two RNA polymerase II (PolII)-related RNA polymerases named PolIV and PolV. A genetic screen designed to reveal factors important for RdDM in a developmental context in Arabidopsis identified DEFECTIVE IN MERISTEM SILENCING 4 (DMS4). Unlike other mutants defective in RdDM, dms4 mutants have a pleiotropic developmental phenotype.DMS4 is similar to yeast IWR1, a conserved putative transcription factor that interacts with PolII subunits. In the transgenic system studied, mutations in DMS4 directly or indirectly affect PolIV-dependent secondary siRNAs, PolV-mediated RdDM, PolV-dependent synthesis of intergenic noncoding RNA, and expression of many PolII-driven genes. These data suggest that DMS4 may be a regulatory factor for multiple RNA polymerases, thus explaining its diverse roles in the plant.