Project description:A) Chromatins were prepared from Cdx2-inducible ES cells cultured for 48 - 60 hours in the Dox+ and Dox- conditions. Chromatin immunoprecipitation (ChIP) was carried out by using anti-FLAG M2 affinity gel. ChIP product was tested by Western blotting using anti-FLAG antibody. Nuclear extract from ES cells cultured for 48 - 60 hours in Dox+ and Dox- condition was used for the Western blot. B) CDX2 ChIP-Seq peaks in the Hoxa7 gene region. UCSC Mouse Mm9 browser view of Hoxa7 gene locus after mapping CDX2 ChIP-Seq tags locations in the wiggle format. CDX2 ChIP-Seq peaks are shown in red color. C) Cdx2 ChIP-Seq result was verified by qPCR. Target genes were indicated in (G). Primers flanking a promoter region of Hbb-b1 and Pou5f1 as well as a gene desert region in chromosome 3 were used as negative controls. Primers flanking of Actb gene promoter were used for normalization. The relative enrichment of CDX2 binding was indicated as fold change. (D) CDX2-binding motifs identified with CisFinder using 200 bp sequences centered at ChIP sites. (F) Potential CDX2-direct target genes based on ChIP-Seq and the alteration of expression by Cdx2-overexpression. (G) Identification of CDX2 target genes by combining information on binding sites with gene expression response to Cdx2 over-expression Chromatin IP against CDX2-Flag fusion protein. MC1 ES cells were genetically modified for ROSA26 locus to have Tet-Off expression cassette for C-terminal FLAG tagged Cdx2. The peaks are obtained from the Eland Multi Alignment file. The number of tags in peaks was compared with the number of tags in the control sample for the same region corrected by the total coverage of tags. See supplemental file of the paper for details.
Project description:We used DamID-seq to analyze the genome-wide binding patterns of the group B Sox proteins Dichaete and SoxNeuro in four species of Drosophila: D. melanogaster, D. simulans, D. yakuba and D. pseudoobscura. Both binding site turnover between species and a comparison of the binding properties of the two partially-redundant transcription factors were analyzed. We found that, despite widespread turnover, genomic intervals that are commonly bound by both Dichaete and SoxNeuro are highly conserved in Drosophila. DamID for Dichaete (Dichaete-Dam) was performed in D. melanogaster, D. simulans, D. yakuba and D. pseudoobscura, while DamID for SoxNeuro (SoxN-Dam) was performed in D. melanogaster and D. simulans. The control experiment, Dam-only, was performed in all species. Three biological replicates were sequenced for each condition in each species.
Project description:We performed CUT&RUN assay with control Ig , anti-Flag and anti-ETO antibodies in Flag-tag AML1-ETO Kasumi-1 cells to determine binding occupancy of AML1-ETO following miR-130a KD. Kasumi-1 cells were transduced in 3 independent replicates for control and miR-130a KD samples with lentiviruses containing GFP reporter gene. GFP+ cells were sorted 3 days post-transduction and the cells were used in the CUT&RUN assay. Three antibodies were used for each biological sample.
Project description:We used DamID-seq to analyze the genome-wide binding patterns of the group B Sox proteins Dichaete and SoxNeuro in four species of Drosophila: D. melanogaster, D. simulans, D. yakuba and D. pseudoobscura. Both binding site turnover between species and a comparison of the binding properties of the two partially-redundant transcription factors were analyzed. We found that, despite widespread turnover, genomic intervals that are commonly bound by both Dichaete and SoxNeuro are highly conserved in Drosophila.
Project description:Due to the enhanced labeling capability of maleimide-based fluorescent probes, lysine-cysteine-lysine (KCK) tags are frequently added to proteins for visualization. In this study, we employed in vitro single-molecule DNA flow-stretching assay as a sensitive way to assess the impact of the KCK-tag on the property of DNA-binding proteins. Using Bacillus subtilis ParB as an example, we show that, although no noticeable changes were detected by in vivo fluorescence imaging and chromatin immunoprecipitation (ChIP) assays, the KCK-tag substantially altered ParB’s DNA compaction rates, its response to nucleotide binding and to the presence of the specific sequence (parS) on the DNA. While it is typically assumed that short peptide tags minimally perturb protein function, our results urge researchers to carefully validate the use of tags for protein labeling. Our comprehensive analysis can be expanded and used as a guide to assess the impacts of other tags on DNA-binding proteins in single-molecule assays.
Project description:Endogenous ZFC3H1 was tagged in 293T cells. Dignam cellular extracts where subjected to sequential FLAG and HA immunoprecipitation and elution. Complexes were then analyzed using mass spectrometry at the taplin mass spectrometry facility. The 2 samples are FLA-HA IP in wild-type 293T cells and Flag-HA IP in FH-ZFC3H1 293T cells.
Project description:Purpose: The goals of this study are to investigate the mRNAs that bind to the FTO protein in the white fat tissue from mice. Methods: 1. White fat from CAG promoter driven transgenic Flag-tagged FTO mice were extracted with RNA protected. 2. Flag-FTO proteins were immunoprecipitated with control IP using IgG. 3. The immunoprecipitated RNAs were deep sequenced and analyzed. Results: We focused on the mRNAs which have functions related to lipid metabolism and homeostasis. Immunoprecipitated RNAs profiles from Flag-FTO IP and IgG IP were generated by deep sequencing.
Project description:We identified the precise genome-wide binding sites for all SR proteins, using iCLIP-seq SR proteins were encoded on stable transgenes, transfected in S2 cells, FLAG-tag immunopurified, and the bound RNA purified and subjected to RNA-seq. The resulting reads (CLIP tags) were aligned to the Drosophila genome and generated 38,695-5,900,000 unique CLIP tags for each SR-protein replicate.