Project description:This SuperSeries is composed of the following subset Series: GSE14559: Timed induction of 50 transcription factors in ES cells reveals a common mechanism to initiate differentiations GSE14586: Cdx2 Binding Sites On Cdx2 Expressing ES Cells GSE16148: Timed induction of 10 transcription factors - ES time series data Refer to individual Series
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: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
Project description:To decipher the structure and behaviors of the transcription factor (TF) network, we created 50 permanent mouse ES cell lines, in each of which one of the 50 transcription factors tagged with FLAG, is inserted into the doxycycline (dox)-repressible ROSA26 locus. Expression profiling reveals Cdx2 as the most potent inducer of transcriptome perturbation in ES cells, followed by Esx1, Sox9, Tcf3, Klf4, and Gata3. Immunoprecipitation (IP) with a FLAG-antibody in Cdx2-induced ES cells, identifies NuRD in CDX2-associated protein complexes; and chromatin-IP-sequencing identifies CDX2-binding sites predominantly in genes up-regulated by CDX2. Compendium analyses of Cdx2- and the other TF-inducible ES cells suggest a central role of the POU5F1/SOX2/NANOG protein complex in a swift-acting control mechanism to down-regulate a common set of genes at the beginning of multi-lineage ES cell differentiations. These ES cell lines will be a valuable resource to study biological networks in ES cells and mice. Keywords: dose response design,genetic modification design,individual genetic characteristic design,reference design,replicate design
Project description:Regulatory proteins can bind to different sets of genomic targets in various cell types or conditions. To reliably characterize such condition-specific regulatory binding we introduce MultiGPS, an integrated machine learning approach for the analysis of multiple related ChIP-seq experiments. MultiGPS is based on a generalized Expectation Maximization framework that shares information across multiple experiments for binding event discovery. We demonstrate that our framework enables the simultaneous modeling of sparse condition-specific binding changes, sequence dependence, and replicate-specific noise sources. MultiGPS encourages consistency in reported binding event locations across multiple-condition ChIP-seq datasets and provides accurate estimation of ChIP enrichment levels at each event. MultiGPSM-bM-^@M-^Ys multi-experiment modeling approach thus provides a reliable platform for detecting differential binding enrichment across experimental conditions. We demonstrate the advantages of MultiGPS with an analysis of Cdx2 binding in three distinct developmental contexts. By accurately characterizing condition-specific Cdx2 binding, MultiGPS enables novel insight into the mechanistic basis of Cdx2 site selectivity. Specifically, the condition-specific Cdx2 sites characterized by MultiGPS are highly associated with pre-existing genomic context, suggesting that such sites are pre-determined by cell-specific regulatory architecture. However, MultiGPS-defined condition-independent sites are not predicted by pre-existing regulatory signals, suggesting that Cdx2 can bind to a subset of locations regardless of genomic environment. In this study, we characterize the binding of Cdx2 in embryonic stem cells, endodermal cells, and progenitor motor neurons using V5- or FLAG-tagged doxycycline inducible Cdx2 ESC lines (iCdx2). Endoderm and progenitor motor neurons are generated from the ES cells using directed differentiation approaches. The cells are then exposed to Dox to express the tagged Cdx2 construct. The genome-wide binding of the induced full-length Cdx2 transcription factor is profiled using ChIP-seq with an anti-V5 or anti-FLAG antibody. We also examine the binding behavior of a truncated version of the Cdx2 protein, where a protein interaction domain contained in the first 59 amino acids has been deleted. An appropriate pseudo-IP control experiment for these ChIP-seq experiments has been previously submitted under accession number GSM766062.
Project description:To decipher the structure and behaviors of the transcription factor (TF) network, we created 50 permanent mouse ES cell lines, in each of which one of the 50 transcription factors tagged with FLAG, is inserted into the doxycycline (dox)-repressible ROSA26 locus. We have obtained time series data for 10 of the transcripts at 24h,48h and 72hr. Expression profiling reveals Cdx2 as the most potent inducer of transcriptome perturbation in ES cells, followed by Esx1, Sox9, Tcf3, Klf4, and Gata3. Immunoprecipitation (IP) with a FLAG-antibody in Cdx2-induced ES cells, identifies NuRD in CDX2-associated protein complexes; and chromatin-IP-sequencing identifies CDX2-binding sites predominantly in genes up-regulated by CDX2. Compendium analyses of Cdx2- and the other TF-inducible ES cells suggest a central role of the POU5F1/SOX2/NANOG protein complex in a swift-acting control mechanism to down-regulate a common set of genes at the beginning of multi-lineage ES cell differentiations. These ES cell lines will be a valuable resource to study biological networks in ES cells and mice. Keywords: dose response design,genetic modification design,individual genetic characteristic design,reference design,replicate design,time series design
Project description:Introgressed variants from other species can be an important source of genetic variation because they may arise rapidly, can include multiple mutations on a single haplotype, and have often been pretested by selection in the species of origin. Although introgressed alleles are generally deleterious, several studies have reported introgression as the source of adaptive alleles-including the rodenticide-resistant variant of Vkorc1 that introgressed from Mus spretus into European populations of Mus musculus domesticus. Here, we conducted bidirectional genome scans to characterize introgressed regions into one wild population of M. spretus from Spain and three wild populations of M. m. domesticus from France, Germany, and Iran. Despite the fact that these species show considerable intrinsic postzygotic reproductive isolation, introgression was observed in all individuals, including in the M. musculus reference genome (GRCm38). Mus spretus individuals had a greater proportion of introgression compared with M. m. domesticus, and within M. m. domesticus, the proportion of introgression decreased with geographic distance from the area of sympatry. Introgression was observed on all autosomes for both species, but not on the X-chromosome in M. m. domesticus, consistent with known X-linked hybrid sterility and inviability genes that have been mapped to the M. spretus X-chromosome. Tract lengths were generally short with a few outliers of up to 2.7 Mb. Interestingly, the longest introgressed tracts were in olfactory receptor regions, and introgressed tracts were significantly enriched for olfactory receptor genes in both species, suggesting that introgression may be a source of functional novelty even between species with high barriers to gene flow.