Project description:To determine the potential molecular mechanisms by which STAT5 signaling control ileal Paneth cell homeostasis, we isolated total RNA from ileal intact crypts of STAT5+/+, STAT5DIEC-/- and STAT5DIEC+++ mice and performed RNA sequencing (RNA-seq). With an average of 22.3 million reads per sample, we observed 27540 transcripts when reads were aligned to the mm10 genome with annotations provided by Ensembl. Transcripts were filtered, requiring at least 3 reads in 50% of samples within at least one condition, leaving 10197 transcripts for analysis. To identify differentially-regulated transcripts, we performed ANOVA (FDR-corrected p<0.05) and required the fold change to exceed 1.5.
Project description:The goals of this study are to compare transcriptomes of mouse Ileal crypts isolated from Adarfl/fl and Adar iΔgut mice at 3 dpi of tamoxifen by RNA-seq.
Project description:In order to systematically map STAT5 genomic binding sites and IL-2 mediated gene expression changes we mapped STAT5 binding sites using chip-on-ChIP in IL-2 dependent Kit225 cells that were either left un-stimulated or stimulated with IL-2 for 30 minutes. Using MAT analysis we identified 1581 STAT5 binding sites. IL2-stimulated Kit225 cells ChIPed with STAT5 antibodies vs. Input
Project description:Polycomb-mediated gene repression plays an important role in adult stem cell maintenance. Direct targets of the Polycomb repressive complex PRC2 in th intestinal epithelium were revealed by performing ChIP-sequencing on crypt samples isolated from wild type murine small intestines. The resulting list of H3K27me3-enriched genes were compared with RNA-sequencing data from wild type and Eed knockout crypts. Crypts were isolated from wild type murine intestinal epithelium and subjected to ChIP using anti-H3K27me3 and anti-H3K27Ac antibodies, after which DNA isolated from extracted immunocomplexes was sequenced.
Project description:Estrogen Receptor alpha (ERα) is a key driver of most breast cancers, and it is the target of endocrine therapies used in the clinic to treat women with ERα positive (ER+) breast cancer. The two methods ChIP-seq (chromatin immunoprecipitation coupled with deep sequencing) and RIME (Rapid Immunoprecipitation of Endogenous Proteins) have greatly improved our understanding of ERα function during breast cancer progression and in response to anti-estrogens. A critical component of both ChIP-seq and RIME protocols is the antibody that is used to pull down the bait protein. To date, most of the ChIP-seq and RIME experiments for the study of ERα have been performed using the sc-543 antibody from Santa Cruz Biotechnology. However, this antibody has been discontinued, thereby severely impacting the study of ERα in normal physiology as well as diseases such as breast cancer and ovarian cancer. Here, we compare the sc-543 antibody with other commercially available antibodies, and we show that 06-935 (EMD Millipore) and ab3575 (Abcam) antibodies can successfully replace the sc-543 antibody for ChIP-seq and RIME experiments.
Project description:Signal Transducers and Activators of Transcription (STATs) are principal transcription factors downstream of cytokine receptors. Although STAT5A is expressed in most tissues it remains to be understood why its premier, non-redundant functions are restricted to prolactin-induced mammary gland development. We report that the ubiquitously expressed Stat5a/b locus is subject to lineage-specific transcriptional control in mammary epithelium. Genome-wide surveys of epigenetic status and transcription factor occupancy uncovered a putative mammary-specific enhancer within the intergenic sequences separating the two Stat5 genes. This region exhibited several hallmarks of genomic enhancers, including DNaseI hypersensitive sites, H3K27 acetylation and binding by GR and MED1. Mammary-specific STAT5 binding was obtained at two canonical STAT5 binding motifs. CRISPR/Cas9-mediated genome editing was used to delete these STAT5 binding sites in mice and determine their biological function. Mutant animals exhibited an 80% reduction of Stat5 levels in mammary epithelium and a concomitant reduction of STAT5-dependent gene expression. Transcriptome analysis identified a class of mammary-restricted genes that was particularly dependent on high STAT5 levels as a result of the intergenic enhancer. Taken together, the mammary-specific enhancer enables a positive feedback circuit that underlies the remarkable abundance of STAT5 and, in turn, controls the efficacy of STAT5-dependent mammary physiology. ChIP-seq for H3K27ac, RNA Pol II, and MED1 in mammary tissues at L1, and ChIP-seq for H3K27ac and GR in mammary tissues at p13. mRNA-seq in WT at L1, line B (GAS2 mutation only) and line C (both GAS1 and GAS2 mutations) at L1 in mammary tissues, and DNase-seq in WT mammary tissues at L1.
Project description:To investigate the role of RAD21 in the transcriptional regulation of global gene expression at early stage of colorectal cancer developments, we peformed the genome-wide analysis to map genomic regions bound by Rad21 in normal small testinal crypts and tumors (adenomas) harvested from Apc Min/+ mice using ChIP-seq. ChIP-seq naalysis identified high confidence RAD21 binding sites unique to normal crypts or adenomas, as well as those common to both tissues. We further performed RNA-seq to profile the changes in gene expression from normal WT crypts to adenomas at the very early stage of adenomagenesis in the context of Rad21 heterozygous loss. mRNA profiles of normal small intestinal crypts (WT) and adenomas from Apc Min/+ and Apc Min/+:Rad21+/- double mutant mouse; Mapping of Rad21 genomic binding sites in normal intestinal crypts (WT) and Apc Min/+ adenomas
Project description:To investigate the role of RAD21 in the transcriptional regulation of global gene expression at early stage of colorectal cancer developments, we peformed the genome-wide analysis to map genomic regions bound by Rad21 in normal small testinal crypts and tumors (adenomas) harvested from Apc Min/+ mice using ChIP-seq. ChIP-seq naalysis identified high confidence RAD21 binding sites unique to normal crypts or adenomas, as well as those common to both tissues. We further performed RNA-seq to profile the changes in gene expression from normal WT crypts to adenomas at the very early stage of adenomagenesis in the context of Rad21 heterozygous loss.
Project description:Although small intestine epithelium has been studied widely in murine, a comprehensive expression landscape in human is still lacking. To explore the cellular diversity of human small intestine epithelium, we sampled the ileal crypts from two patients suffered from right-sided colon cancer while their ilea were relatively normal. scRNA-seq libraries were generated using the 10X v3 Kit, which could guarantee both throughput and quality