Project description:p53 wildtype or complementing DNA binding cooperativity versions (EE, RR, EE/RR) were overexpressed in SAOS cells and 18h later harvested to perform ChIP with an p53-specific antibody. The enriched DNA fragments were purified and identified by high throughput sequencing.
Project description:In E. coli, editing efficiency (EE) with Cas9-mediated recombineering varies across targets due to differences in the level of Cas9:gRNA DNA double-strand break (DSB)-induced cell death. We found that EE with the same gRNA and repair template can also change with target position, cas9 promoter strength, and growth conditions. Incomplete editing, off-target activity, non-targeted mutations, and failure to cleave target DNA even if Cas9 is bound also compromise EE. These effects on EE were gRNA-specific. We propose that differences in the efficiency of Cas9:gRNA-mediated DNA DSBs and differences in rates of dissociation of Cas9:gRNA complexes from target sites account for the observed variations in EE between gRNAs. We show that editing behavior using the same gRNA can be modified by mutating the gRNA spacer, which changes the DNA DSB activity. Finally, we discuss how variable editing with different gRNAs could limit high-throughput applications and provide strategies to overcome these limitations.
Project description:The mouse R178E (EE) mutation of the Trp53 is a p53 mutant protein with native conformation that lacks the ability to form tetramers and thus constitutes a mutant form of p53 that lacks DNA binding cooperativity. Here we want to assess DNA binding ability of the EE mutant in MEFs under untreated conditions and following p53 stabilization with the Mdm2 inhibitor nutlin-3a and compare it to p53 KO and WT mice.
Project description:We used the NanoString mouse nCounter miRNA expression platform to compare the miRNA expression profiles of spelnocytes from placebo-, E2, and EE exposed NZB/WF1 mice. We found that EE exposure has a more profound effect than E2 on miRNA genes expression in splenocytes
Project description:In systemic sclerosis (SSc) evidence suggests abnormal keratinocyte-fibroblast interactions. We investigated the potential epidermal dysfunction in SSc and its effects on dermis homeostasis. Epidermal equivalents (EE) were generated from six healthy donor (HD) and four SSc keratinocytes. Skin and EE expression of proliferation, differentiation, and activation markers was evaluated by immunohistochemistry. The transcriptomic profile of SSc-EE and HD-EE was identified by RNAseq analysis. EE conditioned medium (CM) was used to stimulate fibroblasts, and their production of interleukin (IL)-6, IL-8, matrix metalloproteinase (MMP)-1, type-I collagen (col-I), and fibronectin was assessed by ELISA. Compared to HD, SSc-EE exhibited aberrant differentiation, enhanced expression of activation markers, and lower mitotic rate of basal keratinocytes, reproducing most of the abnormalities observed in SSc epidermis. RNAseq analysis revealed that, compared to HD-EE, SSc-EE were characterized by the downregulation of HOX gene family members and by the upregulation of metabolic and oxidative stress molecular pathways. EE-CM enhanced the fibroblast production of IL-6, IL-8, MMP-1, Col-I, and fibronectin (p<0.05). Except for Col-I and fibronectin, this effect was 2-fold higher in the presence of CM generated form SSc-EE. IL-1 was, at least in part, responsible for keratinocyte-dependent fibroblast activation. SSc-EE recapitulate the in vivo characteristic of SSc epidermis demonstrating that SSc keratinocytes have an intrinsically altered differentiation program possibly due to the downregulation of genes from the HOX family. The increased metabolic and oxidative stress associated with SSc epidermis may participate to dermis chronic inflammation and fibrosis
Project description:Erosive Esophagitis (EE) is one of the common forms of gastroesophageal reflux disease (GERD). We performed transcriptomic analysis (RNA sequencing) of esophageal biopsies from patients with EE and healthy controls to increase understanding of complex cellular and molecular pathways in EE.
Project description:Adult stem cells must coordinate transcriptional programs with external cues to maintain tissue homeostasis. In the Drosophila midgut, intestinal stem cells (ISCs) generate enterocytes (ECs) or enteroendocrine (EE) cells through Notch-dependent fate decisions, but how chromatin regulators influence this balance remains unclear. In this study we identified the Trithorax gene (trx) as a key factor that safeguards ISC lineage fidelity. Trx depletion biases ISCs toward EE differentiation without affecting proliferation, a phenotype exacerbated during aging or DSS-induced damage. Transcriptomic and chromatin profiling revealed the homeobox transcription factor Ptx1 as a Trx-dependent target. Ptx1 knockdown phenocopies Trx loss, whereas Ptx1 overexpression reverts trx-RNAi-induced EE overproduction, establishing Ptx1 as a critical mediator of Trx function. These findings support a model in which Trx constrains the scute–prospero axis through Ptx1-mediated repression, thereby limiting inappropriate EE specification and maintaining ISC plasticity.
Project description:Previous studies reported that mice living in an enriched environment (EE) showed reduced growth of melanoma, colon and breast tumors. Our study extended the potential anti-tumor effect of EE exposure to pancreatic cancer, and firstly provided evidence demonstrating that EE exposure significantly inhibits tumor growth in a syngeneic murine model of pancreatic cancer. To further investigate the mechanisms underlying the EE-induced pancreatic tumor inhibition, we analyzed the gene expression changes in pancreatic tumor xenograft using an integrative transcriptomic and proteomic approach. Our results found that EE largely decreased the expression of genes associated with mitochondrial function at transcriptional level in pancreatic tumor, suggesting mitochondrial dysfunction in tumor cells may play a critical role in EE-induced anti-tumor phenotype.
Project description:In this dataset contains 3 cases of transcriptome information from 3 normal endometrial tissue of patients with uterine leiomyoma (NE-1,NE-2,NE-3), 3 cases from ectopic endometrial tissue samples of patients with endometriosis (EE-1,EE-2,EE-3).
Project description:To investigate the effect of maternal enriched environment (EE) exposure on fetal neurodevelopment compared with standard habitation (SH) We divided pregant mice into SH and EE group and then performed gene expression profiling analysis using data obtained from RNA-seq of cortex and hippocampus of fetal brains from SH group and EE group
