Gene expression regulated by AGN193109 and 4-diehtylaminobenzaldehyde (DEAB) in mIMCD-3 cells
Ontology highlight
ABSTRACT: Retinoic acid (RA) is the bioactive derivative of vitamin A that regulates gene expression by activating RA receptors (RAR). By using a reporter mouse model, we recently showed that endogenous RA/RAR signaling was present in kidney collecting duct cells, and in mouse inner medullary collecting duct cell line (mIMCD-3). In order to unveil target genes of endogenous RA/RAR signaling in kidney collecting duct cells, whole genome microarray analysis was performed on mIMCD-3 cells treated with AGN193109, a pan-RAR antagonist, and 4-diethylaminobenzaldehyde (DEAB), an inhibitor of RA synthesizing enzyme. Specificity of gene expression regulation was confirmed by determining the reversibility of the regulation by simultaneous addtion of exogenous tRA. In the first set of experiment, mIMCD-3 cells were treated with either vehicle (0.1% ethanol and 0.1% DMSO) or 1 μM AGN193109 or 1 μM AGN193109+0.2 μM tRA. Three independent biological experiments were performed. In the second set of experiment, mIMCD-3 cells were treated with either vehicle (0.1% ethanol and 0.1% DMSO) or 25 μM DEAB or 25 μM DEAB+0. 01 μM tRA. Three independent biological experiments were performed. Treatment period for both sets of experiments were 24 hours.
Project description:All-trans retinoic acid (tRA) is the bioactive derivative of vitamin A that regulates gene expression by activating RA receptors (RAR). By using a reporter mouse model, we recently showed that endogenous tRA/RAR signaling was present in kidney collecting duct cells, and in mouse inner medullary collecting duct cell line (mIMCD-3). In order to unveil target genes of endogenous tRA/RAR signaling in kidney collecting duct cells, whole genome microarray analysis was performed on mIMCD-3 cells treated with AGN193109, a pan-RAR antagonist, and 4-diethylaminobenzaldehyde (DEAB), an inhibitor of tRA synthesizing enzyme. Specificity of gene expression regulation was confirmed by determining the reversibility of the regulation by simultaneous addition of exogenous tRA.
Project description:Analysis of expression changes in renal collecting duct epithelial cells by adenoviral mediated Krüppel like transcription factor 5 (KLF5) overexpression. KLF5 is a key regulator of static and inflammatory stage in renal collecting duct epithelial cells. We thought these results provide insights into downstream genes of KLF5 in renal collecting duct epithelial cells. Total RNAs were isolated from adenovirally-mediated KLF5 over expressed cultured mIMCD-3 cells or control adenovirus infected mIMCD-3. We analyzed these two gene expression profiles after 24 hours after infection.
Project description:The affect of aldosterone on the miRNA landscape in mIMCD-3 cells was determined In the study presented here, the affect of aldosterone was examined on the miRNA content in a murine inner medullary collecting duct cell line
Project description:Ascrobate peroxidase (APEX) was expressed as a fusion protein and directed to primary cilia of mouse inner medullary collecting duct (mIMCD-3) cells, where it was used for proximity labeling to biotinylate proteins, which are subsequently enriched by streptavidin chromatography and identified by mass spectrometry. Cilia-APEX proteomics profiling compared the cilia proteomes of wild-type and Arl6-/- cilia (generated by CRISPR/Cas9-mediated genome editing) by spectral counting.
Project description:We aimed to define epithelial-specific genes in the kidney. In the developing mouse kidney at E12.5 epithelial cells are restricted to the ureteric bud, while mesenchymal cells surrounding the ureteric bud are non-epithelial. The mouse renal epithelial cell line mIMCD-3 was used to represent kidney epithelia in vitro. Gene expression was analyzed using Affymetrix microarrays in ureteric bud stalks, ureteric bud tips, and mIMCD-3 cells and compared to metanephric mesenchyme. Affymetrix Mouse Genome 430 2.0 microarrays from mouse E12.5 ureteric bud tips, ureteric bus stalks, and metanephric mesenchymes had previously been published (GEO deposition: GDS1583) (Schmidt-Ott, K. M., Yang, J., Chen, X., Wang, H., Paragas, N., Mori, K., Li, J. Y., Lu, B., Costantini, F., Schiffer, M. et al. (2005). Novel regulators of kidney development from the tips of the ureteric bud. J Am Soc Nephrol 16, 1993-2002.). We added a new microarray analysis from a subclone of mIMCD-3 cells and recalculated expression values in the complete dataset by robust multichip analysis.
Project description:Primary cilia are tiny membrane protrusions emanating from the surface of almost all mammalian cell types. Recently, a picture has emerged of the primary cilium functioning as a cellular antenna that senses extracellular stimuli via receptors, locally processes the signal using cilia-specific signalling pathways, and transduces this information into a cellular response. Components of the cyclic AMP (cAMP) signalling cascade have been proposed to be part of the ciliary signalling pathways. We aimed to shed light on whether ciliary cAMP signaling controls gene expression, and if yes, which gene expression program is particularly targeted by ciliary cAMP signaling. To this end, we targeted an optogenetic tool to increase cAMP levels (bPAC) to the primary cilium in murine, kidney-derived inner medullary collecting duct (mIMCD-3) cells (mIMCD-3 cilia-bPAC cells). As controls, we used cells were bPAC is targeted to the cytosol (mIMCD-3 cyto-bPAC cells) and wild-type cells (mIMCD-3 WT cells). We increased cAMP levels by light stimulation and next analysed gene expression using bulk RNA-sequencing. As an additional control, we also included WT cells treated with the compound Forskolin, which targets endogeneous adenylyl cyclases and thus, in principle, is believed to increase cAMP levels in both compartments, cilium and cell soma.
Project description:Retinoic acid (RA) is a key signal for the specification of the pancreas. Still, the gene regulatory cascade triggered by RA in the endoderm remains poorly characterized. In this study, we investigated this regulatory network in zebrafish by combining RNA-seq, RAR ChIP-seq and ATAC-seq assays. By analysing the effect of RA and of the RA receptor (RAR) antagonist BMS439 on the transcriptome and on the chromatin accessibility of endodermal cells, we identified a large set of genes and regulatory regions regulated by RA signalling. RAR ChIP-seq further defined the direct RAR target genes including the known hox genes as well as several pancreatic regulators like mnx1, insm1b, hnf1ba and gata6. Comparison of our zebrafish data with available murine RAR ChIP-seq data highlighted conserved direct target genes and revealed that some RAR sites are under strong evolutionary constraints. Among them, a novel highly conserved RAR-induced enhancer was identified downstream of the HoxB locus and driving expression in the nervous system and in the gut in a RA-dependant manner. Finally, ATAC-seq data unveiled the role of the RAR-direct targets Hnf1ba and Gata6 in opening chromatin at many regulatory loci upon RA treatment.
Project description:Retinoic acid (RA) is a key signal for the specification of the pancreas. Still, the gene regulatory cascade triggered by RA in the endoderm remains poorly characterized. In this study, we investigated this regulatory network in zebrafish by combining RNA-seq, RAR ChIP-seq and ATAC-seq assays. By analysing the effect of RA and of the RA receptor (RAR) antagonist BMS439 on the transcriptome and on the chromatin accessibility of endodermal cells, we identified a large set of genes and regulatory regions regulated by RA signalling. RAR ChIP-seq further defined the direct RAR target genes including the known hox genes as well as several pancreatic regulators like mnx1, insm1b, hnf1ba and gata6. Comparison of our zebrafish data with available murine RAR ChIP-seq data highlighted conserved direct target genes and revealed that some RAR sites are under strong evolutionary constraints. Among them, a novel highly conserved RAR-induced enhancer was identified downstream of the HoxB locus and driving expression in the nervous system and in the gut in a RA-dependant manner. Finally, ATAC-seq data unveiled the role of the RAR-direct targets Hnf1ba and Gata6 in opening chromatin at many regulatory loci upon RA treatment.
Project description:Retinoic acid (RA) is a key signal for the specification of the pancreas. Still, the gene regulatory cascade triggered by RA in the endoderm remains poorly characterized. In this study, we investigated this regulatory network in zebrafish by combining RNA-seq, RAR ChIP-seq and ATAC-seq assays. By analysing the effect of RA and of the RA receptor (RAR) antagonist BMS439 on the transcriptome and on the chromatin accessibility of endodermal cells, we identified a large set of genes and regulatory regions regulated by RA signalling. RAR ChIP-seq further defined the direct RAR target genes including the known hox genes as well as several pancreatic regulators like mnx1, insm1b, hnf1ba and gata6. Comparison of our zebrafish data with available murine RAR ChIP-seq data highlighted conserved direct target genes and revealed that some RAR sites are under strong evolutionary constraints. Among them, a novel highly conserved RAR-induced enhancer was identified downstream of the HoxB locus and driving expression in the nervous system and in the gut in a RA-dependant manner. Finally, ATAC-seq data unveiled the role of the RAR-direct targets Hnf1ba and Gata6 in opening chromatin at many regulatory loci upon RA treatment.