ChIP-chip of BMP2 or control treated human pulmonary artery endothelial cells with anti-beta-catenin or anti-ppar-gamma antibodies on promoter regions
ABSTRACT: Reduced bone morphogenetic protein receptor (BMPR)2 expression in patients with pulmonary arterial (PA) hypertension (PAH), can impair PA endothelial cell (EC) function. We now characterize, in human PAECs, a novel BMPR2-mediated transcriptionally active complex between peroxisome proliferator-activated receptor (PPAR) gamma and beta-catenin (BC), and show that disruption of this complex impairs BMP mediated HPAEC survival. Using whole genome wide ChIP-Chip promoter analysis we delineate PPARG-BC dependent transcription of target genes that include apelin. Comparison of ppar-gamma and beta-catenin occupancy on promoter regions from human pulmonary artery endothelial cells after either treatment with BMP2 or control. A total of 8 samples were created using NimbleGen human HG18 promoter arrays.
Project description:Expression analysis of genes potentially regulated by BMPRII and beta-catenin. BMPRII has been linked as a genetic factor to the disease pulmonary arterial hypertension. Comparison of total mRNA obtained from human pulmonary artery endothelial cells treated with control, bone morphogentic protein receptor II, or beta-catenin siRNA
Project description:To determine the global transcriptomic changes induced by treatment with the Beta Catenin antagonist BC2059 in AML cells The canonical WNT-β-catenin pathway is essential for self-renewal, growth and survival of AML stem/blast progenitor cells (BPCs). Deregulated WNT signaling inhibits degradation of β-catenin, causing increased nuclear translocation and co-factor activity of β-catenin with the transcriptional regulator TCF4/LEF1 in AML BPCs. Here, we determined the pre-clinical anti-AML activity of the anthraquinone oxime-analog BC2059 (BC), known to attenuate β-catenin levels. BC treatment disrupted the binding of β-catenin with the scaffold protein TBL1 (transducin β-like 1) and proteasomal degradation and decline in the nuclear levels of β-catenin. This was associated with reduced transcriptional activity of TCF4 and expression of its target genes, cyclin D1, c-Myc and survivin. BC treatment dose-dependently induced apoptosis of cultured and primary AML BPCs. Treatment with BC also significantly improved the median survival of immune-depleted mice engrafted with either cultured or primary AML BPCs exhibiting nuclear expression of β-catenin. Co-treatment with the pan-histone deacetylase inhibitor panobinostat and BC synergistically induced apoptosis of cultured and primary AML BPCs, including those expressing FLT3-ITD, as well as further significantly improved the survival of immune-depleted mice engrafted with primary AML BPCs. These findings underscore the promising pre-clinical activity and warrant further testing of BC against human AML, especially those expressing FLT3-ITD. Overall design: 4 samples analyzed: OCI-AML3 cells (untreated); OCI-AML3 cells treated with 100 nM of BC2059 for 8 hours; Primary FLT3-ITD expressing AML cells (untreated); Primary FLT3-ITD expressing AML cells treated with 100 nM of BC2059 for 8 hours.
Project description:Long intergenic noncoding RNAs (lincRNAs) regulate chromatin states and epigenetic inheritance. Here we show that the lincRNA HOTAIR serves as a scaffold for at least two distinct histone modification complexes. A 5’ domain of HOTAIR binds Polycomb Repressive Complex 2 (PRC2) while a 3’ domain of HOTAIR binds the LSD1/CoREST/REST complex. The ability to tether two distinct complexes enables RNA-mediated assembly of PRC2 and LSD1, and coordinates targeting of PRC2 and LSD1 to chromatin for coupled histone H3 lysine 27 methylation and lysine 4 demethylation. Our results suggest that lincRNAs may serve as scaffolds by providing binding surfaces to assemble select histone modification enzymes, and thereby specify the pattern of histone modifications on target genes. LSD1 and SUZ12 co-occupied on 721 genes in human foreskin fibroblasts. Coordinate loss of SUZ12 and LSD1 occupancy was caused by HOTAIR knockdown. Comparison occupancy of LSD1 and SUZ12 of siGFP and siHOTAIR foreskin fibroblasts on human HG18 promoter arrays. Human foreskin fibroblasts were transfected with siGFP or siHOTAIR. The cells were harvested and ChIP analysis with anti-LSD1 and anti-SUZ12 antibodies was performed.
Project description:Histone modification H3K9me2 is associated heterochromatin and gene silencing, but the relationship between DNA methylation and H9K9me2 haven’t been checked in a genome-wide scale. This dataset was generated to compare with genome-wide DNA methylation data. Genome-wide distribution of H3K9me2 in human fibroblasts was mapped using ChIP-chip.
Project description:Coordinate loss of MLL1 and WDR5 occupancy caused by HOTTIP knockdown were observed in distal HOXA genes. These regions correspondingly lost H3K4me3 and H3K4me2, without changes in pan-histone or H3K27me3. human foreskin fibroblasts (CRL2091), anti-H3K4me3 (Abcam ab8580, lot#1016899), anti-H3K4me2 (Abcam ab32356, lot#947550), anti-H3K27me3 (Abcam ab4729, lot#1021724), anti-histone H3 (Abcam ab1791, lot#1025144), anti-MLL1 (gift of R. Roeder, Rockefeller University), and anti-WDR5 (gift of W. Herr, UNIL) Comparison of occupancy of MLL1 and WDR5 of siGFP and siHOTTIP foreskin fibroblasts on HOX tiling array. Human foreskin fibroblasts are transfected with siGFP or siHOTTIP for 72 hrs. The cells are harvesed and ChIP performed with H3K4me3, H4K27me3, H3K4me2, MLL1, WDR5, and histone antibodies.
Project description:The gene encoding the aquaporin-2 water channel is regulated transcriptionally in response to vasopressin. In the renal collecting duct, vasopressin stimulates the nuclear translocation and phosphorylation (at Ser552) of β-catenin, a multifunctional protein that can act as a transcriptional co-regulator in the nucleus. The purpose of this study was to identify β-catenin interacting proteins in nuclei of rat inner medullary collecting duct (IMCD) cells using both experimental and computational approaches. We used chromatin immunoprecipitation coupled to mass spectrometry (ChIP-MS) in nuclei isolated from rat IMCD suspensions to identify β-catenin interacting proteins. We reproducibly (n=4) identified 43 β-catenin binding proteins, which included a number of known β-catenin binding partners as well as novel interacting proteins. Multiple proteins involved in transcriptional regulation were identified (Taf1, Jup, Tdrd3, Cdh1, Cenpj, and several histones). Many of the identified β-catenin binding partners were found in prior studies to translocate to the nucleus in response to vasopressin. There was one DNA-binding transcription factor (TF), viz. Taf1, part of the RNA-polymerase II pre-initiation complex. To identify sequence-specific TFs that may interact with β-catenin but are expressed at abundance levels too low to be detected by MS, Bayes’ Theorem was used to integrate data from several information sources. The analysis identified several TFs with potential binding sites in the Aqp2 gene promoter that could interact with β-catenin in the regulation of Aqp2 gene transcription viz. Jun, Junb, Jund, Atf1, Atf2, Mef2d, Usf1, Max, Pou2f1, and Rxra. The findings provide information necessary for modeling the transcriptional response to vasopressin.
Project description:Model destription:
The model describes the dynamics of murine IRF7 gene expression upon IFN stimulation. The present model comprises known key components and feedback mechanisms. The overall system describes the active IRF7 promoter (Pa) by the binding of IRF7 dimer and ISGF3 to the DNA binding sites ISRE and IRFE, respectively, the transcription and translation of IRF7, and its subsequent phosphorylation and dimerization. IRF7 protein binding to the IRFE binding site in the promoter results in the production of more IRF7 protein, constituting a positive feedback loop.
A New Efficient Approach to Fit Stochastic Models on the Basis of High-throughput Experimental Data Using a Model of IRF7 Gene Expression as Case Study
Luis U. Aguilera, Christoph Zimmer and Ursula Kummer.
Project description:Dysregulation of Wnt signaling is involved in carcinogenesis, mainly through activation of beta-catenin/TCF target genes. Although aberrant activation of several Wnt target genes, including CCND1 and MYC, has been reported as promoting proliferation of cancer cells, it is hardly enough to understand malignant phenotypes of cancer cells with aberrant Wnt signaling. To elucidate the transcriptional regulation of beta-catenin/TCF target genes, we examined binding of beta-catenin (BC) to DNA by chromatin immunoprecipitation linked to genome tiling array (ChIP-on-chip). Overall design: Beta-catenin ChIP vs. Input in SW480 cells.
Project description:Expression analysis of genes potentially regulated by BMPRII and beta-catenin. BMPRII has been linked as a genetic factor to the disease pulmonary arterial hypertension. Overall design: Comparison of total mRNA obtained from human pulmonary artery endothelial cells treated with control, bone morphogentic protein receptor II, or beta-catenin siRNA