Project description:RBP2 is downstream of pRB pathway; We used gene expression profiling experiments to investigate if gene expression changes in cells with RBP2 knockdown significantly overlap with gene expression changes in cells overexpressing pRB, consistent with the data that knockdown of RBP2 phenocopies reintroduction of pRB in SAOS-2 (RB-/-) cells Experiment Overall Design: Knockdown of RBP2 by siRNA to RBP2 (RBP2siRNA) in SAOS-2 (RB-/-) cells in comparison to overexpression of pRB in the SOAS-2 (RB-/-) cells and scrambled version of the RBP2 siRNA (RBP2scsiRNA)
Project description:RBP2 is downstream of pRB pathway We used gene expression profiling experiments to investigate if gene expression changes in cells with RBP2 knockdown significantly overlap with gene expression changes in cells overexpressing pRB, consistent with the data that knockdown of RBP2 phenocopies reintroduction of pRB in SAOS-2 (RB-/-) cells Keywords: epistatic experiment
Project description:RBP2 (JARID1A/RBBP2) enrichment at proximal promoter regions in differentiating promonocytic U937 cells and in the osteosarcoma SAOS-2 cells Keywords: differentiation treatment, ChIP-on-chip analysis
Project description:RBP2 (JARID1A/RBBP2) enrichment at proximal promoter regions in differentiating promonocytic U937 cells and in the osteosarcoma SAOS-2 cells Keywords: differentiation treatment, ChIP-on-chip analysis We sought to determine transcriptional regulation by RBP2 genome-wide by using location analysis. We describe that RBP2 target genes are separated into two functionally distinct classes: differentiation independent and differentiation dependent genes.Three condition experiment, U937 cells treated with 100 nM 12-O-tetradeconoyl-phorbol 13-acetate (TPA) (Sigma) for two different time points (27 hrs and 96 hrs) and untreated (0 hrs time point or no TPA treatment). In addition, processed data for the unrelated SAOS-2 cells are included. Biological replicates: threechromatin immunoprecipitations (ChIP) performed independently in parallel
Project description:Aberrations in epigenetic processes, such as histone methylation, can lead to cancer. Retinoblastoma Binding Protein 2 (RBP2)(also called JARID1A or KDM5A) can demethylate tri- and di-methylated lysine 4 in histone H3, which are epigenetic marks for transcriptionally active chromatin, whereas the MEN1 tumor suppressor promotes H3K4 methylation. Previous studies suggested that inhibition of RBP2 contributed to tumor suppression by pRB. Here we show RBP2 loss promotes cellular differentiation in vitro. We use mouse expression array 430 2.0 array to profile gene expression patterns of Rbp2f/f and Rbp2-/- ES cells in ES cell medium and after 6 days in ES cell medium without LIF.
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.
Project description:Aberrations in epigenetic processes, such as histone methylation, can lead to cancer. Retinoblastoma Binding Protein 2 (RBP2)(also called JARID1A or KDM5A) can demethylate tri- and di-methylated lysine 4 in histone H3, which are epigenetic marks for transcriptionally active chromatin, whereas the MEN1 tumor suppressor promotes H3K4 methylation. Previous studies suggested that inhibition of RBP2 contributed to tumor suppression by pRB. Here we show RBP2 loss promotes cellular differentiation in vitro. We use mouse expression array 430 2.0 array to profile gene expression patterns of Rbp2f/f and Rbp2-/- ES cells in ES cell medium and after 6 days in ES cell medium without LIF. Subconfluent Rbp2f/f and Rbp2-/- ES cells in ES cell medium and after 6 days in ES cell medium without LIF were harvested for RNA isolation using RNeasy mini kit with on-column DNase digestion (Qiagen). Gene expression profiling was performed using Affymetrix GeneChip mouse genome 430 2.0 arrays. Duplicate samples were used