Project description:This SuperSeries is composed of the following subset Series: GSE28444: Expression data for MCF-7 treated with PolII inhibitors, Triptolide or alpha-amanitin GSE28502: Expression data for HUVSMC treated with Triptolide GSE28503: Expression data for knockdown of POLRMT or RNA PolII in MCF-7 cell line Refer to individual Series
Project description:Transcription of mRNA in mammalian is mainly performed by RNA polymerase II (PolII). POLRMT is responsible for the production of cytoplasmic and nuclear form of mitochondrial RNA polymerase. The former (mtRNAP) participates in transcription of RNA in the mitochondria while the latter (spRNAP-IV) is responsible for some mRNA transcription in the nucleus. The nature and amount of genes transcribed by spRNAP-IV still remains unclear. Thus, we scanned for possible candidate genes by using Affymetrix. MCF7 cells were cultured in RPMI medium supplemented with 10% fetal bovine serum. To determine the inhibition of PolII transcription, cells were cultured in the presence of 10 μg/ml of alpha-amanitin or 0.3μM of triptolide (Sigma) for 48 or 24 h, respectively.
Project description:In this experiment we examined higher order chromatin structure during early Drosophila melanogaster development. We performed in situ Hi-C for hand-sorted non-mitotic embryos at nuclear cycle number 12, 13 and 14, and for embryos at 3-4 hours post fertilisation. During this time in development, the zygotic genome is activated and zygotic transcription is taking place for the first time. To assess the impact of transcription on chromatin structure we injected the transcription inhibitors alpha-amanitin or triptolide before zygotic genome activation and performed Hi-C and ChIP-seq for RNA Pol II. Furthermore, we used Hi-C to study genome architecture in embryos lacking the transcription factor Zelda.
Project description:Transcription of mRNA in mammalian is mainly performed by RNA polymerase II (PolII). POLRMT is responsible for the production of cytoplasmic and nuclear form of mitochondrial RNA polymerase. The former (mtRNAP) participates in transcription of RNA in the mitochondria while the latter (spRNAP-IV) is responsible for some mRNA transcription in the nucleus. The nature and amount of genes transcribed by spRNAP-IV still remains unclear. Thus, we scanned for possible candidate genes by using Affymetrix. HUVSMC cell line was treated with or without 0.3 uM of Triptolide (an inhibitor of PolII) for 24 hours to determine which genes are insensitive to triptolide treatment.
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:Transcriptional profiling of HeLa cells treated with α-amanitin for 9 h. Relative abundance to untreated control cells was used to evaluate the biogenesis of lncRNAs by RNA polymerase II. Two-condition experiment, alpha-amanitin-treated vs. untretated HeLa cells. Biological replicates: 4 with dye-swap