Project description:Methods: mRNA profiles of untransfected HeLa cells (wild-type; wt) were compared with mRNA profiles of HeLa cells stably maintaining an S/MAR-based episome. Results: We here report for the first time that episomally maintained S/MAR-based vectors do not alter gene expression profile of the host cell's genome. No global changes in gene expression in episome maintaining cells, compared to non-transfected cells could be observed. To identify differentially expressed genes, false discovery rate (FDR; q-value) cut off was set to 0.01. Significantly differentially expressed genes with q<0.01 and an absolute fold-change of 2 were not detected. For verification, we chose five genes with high fold-change and low q-values (q<0.05) and compared expression levels between untransfected HeLa and HeLa stably maintaining an S/MAR-based within three replicates in qPCR. Conclusions: S/MAR-based replicons used in this study do not code for viral proteins but tend to co-localise with promoter sequences and transcription start sites. Recent observations that cooperatively transcribed promoters can influence each other raise concerns that S/MAR-based replicons have the potential to alter endogenous gene expression. Therefore, we compared the transcriptome of untransfected HeLa cells with HeLa cells stably maintaining an S/MAR-based episome. Setting the FDR to <0.01, we found no significantly differentially expressed genes. This finding is of utmost importance for potential gene therapeutic application of S/MAR-based replicons.
Project description:Bacteria play a critical role in the global sulfur cycle by using enzymes capable of cleaving strong bonds such as C-S. Most described enzymes require oxygen for their activity, but recent work has uncovered anaerobic alternatives. Among these are the methylthio alkane reductases (Mar), a group of nitrogenase-like enzymes that reduce volatile organic sulfur compounds (VOSCs) in the absence of oxygen. The purple non-sulfur bacterium Rhodopseudomonas palustris encodes three Mar homologs, providing a unique system to study their distinct functions. Here, we characterize the activity of these enzymes using genomic (RB-TnSeq) and transcriptomic (RNA-Seq) approaches to assess the essentiality and expression patterns of the different Mar enzymes under sulfate limitation. Growth assays with knockout strains supplied with different VOSCs, combined with measuring the specific reaction by-product, revealed that Mar1 preferentially acts on shorter-chain substrates. Our findings highlight the functional diversification of Mar enzymes and expand our understanding of their role in anaerobic sulfur metabolism.
Project description:The intermediate filament protein Nestin serves as a biomarker for stem cells and has been used to identify subsets of cancer stem-like cells. However, the mechanistic contributions of Nestin to cancer pathogenesis are not understood. Here we report that Nestin binds the hedgehog pathway transcription factor Gli3 to mediate the development of medulloblastomas of the hedgehog subtype. In a mouse model system, Nestin levels increased progressively during medulloblastoma formation resulting in enhanced tumor growth. Conversely, loss of Nestin dramatically inhibited proliferation and promoted differentiation. Mechanistic investigations revealed that the tumor-promoting effects of Nestin were mediated by binding to Gli3, a zinc finger transcription factor that negatively regulates hedgehog signaling. Nestin binding to Gli3 blocked Gli3 phosphorylation and its subsequent proteolytic processing, thereby abrogating its ability to negatively regulate the hedgehog pathway. Our findings show how Nestin drives hedgehog pathway-driven cancers and uncover in Gli3 a therapeutic target to treat these malignancies.
Project description:In this study, in order to identify miRNA targets, a degradome library derived from anthers of the WT and GMS (Genetic Male Sterility) mutant representing three stages of development was constructed and sequenced, resulting in the generation of 24.6 million raw reads. After removal of low quality sequences and adapter sequences, 24.4 million clean reads were obtained and 98% were 20 or 21 nt in length as expected in that normally length distribution peak of degradome fragment is between 20 and 21 nt [Addo-Quaye C, Eshoo TW, Bartel DP, Axtell MJ: Endogenous siRNA and miRNA targets identified by sequencing of the Arabidopsis degradome. Curr Biol 2008, 18:758-762]. Identification of miRNA targets in the WT and GMS muant anthers. Anthers of the WT and GMS mutant representing three stages of development [the meiosis stage (WT: Mar-F-1; mutant: Mar-S-1) and tetrad stage (WT: Mar-F-2; mutant: Mar-S-2), together with the uninucleate microspore stage (WT: Mar-F-3; mutant: Mar-S-3) from the GMS M-bM-^@M-^XDong AM-bM-^@M-^Y mutant and its fertile wild type] were collected during early mornings.
Project description:Non-integrating minimally sized Nano-S/MAR DNA vectors can be used to genetically modify dividing cells in place of integrating vectors such as lentivirus and sleeping beauty. They represent a unique genetic tool, which avoids vector-mediated genetic damage cells and the activation of innate immune responses. Previous work has shown that DNA vectors comprising the mammalian scaffold/matrix attachment region (S/MAR) element can provide persistent mitotic stability over hundreds of cell divisions, resisting epigenetic silencing and thereby allowing sustained transgene expression. The composition of the original S/MAR vectors does present some inherent limitations which reduce their stability and can provoke cellular toxicity. Here, we present a new system, the Nano-S/MAR, which drives higher transgene expression and has improved efficiency of establishment, due to their minimal impact on cellular processes and perturbation of the endogenous transcriptome. We show that these features enable the hitherto challenging genetic modification of patient-derived cells by using Nano-S/MARs to stably restore the tumour suppressor gene SMAD4 to a patient-derived SMAD4 knockout pancreatic cancer line. Nano-S/MAR modification does not alter the molecular or phenotypic integrity of the patient-derived cells in cell culture and xenograft mouse models. In conclusion, we show that this class of DNA vector can be used to persistently modify a wide range of cells, providing sustained high levels of transgene expression while avoiding the risks of insertional mutagenesis and other vector-mediated toxicity.
Project description:Non-integrating minimally sized Nano-S/MAR DNA vectors can be used to genetically modify dividing cells in place of integrating vectors such as lentivirus and sleeping beauty. They represent a unique genetic tool, which avoids vector-mediated genetic damage cells and the activation of innate immune responses. Previous work has shown that DNA vectors comprising the mammalian scaffold/matrix attachment region (S/MAR) element can provide persistent mitotic stability over hundreds of cell divisions, resisting epigenetic silencing and thereby allowing sustained transgene expression. The composition of the original S/MAR vectors does present some inherent limitations which reduce their stability and can provoke cellular toxicity. Here, we present a new system, the Nano-S/MAR, which drives higher transgene expression and has improved efficiency of establishment, due to their minimal impact on cellular processes and perturbation of the endogenous transcriptome. We show that these features enable the hitherto challenging genetic modification of patient-derived cells by using Nano-S/MARs to stably restore the tumour suppressor gene SMAD4 to a patient-derived SMAD4 knockout pancreatic cancer line. Nano-S/MAR modification does not alter the molecular or phenotypic integrity of the patient-derived cells in cell culture and xenograft mouse models. In conclusion, we show that this class of DNA vector can be used to persistently modify a wide range of cells, providing sustained high levels of transgene expression while avoiding the risks of insertional mutagenesis and other vector-mediated toxicity.