A massively parallel reporter assay library to screen short synthetic promoters in mammalian cells
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ABSTRACT:
ORGANISM(S): Mesocricetus auratus Mus musculus synthetic construct Homo sapiens
PROVIDER: GSE271608 | GEO | 2024/10/08
REPOSITORIES: GEO
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Project description:Transcriptional regulatory elements (TREs), including enhancers and promoters, determine the transcription levels of associated genes. We have recently shown that global run-on and sequencing (GRO-seq) with enrichment for 5'-capped RNAs reveals active TREs with high accuracy. Here, we demonstrate that active TREs can be identified by applying sensitive machine-learning methods to standard GRO-seq data. This approach allows TREs to be assayed together with gene expression levels and other transcriptional features in a single experiment. Our prediction method, called discriminative Regulatory Element detection from GRO-seq (dREG), summarizes GRO-seq read counts at multiple scales and uses support vector regression to identify active TREs. The predicted TREs are more strongly enriched for several marks of transcriptional activation, including eQTL, GWAS-associated SNPs, H3K27ac, and transcription factor binding than those identified by alternative functional assays. Using dREG, we survey TREs in eight human cell types and provide new insights into global patterns of TRE function.
2015-02-19 | GSE66031 | GEO
Project description:Transcriptional regulatory elements (TREs), including enhancers and promoters, determine the transcription levels of associated genes. We have recently shown that global run-on and sequencing (GRO-seq) with enrichment for 5'-capped RNAs reveals active TREs with high accuracy. Here, we demonstrate that active TREs can be identified by applying sensitive machine-learning methods to standard GRO-seq data. This approach allows TREs to be assayed together with gene expression levels and other transcriptional features in a single experiment. Our prediction method, called discriminative Regulatory Element detection from GRO-seq (dREG), summarizes GRO-seq read counts at multiple scales and uses support vector regression to identify active TREs. The predicted TREs are more strongly enriched for several marks of transcriptional activation, including eQTL, GWAS-associated SNPs, H3K27ac, and transcription factor binding than those identified by alternative functional assays. Using dREG, we survey TREs in eight human cell types and provide new insights into global patterns of TRE function. We analyzed GRO-seq or PRO-seq data from eight human cell lines. Please note that this study comprises new sample data plus reanalysis of old Sample data submitted by another user. Existing PRO-seq or GRO-seq data was combined as detailed in the GSE66031_readme.txt. See GSM1613181 and GSM1613182 Sample records for data processing information.
2015-02-19 | E-GEOD-66031 | biostudies-arrayexpress
Project description:Tachykinins (TKs) are a family of peptides involved in the peripheral and central regulation of urinary functions through the stimulation of neurokinin (NK) NK1, NK2 and NK3 receptors. Recent evidence indicates that NK1 receptors are required in antigen-induced cystitis. Therefore, determining the regulatory network downstream NK1 receptor activation is a key step toward understanding the role of TKs in inflammation. For this purpose, we used a Transcriptional Regulatory Network Analysis (TRNA) to identify biologically relevant transcriptional regulatory elements (TRE) that underline the NK1-dependent gene expression in bladder responses to inflammation. Gene expression analysis was obtained using the urinary bladder isolated from WT and NK1-R-/- mice that were stimulated with intravesical instillation of saline or antigen challenge (in sensitized mice) in order to promote cystitis. Based on cDNA array results, we selected a cluster of genes that was dependent on NK1 receptors for their up-regulation in response to inflammation. Next, we used PAINT database to retrieve upstream promoter sequences for those NK1-R-dependent genes and to identify TREs on those promoters. Finally, TREs were enriched statistically by selecting only those that were significantly expressed and a regulatory network downstream of NK1 receptor activation was determined. This work indicates an overriding participation of NK1 receptors in bladder inflammation, provides a working model for the involvement of transcription regulators such as AP1, NF-kB, and Nkx-2.5, and evokes testable hypotheses regarding the regulatory network downstream of NK1 receptor activation. Keywords: URINARY BLADDER INFLAMMATION
2005-06-17 | GSE2821 | GEO
Project description:Glycerol offers several advantages as a substrate for biotechnological applications. An important step towards using the popular production host Saccharomyces cerevisiae for glycerol-based bioprocesses have been recent studies in which commonly used S. cerevisiae strains were engineered to grow in synthetic medium containing glycerol as the sole carbon source. In order to boost extensive S. cerevisiae metabolic engineering incentives aiming at the use of glycerol, we realized that promoters with predictable expression levels in synthetic glycerol medium were required. In the current study, we used transcriptome analysis and a yECitrine-based fluorescence reporter assay to select and characterize useful 25 promoters for driving expression of target genes in S. cerevisiae under the given conditions. The promoters of the genes ALD4 and ADH2 showed 4.2- and 3-fold higher activities compared to the well-known strong TEF1 promoter. Moreover, the collection contains promoters with graded activities in synthetic glycerol medium and different degrees of glucose repression. To demonstrate the general applicability of the promoter collection, we successfully used a subset of the characterized promoters with graded activities in order to optimize growth on glycerol in an engineered derivative of CEN.PK, in which glycerol catabolism exclusively occurs via a non-native DHA pathway.
2018-03-31 | GSE109036 | GEO
Project description:Quantitative analysis of the sequence determinants of transcription and translation regulation is of special relevance for systems and synthetic biology applications. Here, we developed a novel generic approach for the fast and efficient analysis of these determinants in vivo. ELM-seq (expression level monitoring by DNA methylation) uses Dam coupled to high-throughput sequencing) as a reporter that can be detected by DNA-seq. We used the genome-reduced bacterium Mycoplasma pneumoniae to show that it is a quantitative reporter. We showed that the methylase activity correlates with protein expression, does not affect cell viability, and has a large dynamic range (~10,000-fold). We applied ELM-seq to randomized libraries of promoters or 5’ untranslated regions. We found that transcription is greatly influenced by the bases around the +1 of the transcript and the Pribnow box, and we also identified several epistatic interactions (including the +1 and the “extended Pribnow”). Regarding translation initiation, we confirmed that the Shine-Dalgarno motif is not relevant, but instead, that RNA secondary structure is the main governing factor. With this in hand, we developed a predictor to help tailor gene expression in M. pneumoniae. The simple ELM-seq methodology will allow identifying and optimizing key sequence determinants for promoter strength and translation. The ELM-seq methodology allows both researchers and companies to identify and optimize in an easy and comprehensive manner, key sequence determinants for promoter strength and translation.
2017-08-29 | PXD005606 | Pride
Project description:The transition from transcription initiation into elongation at promoters of primary response genes (PRG) in metazoan cells is controlled by inducible transcription factors, which utilize P-TEFb to phosphorylate RNA Polymerase II (Pol II) in response to stimuli. Prior to stimulation, a fraction of P-TEFb is recruited to promoters in a catalytically inactive state bound to the 7SK small nuclear ribonucleoprotein (snRNP). However, it remains unclear how and why the 7SK snRNP is assembled at promoters. Here we report that the transcriptional regulator KAP1 directly recruits the 7SK snRNP to facilitate localized release of P-TEFb, promoting rapid Pol II elongation and PRG synthesis in response to stimuli. Collectively, we have discovered and characterized a novel complex, which we term the KEC, which dictates rapid and robust PRG induction upon stimuli.
2015-10-09 | GSE72622 | GEO
Project description:New chemotherapeutics are urgently required to control the tuberculosis pandemic fueled by the emergence of multidrug- and extensively-drug-resistant Mycobacterium tuberculosis strains and the bacterium`s catastrophic alliance with HIV. Here we describe a novel trehalose-to-α-glucan pathway in M. tuberculosis comprising four enzymatic steps mediated by TreS, Pep2, GlgB, and GlgE, identified as an essential maltosyltransferase capable of utilizing maltose 1-phosphate. Using traditional and chemical reverse genetics, we show that GlgE inactivation causes rapid death of M. tuberculosis in vitro and in mice, through self-poisoning by maltose 1-phosphate accumulation driven by a self-amplifying feedback loop promoting pleiotropic phosphosugar-induced stress responses. Moreover, this α-glucan pathway exhibited a synthetic lethal interaction with the glucosyltransferase Rv3032 involved in biosynthesis of specialized α-glucan derivatives. The unique combination of gene essentiality within a synthetic lethal pathway validates GlgE as a new class of drug targets, revealing novel synergistic mechanisms to induce death in M. tuberculosis. Transcriptional profiling was performed to characterize the lethality induced by maltose 1-phosphate accumulation. Triplicate 10 mL cultures of the conditional lethal Mtb mutant strain H37Rv Delta treS Delta glgE (pMV361::treS) and of the vector control strain H37Rv Delta treS Delta glgE (pMV361) were grown in liquid culture to log-phase in the presence of 5 mM validamycin A (VA) to suppress M1P formation. Subsequently, cells were washed to remove the inhibitor; after 48 h of starvation for VA cultures were harvested. Keywords: tuberculosis, trehalose, compound treatment design, genetic modification design, and stimulus or stress design Three biological replicates with one dye-flip
2010-05-14 | E-GEOD-18575 | biostudies-arrayexpress
Project description:Rantasalo2015-Synthetic_expresion_modulator_constitutiveSTF_VP16
This model is part of a family of models describing a modular synthetic expression system that modulates the expression level of a gene in S. Cerevisae. The whole family of models is described in the article:
Synthetic transcription amplifier system for orthogonal control of gene expression in Saccharomyces cerevisiae, by
Anssi Rantasalo, Elena Czeizler, Riitta Virtanen, Juho Rousu, Harri Lähdesmäki, Merja Penttilä, Jussi Jäntti and Dominik Mojzita (submitted).
The family comprises 5 different models corresponding to the synthetic systems using:
1) the methionine induced synthetic transcription factor sTF-VP16 construct,
2) the methionine induced synthetic transcription factor sTF-B42 construct,
3) the constitutive sTF-VP16 construct,
4) the constitutive sTF-B42 construct, and
5) the constitutive sTF-VP16 construct with a different core promoter for the reporter gene.
The computational models were designed, developed and implemented by Elena Czeizler, Harri Lahdesmaki and Juho Rousu and they are all hosted separately in the Biomodels database.
The only difference between the models corresponding to the constitutive and the methionine-induced systems stands in the sTF transcription process. The only difference between the models for the systems using the sTF16 or sTF42 constructs stands in the kinetic rates associated to 2 reactions: i) the association of the polymerase with the sTF’s bound to their specific DNA sites and ii) the degradation rate of the sTF proteins corresponding to the two transcription factors sTF16 or sTF42. The first 4 models correspond to systems using the pBID2-EP core promoter for the reporter mCherry gene. Starting from the model associated to the constitutive system using the sTF16 construct we derived the 5th model corresponding to the case when the core promoter for the reporter mCherry gene is switched from pBID2-EP to pBID2-ED. The only difference between these two last models stands in the kinetic rates for the association of polymerase bound to sTF and the core promoter.
Since the 5 considered models have many parts in common, the values for the kinetic parameters corresponding to these common parts are identical in all of them.
The current model describes the gene expression modulation system using the constitutive synthetic transcription factor sTF-VP16 composed of a LexA DNA binding domain, the Herpes simplex virus transactivation domain (VP16) and a 6×His-tag. In this model, the number of boxes to which this sTF can bind (encoded in the species B) is set to 2. In the experimental setups, the number of binding boxes varied between 0 and 16 (8 binding sites on each of the 2 DNA constructs), which can be easily modified in the model by changing the initial value for B. The core promoter used for the reporter mCherry gene is pBID-EP.
All model analysis and simulations were done by using the software COPASI (Hoops, S., Sahle, S., Gauges, R., Lee, C., Pahle, J., Simus, N., Singhal, M., Xu, L., Mendes, P. and Kummer, U. (2006) COPASI- A COmplex PAthway SImulator. Bioinformatics, 22, 3067-3074.)
2016-02-23 | MODEL1510230001 | BioModels
Project description:Rantasalo2015-Synthetic_expresion_modulator_constitutiveSTF_VP16_pBID2-EDcorePromoter
This model is part of a family of models describing a modular synthetic expression system that modulates the expression level of a gene in S. Cerevisae. The whole family of models is described in the article:
Synthetic transcription amplifier system for orthogonal control of gene expression in Saccharomyces cerevisiae, by
Anssi Rantasalo, Elena Czeizler, Riitta Virtanen, Juho Rousu, Harri Lähdesmäki, Merja Penttilä, Jussi Jäntti and Dominik Mojzita (submitted).
The family comprises 5 different models corresponding to the synthetic systems using:
1) the methionine induced synthetic transcription factor sTF-VP16 construct,
2) the methionine induced synthetic transcription factor sTF-B42 construct,
3) the constitutive sTF-VP16 construct,
4) the constitutive sTF-B42 construct, and
5) the constitutive sTF-VP16 construct with a different core promoter for the reporter gene.
The computational models were designed, developed and implemented by Elena Czeizler, Harri Lahdesmaki and Juho Rousu and they are all hosted separately in the Biomodels database.
The only difference between the models corresponding to the constitutive and the methionine-induced systems stands in the sTF transcription process. The only difference between the models for the systems using the sTF16 or sTF42 constructs stands in the kinetic rates associated to 2 reactions: i) the association of the polymerase with the sTF’s bound to their specific DNA sites and ii) the degradation rate of the sTF proteins corresponding to the two transcription factors sTF16 or sTF42. The first 4 models correspond to systems using the pBID2-EP core promoter for the reporter mCherry gene. Starting from the model associated to the constitutive system using the sTF16 construct we derived the 5th model corresponding to the case when the core promoter for the reporter mCherry gene is switched from pBID2-EP to pBID2-ED. The only difference between these two last models stands in the kinetic rates for the association of polymerase bound to sTF and the core promoter.
Since the 5 considered models have many parts in common, the values for the kinetic parameters corresponding to these common parts are identical in all of them.
The current model describes the gene expression modulation system using the constitutive synthetic transcription factor sTF-VP16 composed of a LexA DNA binding domain, the Herpes simplex virus transactivation domain (VP16) and a 6×His-tag. In this model, the number of boxes to which this sTF can bind (encoded in the species B) is set to 2. In the experimental setups, the number of binding boxes varied between 0 and 16 (8 binding sites on each of the 2 DNA constructs), which can be easily modified in the model by changing the initial value for B. The core promoter used for the reporter mCherry gene is pBID2-ED.
All model analysis and simulations were done by using the software COPASI (Hoops, S., Sahle, S., Gauges, R., Lee, C., Pahle, J., Simus, N., Singhal, M., Xu, L., Mendes, P. and Kummer, U. (2006) COPASI- A COmplex PAthway SImulator. Bioinformatics, 22, 3067-3074.)
2016-02-23 | MODEL1510230003 | BioModels
Project description:Rantasalo2015-Synthetic_expresion_modulator_constitutiveSTF_B42
This model is part of a family of models describing a modular synthetic expression system that modulates the expression level of a gene in S. Cerevisae. The whole family of models is described in the article:
Synthetic transcription amplifier system for orthogonal control of gene expression in Saccharomyces cerevisiae, by
Anssi Rantasalo, Elena Czeizler, Riitta Virtanen, Juho Rousu, Harri Lähdesmäki, Merja Penttilä, Jussi Jäntti and Dominik Mojzita (submitted).
The family comprises 5 different models corresponding to the synthetic systems using:
1) the methionine induced synthetic transcription factor sTF-VP16 construct,
2) the methionine induced synthetic transcription factor sTF-B42 construct,
3) the constitutive sTF-VP16 construct,
4) the constitutive sTF-B42 construct, and
5) the constitutive sTF-VP16 construct with a different core promoter for the reporter gene.
All 5 computational models were developed by Elena Czeizler and Harri Lahdesmaki and they are all hosted separately in the Biomodels database.
The only difference between the models corresponding to the constitutive and the methionine-induced systems stands in the sTF transcription process. The only difference between the models for the systems using the sTF16 or sTF42 constructs stands in the kinetic rates associated to 2 reactions: i) the association of the polymerase with the sTF’s bound to their specific DNA sites and ii) the degradation rate of the sTF proteins corresponding to the two transcription factors sTF16 or sTF42. The first 4 models correspond to systems using the pBID2-EP core promoter for the reporter mCherry gene. Starting from the model associated to the constitutive system using the sTF16 construct we derived the 5th model corresponding to the case when the core promoter for the reporter mCherry gene is switched from pBID2-EP to pBID2-ED. The only difference between these two last models stands in the kinetic rates for the association of polymerase bound to sTF and the core promoter.
Since the 5 considered models have many parts in common, the values for the kinetic parameters corresponding to these common parts are identical in all of them.
The current model describes the gene expression modulation system using the constitutive synthetic transcription factor sTF-B42 composed of a LexA DNA binding domain, the SV40 nuclear localization signal and the B42 activation domain. In this model, the number of boxes to which this sTF can bind (encoded in the species B) is set to 2. In the experimental setups, the number of binding boxes varied between 0 and 16 (8 binding sites on each of the 2 DNA constructs), which can be easily modified in the model by changing the initial value for B. The core promoter used for the reporter mCherry gene is pBID-EP.
All model analysis and simulations were done by using the software COPASI (Hoops, S., Sahle, S., Gauges, R., Lee, C., Pahle, J., Simus, N., Singhal, M., Xu, L., Mendes, P. and Kummer, U. (2006) COPASI- A COmplex PAthway SImulator. Bioinformatics, 22, 3067-3074.)
2016-02-23 | MODEL1510230002 | BioModels