scholarly journals Measuring cis-regulatory energetics in living cells using allelic manifolds

2018 ◽  
Author(s):  
Talitha Forcier ◽  
Andalus Ayaz ◽  
Manraj S. Gill ◽  
Daniel Jones ◽  
Rob Phillips ◽  
...  
Keyword(s):  
Gene Expression ◽  
Living Cells ◽  
Massively Parallel ◽  
Regulatory Sequences ◽  
Allelic Series ◽  
Cooperative Interactions ◽  
Biophysical Models ◽  
Prior Literature ◽  
Reporter Assays ◽  
Classical Genetics

AbstractGene expression in all organisms is controlled by cooperative interactions between DNA-bound transcription factors (TFs), but quantitatively measuring TF-DNA and TF-TF interactions remains difficult. Here we introduce a strategy for precisely measuring the Gibbs free energy of such interactions in living cells. This strategy centers on the measurement and modeling of “allelic manifolds”, a multidimensional generalization of the classical genetics concept of allelic series. Allelic manifolds are measured using reporter assays performed on strategically designed cis-regulatory sequences. Quantitative biophysical models are then fit to the resulting data. We used this strategy to study regulation by twoEscherichia coliTFs, CRP and σ70RNA polymerase. Doing so, we consistently obtained energetic measurements precise to ~ 0.1 kcal/mol. We also obtained multiple results that deviate from the prior literature. Our strategy is compatible with massively parallel reporter assays in both prokaryotes and eukaryotes, and should therefore be highly scalable and broadly applicable.

scholarly journals Measuring cis-regulatory energetics in living cells using allelic manifolds

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Talitha L Forcier ◽  
Andalus Ayaz ◽  
Manraj S Gill ◽  
Daniel Jones ◽  
Rob Phillips ◽  
...  
Keyword(s):  
Living Cells ◽  
Massively Parallel ◽  
Editorial Note ◽  
Regulatory Sequences ◽  
Allelic Series ◽  
Cooperative Interactions ◽  
Biophysical Models ◽  
Prior Literature ◽  
Reporter Assays ◽  
Classical Genetics

Gene expression in all organisms is controlled by cooperative interactions between DNA-bound transcription factors (TFs), but quantitatively measuring TF-DNA and TF-TF interactions remains difficult. Here we introduce a strategy for precisely measuring the Gibbs free energy of such interactions in living cells. This strategy centers on the measurement and modeling of ‘allelic manifolds’, a multidimensional generalization of the classical genetics concept of allelic series. Allelic manifolds are measured using reporter assays performed on strategically designed cis-regulatory sequences. Quantitative biophysical models are then fit to the resulting data. We used this strategy to study regulation by two Escherichia coli TFs, CRP andσ70RNA polymerase. Doing so, we consistently obtained energetic measurements precise to∼0.1kcal/mol. We also obtained multiple results that deviate from the prior literature. Our strategy is compatible with massively parallel reporter assays in both prokaryotes and eukaryotes, and should therefore be highly scalable and broadly applicable.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that minor issues remain unresolved (<xref ref-type="decision-letter" rid="SA1">see decision letter</xref>).

scholarly journals The inducible lac operator-repressor system is functional in zebrafish cells

2020 ◽  
Author(s):  
Sierra S. Nishizaki ◽  
Torrin L. McDonald ◽  
Gregory A. Farnum ◽  
Monica J. Holmes ◽  
Melissa L. Drexel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Model Organism ◽  
Firefly Luciferase ◽  
Regulatory Elements ◽  
Regulatory Sequences ◽  
Flexible Tool ◽  
Lac Operator ◽  
Promising Tool ◽  
Reporter Assays ◽  
Spatio Temporal

AbstractBackgroundZebrafish are a foundational model organism for studying the spatio-temporal activity of genes and their regulatory sequences. A variety of approaches are currently available for editing genes and modifying gene expression in zebrafish, including RNAi, Cre/lox, and CRISPR-Cas9. However, the lac operator-repressor system, a component of the E. coli lac operon which has been adapted for use in many other species and is a valuable, flexible tool for studying the inducible modulation of gene expression, has not previously been tested in zebrafish.ResultsHere we demonstrate that the lac operator-repressor system robustly decreases expression of firefly luciferase in cultured zebrafish fibroblast cells. Our work establishes the lac operator-repressor system as a promising tool for the manipulation of gene expression in whole zebrafish.ConclusionsOur results lay the groundwork for the development of lac-based reporter assays in zebrafish, and adds to the tools available for investigating dynamic gene expression in embryogenesis. We believe that this work will catalyze the development of new reporter assay systems to investigate uncharacterized regulatory elements and their cell-type specific activities.

scholarly journals A systematic approach for dissecting the molecular mechanisms of transcriptional regulation in bacteria

2017 ◽  
Author(s):  
Nathan M. Belliveau ◽  
Stephanie L. Barnes ◽  
William T. Ireland ◽  
Daniel L. Jones ◽  
Mike J. Sweredoski ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Regulatory Sequences ◽  
Bacterial Genomes ◽  
Information Theoretic ◽  
E Coli ◽  
Biophysical Models ◽  
Wide Range ◽  
Reporter Assays ◽  
Nucleotide Resolution ◽  
Almost All

Gene regulation is one of the most ubiquitous processes in biology. But while the catalog of bacterial genomes continues to expand rapidly, we remain ignorant about how almost all of the genes in these genomes are regulated. At present, characterizing the molecular mechanisms by which individual regulatory sequences operate requires focused efforts using low-throughput methods. Here we show how a combination of massively parallel reporter assays, mass spectrometry, and information-theoretic modeling can be used to dissect bacterial promoters in a systematic and scalable way. We demonstrate this method on both well-studied and previously uncharacterized promoters in the enteric bacterium Escherichia coli. In all cases we recover nucleotide-resolution models of promoter mechanism. For some promoters, including previously unannotated ones, the approach allowed us to further extract quantitative biophysical models describing input-output relationships. This method opens up the possibility of exhaustively dissecting the mechanisms of promoter function in E. coli and a wide range of other bacteria.

scholarly journals Systematic approach for dissecting the molecular mechanisms of transcriptional regulation in bacteria

2018 ◽  
Vol 115 (21) ◽  
pp. E4796-E4805 ◽  
Author(s):  
Nathan M. Belliveau ◽  
Stephanie L. Barnes ◽  
William T. Ireland ◽  
Daniel L. Jones ◽  
Michael J. Sweredoski ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Regulatory Sequences ◽  
Bacterial Genomes ◽  
Information Theoretic ◽  
E Coli ◽  
Biophysical Models ◽  
Wide Range ◽  
Reporter Assays ◽  
Nucleotide Resolution ◽  
Almost All

Gene regulation is one of the most ubiquitous processes in biology. However, while the catalog of bacterial genomes continues to expand rapidly, we remain ignorant about how almost all of the genes in these genomes are regulated. At present, characterizing the molecular mechanisms by which individual regulatory sequences operate requires focused efforts using low-throughput methods. Here, we take a first step toward multipromoter dissection and show how a combination of massively parallel reporter assays, mass spectrometry, and information-theoretic modeling can be used to dissect multiple bacterial promoters in a systematic way. We show this approach on both well-studied and previously uncharacterized promoters in the enteric bacterium Escherichia coli. In all cases, we recover nucleotide-resolution models of promoter mechanism. For some promoters, including previously unannotated ones, the approach allowed us to further extract quantitative biophysical models describing input–output relationships. Given the generality of the approach presented here, it opens up the possibility of quantitatively dissecting the mechanisms of promoter function in E. coli and a wide range of other bacteria.

scholarly journals Predicting gene expression in massively parallel reporter assays: A comparative study

2017 ◽  
Vol 38 (9) ◽  
pp. 1240-1250 ◽  
Author(s):  
Anat Kreimer ◽  
Haoyang Zeng ◽  
Matthew D. Edwards ◽  
Yuchun Guo ◽  
Kevin Tian ◽  
...  
Keyword(s):  
Gene Expression ◽  
Comparative Study ◽  
Massively Parallel ◽  
Reporter Assays

scholarly journals MPRAnator: a web-based tool for the design of Massively Parallel Reporter Assay experiments.

2015 ◽  
Author(s):  
Ilias Georgakopoulos-Soares ◽  
Naman Jain ◽  
Jesse Gray ◽  
Martin Hemberg
Keyword(s):  
High Throughput ◽  
Regulatory Elements ◽  
Massively Parallel ◽  
Regulatory Sequences ◽  
Reporter Assay ◽  
Sequencing Technologies ◽  
Fine Control ◽  
Reporter Assays ◽  
Dna Regulatory Elements ◽  
Massively Parallel Reporter Assay

DNA regulatory elements contain short motifs where transcription factors (TFs) can bind to modulate gene expression. Although the broad principles of TF regulation are well understood, the rules that dictate how combinatorial TF binding translates into transcriptional activity remain largely unknown. With the rapid advances in DNA synthesis and sequencing technologies and the continuing decline in the associated costs, high-throughput experiments can be performed to investigate the regulatory role of thousands of oligonucleotide sequences simultaneously. Nevertheless, designing high-throughput reporter assay experiments such as Massively Parallel Reporter Assays (MPRAs) and similar methods remains challenging. We introduce MPRAnator, a set of tools that facilitate rapid design of MPRA experiments. With MPRA Motif design, a set of variables provides fine control of how motifs are placed into sequences therefore allowing the user to investigate the rules that govern TF occupancy. MPRA SNP design can be used to investigate the functional effects of single or combinations of SNPs at regulatory sequences. Finally, the Transmutation tool allows for the design of negative controls by permitting scrambling, reversing, complementing or introducing multiple random mutations in the input sequences or motifs.

scholarly journals MPRAnalyze - A statistical framework for Massively Parallel Reporter Assays

2019 ◽  
Author(s):  
Tal Ashuach ◽  
David Sebastian Fischer ◽  
Anat Kreimer ◽  
Nadav Ahituv ◽  
Fabian Theis ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Linear Models ◽  
Simulated Data ◽  
R Package ◽  
Massively Parallel ◽  
Published Data ◽  
Regulatory Sequence ◽  
Regulatory Sequences ◽  
Statistical Framework ◽  
Reporter Assays

AbstractMassively parallel reporter assays (MPRAs) are a technique that enables testing thousands of regulatory DNA sequences and their variants in a single, quantitative experiment. Despite growing popularity, there is lack of statistical methods that account for the different sources of uncertainty inherent to these assays, thus effectively leveraging their promise. Development of such methods could help enhance our ability to identify regulatory sequences in the genome, understand their function under various setting, and ultimately gain a better understanding of how the regulatory code and its alteration lead to phenotypic consequence.Here we present MPRAnalyze: a statistical framework dedicated to analyzing MPRA count data. MPRAnalyze addresses the major questions that are posed in the context of MPRA experiments: estimating the magnitude of the effect of a regulatory sequence in a single condition setting, and comparing differential activity of regulatory sequences across multiple conditions. The framework uses a nested construction of generalized linear models to account for uncertainty in both DNA and RNA observations, controls for various sources of unwanted variation, and incorporates negative controls for robust hypothesis testing, thereby providing clear quantitative answers in complex experimental settings.We demonstrate the robustness, accuracy and applicability of MPR-Analyze on simulated data and published data sets and compare it against the existing analysis methodologies. MPRAnalyze is implemented as an R package and is publicly available through Bioconductor [1].

scholarly journals QuASAR-MPRA: Accurate allele-specific analysis for massively parallel reporter assays

2017 ◽  
Author(s):  
Cynthia A. Kalita ◽  
Gregory A. Moyerbrailean ◽  
Christopher Brown ◽  
Xiaoquan Wen ◽  
Francesca Luca ◽  
...  
Keyword(s):  
Data Analysis ◽  
High Throughput ◽  
Regulatory Elements ◽  
Massively Parallel ◽  
Regulatory Function ◽  
Regulatory Sequences ◽  
Analysis Tools ◽  
Specific Analysis ◽  
Allele Specific ◽  
Reporter Assays

ABSTRACTMotivationThe majority of the human genome is composed of non-coding regions containing regulatory elements such as enhancers, which are crucial for controlling gene expression. Many variants associated with complex traits are in these regions, and may disrupt gene regulatory sequences. Consequently, it is important to not only identify true enhancers but also to test if a variant within an enhancer affects gene regulation. Recently, allele-specific analysis in high-throughput reporter assays, such as massively parallel reporter assays (MPRA), have been used to functionally validate non-coding variants. However, we are still missing high-quality and robust data analysis tools for these datasets.ResultsWe have further developed our method for allele-specific analysis QuASAR (quantitative allele-specific analysis of reads) to analyze allele-specific signals in barcoded read counts data from MPRA. Using this approach, we can take into account the uncertainty on the original plasmid proportions, over-dispersion, and sequencing errors. The provided allelic skew estimate and its standard error also simplifies meta-analysis of replicate experiments. Additionally, we show that a beta-binomial distribution better models the variability present in the allelic imbalance of these synthetic reporters and results in a test that is statistically well calibrated under the null. Applying this approach to the MPRA data by Tewheyet al.(2016), we found 602 SNPs with significant (FDR 10%) allele-specific regulatory function in LCLs. We also show that we can combine MPRA with QuASAR estimates to validate existing experimental and computational annotations of regulatory variants. Our study shows that with appropriate data analysis tools, we can improve the power to detect allelic effects in high throughput reporter assays.Availabilityhttp://github.com/piquelab/QuASAR/tree/master/[email protected];[email protected]

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