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]

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 Recent advances in high-throughput approaches to dissect enhancer function

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 939 ◽  
Author(s):  
David Santiago-Algarra ◽  
Lan T.M. Dao ◽  
Lydie Pradel ◽  
Alexandre España ◽  
Salvatore Spicuglia
Keyword(s):  
High Throughput ◽  
Regulatory Elements ◽  
Genetic Alterations ◽  
Regulatory Sequences ◽  
Transcription Start ◽  
Enhancer Activity ◽  
Recent Advances ◽  
Reporter Assays ◽  
Enhancer Function ◽  
Higher Eukaryotes

The regulation of gene transcription in higher eukaryotes is accomplished through the involvement of transcription start site (TSS)-proximal (promoters) and -distal (enhancers) regulatory elements. It is now well acknowledged that enhancer elements play an essential role during development and cell differentiation, while genetic alterations in these elements are a major cause of human disease. Many strategies have been developed to identify and characterize enhancers. Here, we discuss recent advances in high-throughput approaches to assess enhancer activity, from the well-established massively parallel reporter assays to the recent clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based technologies. We highlight how these approaches contribute toward a better understanding of enhancer function, eventually leading to the discovery of new types of regulatory sequences, and how the alteration of enhancers can affect transcriptional regulation.

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

2017 ◽  
Vol 34 (5) ◽  
pp. 787-794 ◽  
Author(s):  
Cynthia A Kalita ◽  
Gregory A Moyerbrailean ◽  
Christopher Brown ◽  
Xiaoquan Wen ◽  
Francesca Luca ◽  
...  
Keyword(s):  
Massively Parallel ◽  
Specific Analysis ◽  
Allele Specific ◽  
Reporter Assays

scholarly journals Meta‐analysis of massively parallel reporter assays enables prediction of regulatory function across cell types

2019 ◽  
Vol 40 (9) ◽  
pp. 1299-1313 ◽  
Author(s):  
Anat Kreimer ◽  
Zhongxia Yan ◽  
Nadav Ahituv ◽  
Nir Yosef
Keyword(s):  
Meta Analysis ◽  
Cell Types ◽  
Massively Parallel ◽  
Regulatory Function ◽  
Reporter Assays

scholarly journals Transposable elements as a potent source of diverse cis -regulatory sequences in mammalian genomes

2020 ◽  
Vol 375 (1795) ◽  
pp. 20190347 ◽  
Author(s):  
Vasavi Sundaram ◽  
Joanna Wysocka
Keyword(s):  
Gene Regulation ◽  
Transposable Elements ◽  
Regulatory Networks ◽  
Regulatory Elements ◽  
Regulatory Function ◽  
Specific Gene ◽  
Regulatory Sequences ◽  
Dependent Manner ◽  
Unique Contribution ◽  
Regulatory Potential

Eukaryotic gene regulation is mediated by cis -regulatory elements, which are embedded within the vast non-coding genomic space and recognized by the transcription factors in a sequence- and context-dependent manner. A large proportion of eukaryotic genomes, including at least half of the human genome, are composed of transposable elements (TEs), which in their ancestral form carried their own cis -regulatory sequences able to exploit the host trans environment to promote TE transcription and facilitate transposition. Although not all present-day TE copies have retained this regulatory function, the preexisting regulatory potential of TEs can provide a rich source of cis -regulatory innovation for the host. Here, we review recent evidence documenting diverse contributions of TE sequences to gene regulation by functioning as enhancers, promoters, silencers and boundary elements. We discuss how TE-derived enhancer sequences can rapidly facilitate changes in existing gene regulatory networks and mediate species- and cell-type-specific regulatory innovations, and we postulate a unique contribution of TEs to species-specific gene expression divergence in pluripotency and early embryogenesis. With advances in genome-wide technologies and analyses, systematic investigation of TEs' cis -regulatory potential is now possible and our understanding of the biological impact of genomic TEs is increasing. This article is part of a discussion meeting issue ‘Crossroads between transposons and gene regulation’.

scholarly journals Bmp2 Transcription in Osteoblast Progenitors Is Regulated by a Distant 3′ Enhancer Located 156.3 Kilobases from the Promoter

2007 ◽  
Vol 27 (8) ◽  
pp. 2934-2951 ◽  
Author(s):  
Ronald L. Chandler ◽  
Kelly J. Chandler ◽  
Karen A. McFarland ◽  
Douglas P. Mortlock
Keyword(s):  
Risk Factor ◽  
Osteoblast Differentiation ◽  
Expression Patterns ◽  
Artificial Chromosome ◽  
Regulatory Elements ◽  
Bone Morphogenetic Protein 2 ◽  
Regulatory Function ◽  
Regulatory Sequences ◽  
Bac Clone ◽  
Osteoblast Progenitor

ABSTRACT Bone morphogenetic protein 2 (encoded by Bmp2) has been implicated as an important signaling ligand for osteoblast differentiation and bone formation and as a genetic risk factor for osteoporosis. To initially survey a large genomic region flanking the mouse Bmp2 gene for cis-regulatory function, two bacterial artificial chromosome (BAC) clones that extend far upstream and downstream of the gene were engineered to contain a lacZ reporter cassette and tested in transgenic mice. Each BAC clone directs a distinct subset of normal Bmp2 expression patterns, suggesting a modular arrangement of distant Bmp2 regulatory elements. Strikingly, regulatory sequences required for Bmp2 expression in differentiating osteoblasts, as well as tooth buds, hair placodes, kidney, and other tissues, are located more than 53 kilobases 3′ to the promoter. By testing BACs with engineered deletions across this distant 3′ region, we parsed these regulatory elements into separate locations and more closely refined the location of the osteoblast progenitor element. Finally, a conserved osteoblast progenitor enhancer was identified within a 656-bp sequence located 156.3 kilobases 3′ from the promoter. The identification of this enhancer should permit further investigation of upstream regulatory mechanisms that control Bmp2 transcription during osteoblast differentiation and are relevant to further studies of Bmp2 as a candidate risk factor gene for osteoporosis.

scholarly journals Massively parallel reporter perturbation assay uncovers temporal regulatory architecture during neural differentiation

2021 ◽  
Author(s):  
Anat Kreimer ◽  
Tal Ashuach ◽  
Fumitaka Inoue ◽  
Alex Khodaverdian ◽  
Nir Yosef ◽  
...  
Keyword(s):  
Neural Differentiation ◽  
Cellular Differentiation ◽  
Early Time ◽  
Regulatory Elements ◽  
Fine Tuning ◽  
Massively Parallel ◽  
Regulatory Architecture ◽  
Cellular Environment ◽  
Sequence Components ◽  
Reporter Assays

AbstractGene regulatory elements play a key role in orchestrating gene expression during cellular differentiation, but what determines their function over time remains largely unknown. Here, we performed perturbation-based massively parallel reporter assays at seven early time points of neural differentiation to systematically characterize how regulatory elements and motifs within them guide cellular differentiation. By perturbing over 2,000 putative DNA binding motifs in active regulatory regions, we delineated four categories of functional elements, and observed that activity direction is mostly determined by the sequence itself, while the magnitude of effect depends on the cellular environment. We also find that fine-tuning transcription rates is often achieved by a combined activity of adjacent activating and repressing elements. Our work provides a blueprint for the sequence components needed to induce different transcriptional patterns in general and specifically during neural differentiation.

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.

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