Decoding enhancers using massively parallel reporter assays

Inoue, Fumitaka; Ahituv, Nadav

doi:10.1016/j.ygeno.2015.06.005

Cited by 230 publications

(233 citation statements)

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“…Deciphering the motif grammar that Cold Spring Harbor Laboratory Press on May 9, 2018 -Published by genome.cshlp.org Downloaded from distinguishes activated and repressed cis-regulatory elements should be an important goal of future studies. To this end, statistical learning models taking into account motif orientation and spacing could be used to guide the design of massively parallel reporter assays based on synthetic regulatory elements or more targeted in vitro assays (Fiore and Cohen 2016;Inoue and Ahituv 2015).…”

Section: Discussionmentioning

confidence: 99%

Thrombopoietin signaling to chromatin elicits rapid and pervasive epigenome remodeling within poised chromatin architectures

et al. 2018

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Thrombopoietin (TPO) is a critical cytokine regulating hematopoietic stem cell maintenance and differentiation into the megakaryocytic lineage. However, the transcriptional and chromatin dynamics elicited by TPO signaling are poorly understood. Here, we study the immediate early transcriptional and cis-regulatory responses to TPO in hematopoietic stem/progenitor cells (HSPCs) and use this paradigm of cytokine signaling to chromatin to dissect the relation between cisregulatory activity and chromatin architecture. We show that TPO profoundly alters the transcriptome of HSPCs, with key hematopoietic regulators being transcriptionally repressed within 30 minutes of TPO. By examining cis-regulatory dynamics and chromatin architectures, we demonstrate that these changes are accompanied by rapid and extensive epigenome remodeling of cis-regulatory landscapes that is spatially coordinated within topologically associating domains (TADs). Moreover, TPO-responsive enhancers are spatially clustered and engage in preferential homotypic intra-and inter-TAD interactions that are largely refractory to TPO signaling. By further examining the link between cis-regulatory dynamics and chromatin looping, we show that rapid modulation of cis-regulatory activity is largely independent of chromatin looping dynamics. Finally, we show that, although activated and repressed cis-regulatory elements share remarkably similar DNA sequence compositions, transcription factor binding patterns accurately predict rapid cis-regulatory responses to TPO.

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Section: Discussionmentioning

confidence: 99%

Thrombopoietin signaling to chromatin elicits rapid and pervasive epigenome remodeling within poised chromatin architectures

et al. 2018

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“…Some aspects of MAVEs, namely library synthesis and sequencing, are relatively well established and have been described extensively. 12,13,[31][32][33] Here, we focus on the aspects that are critical for the goal of prospectively interpreting human genetic variation.…”

Section: Annotating Every Possible Variant In Disease-related Functiomentioning

confidence: 99%

“…Several versions of this technology have been developed to improve throughput, assayed sequence length, nucleotide variant testing, genomic integration, and more. 13,[55][56][57] For example, MPRAs have been used to quantify the effects of more than 100,000 variants of three liver enhancers. 58 MPRAs have also been used to simultaneously test thousands of variants associated with eQTLs 22 or variants in linkage disequilibrium with lead SNPs from GWASs for red blood cell traits.…”

Section: Annotating Every Possible Variant In Disease-related Functiomentioning

confidence: 99%

“…This linkage enables the use of DNA sequencing for scoring each variant simultaneously. For example, massively parallel reporter assays (MPRAs) query the effects of regulatory DNA variants on the expression of reporter genes, 13 whereas splicing assays reveal variant effects on mRNA processing. 14,15 The effect of variants on mRNA stability and translation can also be measured by multiplex assays.…”

Section: Introductionmentioning

confidence: 99%

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Variant Interpretation: Functional Assays to the Rescue

Starita

Ahituv

Dunham

et al. 2017

The American Journal of Human Genetics

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Classical genetic approaches for interpreting variants, such as case-control or co-segregation studies, require finding many individuals with each variant. Because the overwhelming majority of variants are present in only a few living humans, this strategy has clear limits. Fully realizing the clinical potential of genetics requires that we accurately infer pathogenicity even for rare or private variation. Many computational approaches to predicting variant effects have been developed, but they can identify only a small fraction of pathogenic variants with the high confidence that is required in the clinic. Experimentally measuring a variant's functional consequences can provide clearer guidance, but individual assays performed only after the discovery of the variant are both time and resource intensive. Here, we discuss how multiplex assays of variant effect (MAVEs) can be used to measure the functional consequences of all possible variants in disease-relevant loci for a variety of molecular and cellular phenotypes. The resulting large-scale functional data can be combined with machine learning and clinical knowledge for the development of ''lookup tables'' of accurate pathogenicity predictions. A coordinated effort to produce, analyze, and disseminate large-scale functional data generated by multiplex assays could be essential to addressing the variant-interpretation crisis.

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“…Unfortunately our ability to explore the functional aspects of diverse sequences remains minuscule. Multiplexed functional assays are a new set of techniques (also called Massively Parallel Reporter Assays 1 and Multiplexed Assays for Variant Effects 2 ) that link a function to a next generation sequencing (NGS)-based output to allow testing of thousands to millions of sequences for function in a pooled manner. Mutagenesis and deep mutational scanning combined with multiplexed functional assays are now able to systematically probe all single and double amino acid substitutions for a protein's function 3,4 .…”

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confidence: 99%

Multiplexed Gene Synthesis in Emulsions for Exploring Protein Functional Landscapes

Plesa

Sidore

Lubock

et al. 2017

Preprint

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Next-generation sequencing has engendered an expanding suite of functional assays that can test sequence-function relationships at unprecedented scales in pooled formats (multiplex). Such assays are currently constrained by the short length of oligonucleotide (oligo) pools, which limit potential applications. Here we report a simple, low-cost, and scalable method called DropSynth that assembles gene libraries from oligo pools for use in multiplexed functional assays. DropSynth utilizes a library of barcoded beads to isolate and concentrate oligos needed for a gene's synthesis in a pooled format. These bead-bound oligos are then emulsified, processed, and assembled into genes within the emulsion droplets. We synthesized~1000 phylogenetically diverse orthologs of phosphopantetheine adenylyltransferase (PPAT) and tested their fitness in a multiplexed functional assay. While the majority of orthologs complement, those that do not are broadly distributed across the phylogenetic tree. Synthetic errors in our assemblies allow us to explore local landscapes around the designed orthologs revealing constrained mutations for complementing orthologs as well as gain-of-function mutations for low-fitness orthologs. This broad mutational scanning approach is complementary to deep mutational scanning and helps us understand proteins by probing evolutionarily divergent sequences that share function.peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/163550 doi: bioRxiv preprint first posted online Jul. 14, 2017;The rapid progress in DNA sequencing has provided a glimpse of the vast diversity of sequence and function that nature has explored. Unfortunately our ability to explore the functional aspects of diverse sequences remains minuscule. ) that link a function to a next generation sequencing (NGS)-based output to allow testing of thousands to millions of sequences for function in a pooled manner. Mutagenesis and deep mutational scanning combined with multiplexed functional assays are now able to systematically probe all single and double amino acid substitutions for a protein's function 3,4 . Despite the power of these approaches, the sequence space explored in such experiments is minute when compared to the evolutionary distance between even highly-homologous protein sequences. Our ability to sample a broad diversity of sequences is bottlenecked by two limitations in existing approaches for producing the libraries to be tested. First, low-cost microarray-based oligo libraries allow for large libraries of designed <200 nucleotide (nt) sequences 5 , which is far below the typical length of a protein and limits many other applications. Secondly, gene synthesis is capable of creating long-length sequences, but its costs currently make it prohibitively expensive for sampling large libraries of designed sequences [6][7][8][9] .Here we develop a new gene synthesis method we term DropSynth, a multiplexed...

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Decoding enhancers using massively parallel reporter assays

Cited by 230 publications

References 50 publications

Thrombopoietin signaling to chromatin elicits rapid and pervasive epigenome remodeling within poised chromatin architectures

Thrombopoietin signaling to chromatin elicits rapid and pervasive epigenome remodeling within poised chromatin architectures

Variant Interpretation: Functional Assays to the Rescue

Multiplexed Gene Synthesis in Emulsions for Exploring Protein Functional Landscapes

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