Direct Identification of Hundreds of Expression-Modulating Variants using a Multiplexed Reporter Assay
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MPRAs have been applied to assess biochemically annotated candidate enhancers, candidate enhancers harbouring variants that potentially mediate eQTLs, and even scans of the entire human genome.
“MPRAs have been applied to assess biochemically annotated candidate enhancers77,176-178, candidate enhancers harbouring variants that potentially mediate eQTLs179-182, and even scans of the entire human genome108,183.”
From Towards a comprehensive catalogue of validated and target-linked human enhancers (2020) · cites this paper as [180]
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“In addition, ExPecto can accurately predict causal eQTLs when evaluated with data from in vitro massively parallel reporter assays (MPRA) in lymphoblastoid cells 17 .”
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“2017;Chaudhari and Cohen 2018) for enhancer activity or the functional impact of single-nucleotide polymorphisms (SNPs) (Kwasnieski et al. 2012; Reddy et al. 2012; Vockley et al. 2015; Tewhey et al. 2016; Ulirsch et al. 2016; for reviews, see Maston et al. 2012; Spitz and Furlong 2012; Yáñez-Cuna et al. 2013; Levine e…”
Wang, X., He, L., Goggin, S. M., Saadat, A., Wang, L., Sinnott-Armstrong, N., Claussnitzer, M., & Kellis, M. (2018) · Nature Communications · doi:10.1038/s41467-018-07746-1 · Cited by 159 · see citing works
Linear models enable powerful differential activity analysis in massively parallel reporter assays
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“There has been some use of DESeq2 to identify (filter) elements with regulatory activity (differential expression of RNA relative to DNA) [19, 32].”
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“To investigate this,we integratedourZoonomiaderived phyloP scores with >35,000 assayed variants from existing 3′ untranslated region (3′UTR) (41) and eQTL (42) MPRAs.”
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“Comparative studies Another common use for MPRAs is comparative studies, looking for differential transcription induced by a putative regulatory sequence between different cell types, stimuli, or other experimental covariates [11, 16].”
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“Consistent with previous MPRA studies (17, 19, 22), most of these fragments showed modest activity; only 15.2% of active fragments showed an average activity higher than 2.”
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“First, while most non-coding mutations are not expression-modulating [23, 24], we cannot rule out within-species sequence variation between the tissues tested and reference genomes contributing to functional differences.”
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“The constructs targeted for MPRA in this eQTL challenge were designed based on the existence of previous experimental evidence that these loci were eQTLs in LCLs (1000 Genomes Project Consortium et al., 2012; Lappalainen et al., 2013; Tewhey et al., 2016).”
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“Though some recent experimental methods such as massively parallel report assay [14, 15] were successful in identifying expression-modulating variants, they are not ready to be applied in diverse human populations.”
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“Tewhey et al. 76 applied a new version of the MPRA to identify variants with effects on gene expression.”
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“Importantly, the sequences of BCs and/or the associated DNA regions of interest (hereafter ROIs) in MPRA plasmid libraries are frequently not known a priori, as random oligonucleotides are used to clone these elements [5, 10, 13].”
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“It has been applied to assess thousands of human polymorphisms with the identification of up to 842 variants which affect gene expression across different GWAS traits, including the regulation in the expression of STX4 in PD (Tewhey et al., 2018).”
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“…necessary to further investigate candidate genetic variants such as rs7604640 (Figure 4B) in future studies using massively parallel reporter assays(Tewhey et al., 2016) or self-transcribing active regulatory region sequencing(Vockley et al., 2015; Kalita et al., 2018) that can measure regulatory…”
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“…progress in determining whether germline SNVs associated with genetic disorders affect Frontiers in Genetics | www.frontiersin.org 6 February 2018 | Volume 9 | Article 16 transcriptional regulation (Figure 4B) (Melnikov et al., 2012; Mogno et al., 2013; Tewhey et al., 2016; Ulirsch et al., 2016).”
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“We calculated dCt of the System in accordance with previously published work (Tewhey et al. 2018) and recovered at a density of 3 × 10 6 cells/mL in pre-warmed RPMI including 15% FBS without antibiotics.”
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“S8)(34, 35).”
Self-citationDong, S.-C., & Boyle, A. (2021) · bioRxiv · doi:10.1093/nar/gkab924 · Cited by 17 · see citing works
“In addition, massively parallel reporter assays (MPRA) identify regulatory variants that affect gene expression levels directly (14–16).”
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“We also observed that while many of the important features are predicted for the same type of cell line as the one the MPRA experiment was performed on (lymphoblastoid cell lines), many features predicted for other cell lines, such as K562 and H1-hESC, also proved to be highly informative.”
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“Since no phenotype has been recorded for the variants residing in FANTOM5 enhancers or for the MPRA hits, we only performed an unweighted PINES analysis.”
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“We further experimentally measured the in vivo transcriptional activities of the DeepCROSS-generated RSs by MPRA experiment 18 (Fig.”
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Omelina, E., Letiagina, A. E., Boldyreva, L., Ogienko, A., Galimova, Y., Yarinich, L., Pindyurin, A., & Andreyeva, E. N. (2022) · International Journal of Molecular Sciences · doi:10.3390/ijms232415485 · Cited by 2 · see citing works
Clark, C., Yang, G., Alarcon, C., Mishra, M., & Perera, M. (2026) · Research Square · doi:10.21203/rs.3.rs-9611937/v1
“In addition, massively parallel reporter assays (MPRAs) in pharmacologically relevant liver-derived cell types, such as HepG2, offer a powerful orthogonal strategy to prioritize functional regulatory variants by directly testing their effects on gene expression 18 .”
- Authors
- Tewhey, R., Kotliar, D., Park, D. S., Liu, B., Winnicki, S., Reilly, S. K., Andersen, K. G., Mikkelsen, T. S., Lander, E. S., Schaffner, S. F., & Sabeti, P. C.
- Corresponding author
- Tewhey, Ryan
- Sabeti, Pardis C.
- ORCID
- Venue
- Cell
- Publisher
- Elsevier BV
- Published
- 2018-02
- Volume
- 172
- Issue
- 5
- Pages
- 1132-1134
- ISSN
- 0092-8674 (ISSN-L)
- OpenAlex
- W2901757429
- Cited by
- 51
- References
- 0
- Citations by year
- 2026: 1 · 2025: 2 · 2024: 8 · 2023: 7 · 2022: 2 · 2021: 12 · 2020: 9 · 2019: 8 · 2018: 2
- FWCI
- 4.74
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- Locations
- Cell (publishedVersion)
- Funders
- National Institute of General Medical Sciences (T32GM007753)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetic Associations and Epidemiology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
- Health Sciences › Medicine › Rheumatology › Spondyloarthritis Studies and Treatments
- Keywords
- EP4 receptor, ankylosing spondylitis, expression quantitative trait loci, allele-specific expression, gene expression regulation, transcription factor binding, massively parallel reporter assay, data correction
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