MPRA

SUMMARY: Massively parallel reporter assay (MPRA) is an important technology to evaluate the impact of genetic variants on gene regulation. Here, we present MPRAVarDB, an online database and web server, for exploring regulatory effects of genetic variants. MPRAVarDB harbors 18 MPRA experiments designed to assess the regulatory effects of genetic variants associated with GWAS loci, eQTLs and various genomic features, resulting in a total of 242,818 variants tested across more than 30 cell lines and 30 human diseases or traits...

BACKGROUND: The rise of large-scale multi-species genome sequencing projects promises to shed new light on how genomes encode gene regulatory instructions. To this end, new algorithms are needed that can leverage conservation to capture regulatory elements while accounting for their evolution. RESULTS: Here, we introduce species-aware DNA language models, which we trained on more than 800 species spanning over 500 million years of evolution. Investigating their ability to predict masked nucleotides from context, we show that DNA language models distinguish transcription factor and RNA-binding protein motifs from background non-coding sequence...

A technique called mSTARR-seq sheds light on how DNA methylation may shape responses to external stimuli by altering the activity of sequences that control gene expression.

Genome-wide association studies have shown that common genetic variants associated with complex diseases are mostly located in non-coding regions, which may not be causal. In addition, the limited number of validated non-coding functional variants makes it difficult to develop an effective supervised learning model. Therefore, improving the accuracy of predicting non-coding causal variants has become critical. This study aims to build a transfer learning-based machine learning method for predicting regulatory variants to overcome the problem of limited sample size...

Genome wide association studies (GWAS) have identified a number of genomic loci that are associated with Parkinson's disease (PD) risk. However, the majority of these variants lie in non-coding regions, and thus the mechanisms by which they influence disease development, and/or potential subtypes, remain largely elusive. To address this, we used a massively parallel reporter assay (MPRA) to screen the regulatory function of 5254 variants that have a known or putative connection to PD. We identified 138 loci with enhancer activity, of which 27 exhibited allele-specific regulatory activity in HEK293 cells...

Previously, we showed that a massively parallel reporter assay, mSTARR-seq, could be used to simultaneously test for both enhancer-like activity and DNA methylation-dependent enhancer activity for millions of loci in a single experiment (Lea et al., 2018). Here, we apply mSTARR-seq to query nearly the entire human genome, including almost all CpG sites profiled either on the commonly used Illumina Infinium MethylationEPIC array or via reduced representation bisulfite sequencing. We show that fragments containing these sites are enriched for regulatory capacity, and that methylation-dependent regulatory activity is in turn sensitive to the cellular environment...

We designed a massively parallel reporter assay (MPRA) in an Epstein-Barr virus transformed B cell line to directly characterize the potential for histone post-translational modifications, i.e., histone quantitative trait loci (hQTLs), expression QTLs (eQTLs), and variants on SLE and autoimmune (AI) disease risk haplotypes to modulate regulatory activity in an allele dependent manner. Our study demonstrates that hQTLs, as a group, are more likely to modulate regulatory activity in an MPRA compared to other variant classes tested, including a set of eQTLs previously shown to interact with hQTLs and tested AI risk variants...

Rare coding mutations cause ∼45% of congenital heart disease (CHD). Noncoding mutations that perturb cis-regulatory elements (CREs) likely contribute to the remaining cases, but their identification has been problematic. Using a lentiviral massively parallel reporter assay (lentiMPRA) in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we functionally evaluated 6,590 noncoding de novo variants (ncDNVs) prioritized from the whole-genome sequencing of 750 CHD trios. A total of 403 ncDNVs substantially affected cardiac CRE activity...

The breakthrough high-throughput measurement of the cis-regulatory activity of millions of randomly generated promoters provides an unprecedented opportunity to systematically decode the cis-regulatory logic that determines the expression values. We developed an end-to-end transformer encoder architecture named Proformer to predict the expression values from DNA sequences. Proformer used a Macaron-like Transformer encoder architecture, where two half-step feed forward (FFN) layers were placed at the beginning and the end of each encoder block, and a separable 1D convolution layer was inserted after the first FFN layer and in front of the multi-head attention layer...

Although genome-wide association studies (GWAS) have identified loci associated with alcohol consumption and alcohol use disorder (AUD), they do not identify which variants are functional. To approach this, we evaluated the impact of variants in 3' untranslated regions (3'-UTRs) of genes in loci associated with substance use and neurological disorders using a massively parallel reporter assay (MPRA) in neuroblastoma and microglia cells. Functionally impactful variants explained a higher proportion of heritability of alcohol traits than non-functional variants...

Regulation of gene expression through enhancers is one of the major processes shaping the structure and function of the human brain during development. High-throughput assays have predicted thousands of enhancers involved in neurodevelopment, and confirming their activity through orthogonal functional assays is crucial. Here, we utilized Massively Parallel Reporter Assays (MPRAs) in stem cells and forebrain organoids to evaluate the activity of ~ 7000 gene-linked enhancers previously identified in human fetal tissues and brain organoids...

Dozens of variants in the gene for the homeodomain transcription factor (TF) cone-rod homeobox (CRX) are linked with human blinding diseases that vary in their severity and age of onset. How different variants in this single TF alter its function in ways that lead to a range of phenotypes is unclear. We characterized the effects of human disease-causing variants on CRX cis -regulatory function by deploying massively parallel reporter assays (MPRAs) in mouse retina explants carrying knock-ins of two variants, one in the DNA-binding domain (p...

UNLABELLED: For the vast majority of genes in sequenced genomes, there is limited understanding of how they are regulated. Without such knowledge, it is not possible to perform a quantitative theory-experiment dialogue on how such genes give rise to physiological and evolutionary adaptation. One category of high-throughput experiments used to understand the sequence-phenotype relationship of the transcriptome is massively parallel reporter assays (MPRAs). However, to improve the versatility and scalability of MPRA pipelines, we need a "theory of the experiment" to help us better understand the impact of various biological and experimental parameters on the interpretation of experimental data...

For the vast majority of genes in sequenced genomes, there is limited understanding of how they are regulated. Without such knowledge, it is not possible to perform a quantitative theory-experiment dialogue on how such genes give rise to physiological and evolutionary adaptation. One category of high-throughput experiments used to understand the sequence-phenotype relationship of the transcriptome is massively parallel reporter assays (MPRAs). However, to improve the versatility and scalability of MPRA pipelines, we need a "theory of the experiment" to help us better understand the impact of various biological and experimental parameters on the interpretation of experimental data...

The advent of perturbation-based massively parallel reporter assays (MPRAs) technique has facilitated the delineation of the roles of non-coding regulatory elements in orchestrating gene expression. However, computational efforts remain scant to evaluate and establish guidelines for sequence design strategies for perturbation MPRAs. In this study, we propose a framework for evaluating and comparing various perturbation strategies for MPRA experiments. Within this framework, we benchmark three different perturbation approaches from the perspectives of alteration in motif-based profiles, consistency of MPRA outputs, and robustness of models that predict the activities of putative regulatory motifs...

Streptomycin-resistant (SM-resistant) Mycobacterium tuberculosis (M. tuberculosis) is a major concern in tuberculosis (TB) treatment. However, the mechanisms underlying streptomycin resistance remain unclear. This study primarily aimed to perform preliminary screening of genes associated with streptomycin resistance through conjoint analysis of multiple genomics. Genome-wide methylation, transcriptome, and proteome analyses were used to elucidate the associations between specific genes and streptomycin resistance in M...

Eosinophilic esophagitis (EoE) is a rare atopic disorder associated with esophageal dysfunction, including difficulty swallowing, food impaction, and inflammation, that develops in a small subset of people with food allergies. Genome-wide association studies (GWASs) have identified 9 independent EoE risk loci reaching genome-wide significance (p < 5 × 10-8 ) and 27 additional loci of suggestive significance (5 × 10-8  < p < 1 × 10-5 )...

The majority of disease-associated variants identified through genome-wide association studies are located outside of protein-coding regions. Prioritizing candidate regulatory variants and gene targets to identify potential biological mechanisms for further functional experiments can be challenging. To address this challenge, we developed FORGEdb ( https://forgedb.cancer.gov/ ; https://forge2.altiusinstitute.org/files/forgedb.html ; and https://doi.org/10.5281/zenodo.10067458 ), a standalone and web-based tool that integrates multiple datasets, delivering information on associated regulatory elements, transcription factor binding sites, and target genes for over 37 million variants...

UNLABELLED: The 5' UTRs of mRNAs are critical for translation regulation, but their in vivo regulatory features are poorly characterized. Here, we report the regulatory landscape of 5' UTRs during early zebrafish embryogenesis using a massively parallel reporter assay of 18,154 sequences coupled to polysome profiling. We found that the 5' UTR is sufficient to confer temporal dynamics to translation initiation, and identified 86 motifs enriched in 5' UTRs with distinct ribosome recruitment capabilities...

Massively parallel reporter assays (MPRAs) represent a set of high-throughput technologies that measure the functional effects of thousands of sequences/variants on gene regulatory activity. There are several different variations of MPRA technology and they are used for numerous applications, including regulatory element discovery, variant effect measurement, saturation mutagenesis, synthetic regulatory element generation or characterization of evolutionary gene regulatory differences. Despite their many designs and uses, there is no comprehensive database that incorporates the results of these experiments...