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Background: STARR-seq and other massively-parallel reporter assays are widely used to discover functional enhancers in transfected cell models, which can be confounded by plasmid vector-induced type-I interferon immune responses and lack the multicellular environment and endogenous chromatin state of complex mammalian tissues. Results: We describe HDI-STARR-seq, which combines STARR-seq plasmid library delivery to the liver, by hydrodynamic tail vein injection (HDI), with reporter RNA transcriptional initiation driven by a minimal Albumin promoter, which we show is essential for mouse liver STARR-seq enhancer activity assayed 7 days after HDI. Importantly, little or no vector-induced innate type-I interferon responses were observed. Comparisons of HDI-STARR-seq activity between male and female mouse livers and in livers from males treated with an activating ligand of the transcription factor (TF) CAR (Nr1i3) identified many condition-dependent enhancers linked to condition-specific gene expression. Further, thousands of active liver enhancers were identified using a high complexity STARR-seq library comprised of ~ 50,000 genomic regions released by DNase-I digestion of mouse liver nuclei. When compared to stringently inactive library sequences, the active enhancer sequences identified were highly enriched for liver open chromatin regions with activating histone marks (H3K27ac, H3K4me1, H3K4me3), were significantly closer to gene transcriptional start sites, and were significantly depleted of repressive (H3K27me3, H3K9me3) and transcribed region histone marks (H3K36me3). Conclusion: HDI-STARR-seq offers substantial improvements over current methodologies for large scale, functional profiling of enhancers, including condition-dependent enhancers, in liver tissue in vivo, and can be adapted to characterize enhancer activities in a variety of species and tissues by selecting suitable tissue- and species-specific promoter sequences. [ABSTRACT FROM AUTHOR]

Using machine learning (ML), we interrogated the function of all human-chimpanzee variants in 2,645 human accelerated regions (HARs), finding 43% of HARs have variants with large opposing effects on chromatin state and 14% on neurodevelopmental enhancer activity. This pattern, consistent with compensatory evolution, was confirmed using massively parallel reporter assays in chimpanzee and human neural progenitor cells. The species-specific enhancer activity of HARs was accurately predicted from the presence and absence of transcription factor footprints in each species. Despite these striking cis effects, activity of a given HAR sequence was nearly identical in human and chimpanzee cells. This suggests that HARs did not evolve to compensate for changes in the trans environment but instead altered their ability to bind factors present in both species. Thus, ML prioritized variants with functional effects on human neurodevelopment and revealed an unexpected reason why HARs may have evolved so rapidly.

Cis-regulatory elements (CREs) play a pivotal role in orchestrating interactions with trans-regulatory factors such as transcription factors, RNA-binding proteins, and noncoding RNAs. These interactions are fundamental to the molecular architecture underpinning complex and diverse biological functions in living organisms, facilitating a myriad of sophisticated and dynamic processes. The rapid advancement in the identification and characterization of these regulatory elements has been marked by initiatives such as the Encyclopedia of DNA Elements (ENCODE) project, which represents a significant milestone in the field. Concurrently, the development of CRE detection technologies, exemplified by massively parallel reporter assays, has progressed at an impressive pace, providing powerful tools for CRE discovery. The exponential growth of multimodal functional genomic data has necessitated the application of advanced analytical methods. Deep learning algorithms, particularly large language models, have emerged as invaluable tools for deconstructing the intricate nucleotide sequences governing CRE function. These advancements facilitate precise predictions of CRE activity and enable the de novo design of CREs. A deeper understanding of CRE operational dynamics is crucial for harnessing their versatile regulatory properties. Such insights are instrumental in refining gene therapy techniques, enhancing the efficacy of selective breeding programs, pushing the boundaries of genetic innovation, and opening new possibilities in microbial synthetic biology. [ABSTRACT FROM AUTHOR]

The aim of this paper is to analyse the habits of researchers and librarians when promoting their work through repositories and academic social networks in the fields of Information, Documentation and Communication. Repositories are also used for preservation and early publication (preprints) of journal articles. Methodology: A bibliographic review, an online survey, interviews with experts, and an analysis of the evolution of some repositories have been carried out. Results: Thematic repositories, which were widely used 20 years ago, are now facing competition from institutional repositories (IR) and academic social networks such as ResearchGate, Academia.edu and others. A continued decrease in the number of deposits in the e-LIS (Eprints in Library and Information Science) and MPRA (Munich Personal RePEc Archive) repositories has been observed in recent years, which seems to be due to the fact that many universities have published mandates that force professors to archive their work in their IR. Another important cause of the decrease in deposits is the greater availability of open access journals in recent years, which has made it less necessary to archive articles in repositories. Academic social networks, on the other hand, are perceived as more dynamic, allowing interaction with other authors, and not requiring the introduction of so much metadata to upload documents. [ABSTRACT FROM AUTHOR]

The notochord is a defining feature of all chordates. The transcription factors Zic and ETS regulate enhancer activity within the notochord. We conduct high-throughput screens of genomic elements within developing Ciona embryos to understand how Zic and ETS sites encode notochord activity. Our screen discovers an enhancer located near Lama, a gene critical for notochord development. Reversing the orientation of an ETS site within this enhancer abolishes expression, indicating that enhancer grammar is critical for notochord activity. Similarly organized clusters of Zic and ETS sites occur within mouse and human Lama1 introns. Within a Brachyury (Bra) enhancer, FoxA and Bra, in combination with Zic and ETS binding sites, are necessary and sufficient for notochord expression. This binding site logic also occurs within other Ciona and vertebrate Bra enhancers. Collectively, this study uncovers the importance of grammar within notochord enhancers and discovers signatures of enhancer logic and grammar conserved across chordates.

AU-rich elements (AREs) are 3' UTR cis-regulatory elements that regulate the stability of mRNAs. Consensus ARE motifs have been determined, but little is known about how differences in 3' UTR sequences that conform to these motifs affect their function. Here, we use functional annotation of sequences from 3' UTRs (fast-UTR), a massively parallel reporter assay (MPRA), to investigate the effects of 41,288 3' UTR sequence fragments from 4653 transcripts on gene expression and mRNA stability in Jurkat and Beas2B cells. Our analyses demonstrate that the length of an ARE and its registration (the first and last nucleotides of the repeating ARE motif) have significant effects on gene expression and stability. Based on this finding, we propose improved ARE classification and concomitant methods to categorize and predict the effect of AREs on gene expression and stability. Finally, to investigate the advantages of our general experimental design we examine other motifs including constitutive decay elements (CDEs), where we show that the length of the CDE stem-loop has a significant impact on steady-state expression and mRNA stability. We conclude that fast-UTR, in conjunction with our analytical approach, can produce improved yet simple sequence-based rules for predicting the activity of human 3' UTRs.

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.

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). In the current study, we perform linkage disequilibrium (LD) expansion of these loci to nominate a set of 531 variants that are potentially causal. To systematically interrogate the gene regulatory activity of these variants, we designed a massively parallel reporter assay (MPRA) containing the alleles of each variant within their genomic sequence context cloned into a GFP reporter library. Analysis of reporter gene expression in TE-7, HaCaT, and Jurkat cells revealed cell-type-specific gene regulation. We identify 32 allelic enhancer variants, representing 6 genome-wide significant EoE loci and 7 suggestive EoE loci, that regulate reporter gene expression in a genotype-dependent manner in at least one cellular context. By annotating these variants with expression quantitative trait loci (eQTL) and chromatin looping data in related tissues and cell types, we identify putative target genes affected by genetic variation in individuals with EoE. Transcription factor enrichment analyses reveal possible roles for cell-type-specific regulators, including GATA3. Our approach reduces the large set of EoE-associated variants to a set of 32 with allelic regulatory activity, providing functional insights into the effects of genetic variation in this disease. [Display omitted] Shook et al. developed and applied a massively parallel reporter assay for the allergic disease eosinophilic esophagitis (EoE). They identified common polymorphisms associated with EoE risk that also impacted transcriptional regulation. This study is an important step toward understanding how EoE genetic risk variants lead to genotype-dependent biology. [ABSTRACT FROM AUTHOR]

Neurodevelopmental disorders (NDDs) are complex disorders with variable clinical features caused by a wide range of genetic variants which disrupt genes with pleiotropic effects. Model organisms allow for recapitulation of phenotypes related to the disorder and characterization of molecular mechanisms at varying throughput and resolution. Human induced pluripotent stem cells allow for evaluating patient-specific and cell-type-specific effects. The massively parallel reporter assay (MPRA) uses high-throughput sequencing to screen thousands of genomic fragments containing patient-specific noncoding mutations to quantify their regulatory effects. Single-cell RNA sequencing and its combination with CRISPR-based screens – such as CRISPR droplet sequencing (CROP-seq) and Perturb-seq – provide promising platforms to understand gene function during brain development. Neurodevelopmental disorders (NDDs) are associated with a wide range of clinical features, affecting multiple pathways involved in brain development and function. Recent advances in high-throughput sequencing have unveiled numerous genetic variants associated with NDDs, which further contribute to disease complexity and make it challenging to infer disease causation and underlying mechanisms. Herein, we review current strategies for dissecting the complexity of NDDs using model organisms, induced pluripotent stem cells, single-cell sequencing technologies, and massively parallel reporter assays. We further highlight single-cell CRISPR-based screening techniques that allow genomic investigation of cellular transcriptomes with high efficiency, accuracy, and throughput. Overall, we provide an integrated review of experimental approaches that can be applicable for investigating a broad range of complex disorders. [ABSTRACT FROM AUTHOR]

Enhancers are cis-regulatory elements that play critical regulatory roles in modulating developmental transcription programs and driving cell-type-specific and context-dependent gene expression in the brain. The development of massively parallel reporter assays (MPRAs) has enabled high-throughput functional screening of candidate DNA sequences for enhancer activity. Tissue-specific screening of in vivo enhancer function at scale has the potential to greatly expand our understanding of the role of non-coding sequences in development, evolution, and disease. Here, we adapted a self-transcribing regulatory element MPRA strategy for delivery to early postnatal mouse brain via recombinant adeno-associated virus (rAAV). We identified and validated putative enhancers capable of driving reporter gene expression in mouse forebrain, including regulatory elements within an intronic CACNA1C linkage disequilibrium block associated with risk in neuropsychiatric disorder genetic studies. Paired screening and single enhancer in vivo functional testing, as we show here, represents a powerful approach towards characterizing regulatory activity of enhancers and understanding how enhancer sequences organize gene expression in the brain.

The Neanderthal and Denisovan genomes enabled the discovery of sequences that differ between modern and archaic humans, the majority of which are noncoding. However, our understanding of the regulatory consequences of these differences remains limited, in part due to the decay of regulatory marks in ancient samples. Here, we used a massively parallel reporter assay in embryonic stem cells, neural progenitor cells, and bone osteoblasts to investigate the regulatory effects of the 14,042 single-nucleotide modern human-specific variants. Overall, 1791 (13%) of sequences containing these variants showed active regulatory activity, and 407 (23%) of these drove differential expression between human groups. Differentially active sequences were associated with divergent transcription factor binding motifs, and with genes enriched for vocal tract and brain anatomy and function. This work provides insight into the regulatory function of variants that emerged along the modern human lineage and the recent evolution of human gene expression.

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. In particular, regulatory responses to interferon alpha (IFNA) stimulation are strongly attenuated by methyl marks, indicating widespread DNA methylation-environment interactions. In agreement, methylation-dependent responses to IFNA identified via mSTARR-seq predict methylation-dependent transcriptional responses to challenge with influenza virus in human macrophages. Our observations support the idea that pre-existing DNA methylation patterns can influence the response to subsequent environmental exposures—one of the tenets of biological embedding. However, we also find that, on average, sites previously associated with early life adversity are not more likely to functionally influence gene regulation than expected by chance.

Despite the recent explosion in genome-wide studies in chromatin and gene regulation, we are still far from extracting a set of genetic rules that can predict the function of the regulatory genome. One major reason for this deficiency is that gene regulation is a multi-layered process that involves an enormous variable space, which cannot be fully explored using native genomes. This problem can be partially solved by introducing synthetic DNA libraries into cells, a method that can test the regulatory roles of thousands to millions of sequences with limited variables. Here, we review recent applications of this method to study transcription factor (TF) binding, nucleosome positioning, and transcriptional activity. We discuss the design principles, experimental procedures, and major findings from these studies and compare the pros and cons of different approaches. [ABSTRACT FROM AUTHOR]

To realize the promise of CRISPR-Cas9-based genetics, approaches are needed to quantify a specific, molecular phenotype across genome-wide libraries of genetic perturbations. We addressed this challenge by profiling transcriptional, translational, and posttranslational reporters using CRISPR interference (CRISPRi) with barcoded expression reporter sequencing (CiBER-seq). Our barcoding approach allowed us to connect an entire library of guides to their individual phenotypic consequences using pooled sequencing. CiBER-seq profiling fully recapitulated the integrated stress response (ISR) pathway in yeast. Genetic perturbations causing uncharged transfer RNA (tRNA) accumulation activated ISR reporter transcription. Notably, tRNA insufficiency also activated the reporter, independent of the uncharged tRNA sensor. By uncovering alternate triggers for ISR activation, we illustrate how precise, comprehensive CiBER-seq profiling provides a powerful and broadly applicable tool for dissecting genetic networks.

Hematopoietic stem cell gene therapy is a promising approach for treating disorders of the hematopoietic system. Identifying combinations of cis-regulatory elements that do not impede packaging or transduction efficiency when included in lentiviral vectors has proven challenging. In this study, we deploy LV-MPRA (lentiviral vector-based, massively parallel reporter assay), an approach that simultaneously analyzes thousands of synthetic DNA fragments in parallel to identify sequence-intrinsic and lineage-specific enhancer function at near-base-pair resolution. We demonstrate the power of LV-MPRA in elucidating the boundaries of previously unknown intrinsic enhancer sequences of the human β-globin locus control region. Our approach facilitated the rapid assembly of novel therapeutic βAS3-globin lentiviral vectors harboring strong lineage-specific recombinant control elements capable of correcting a mouse model of sickle cell disease. LV-MPRA can be used to map any genomic locus for enhancer activity and facilitates the rapid development of therapeutic vectors for treating disorders of the hematopoietic system or other specific tissues and cell types.

The development and viability of an organism relies on tissue-specific gene programs. The genome regulatory elements play a key role in the regulation of such programs, whereas its disfunction can lead to the establishment of various pathologies, including cancer, congenital disorders, and autoimmune diseases. The development of high-throughput approaches in genomics has led to the emergence of massively parallel reporter assays (MPRA), which enable genome-wide screening and functional verification of regulatory elements. Although MPRA was originally used for investigation of fundamental aspects of epigenetics, it also has a great potential for clinical and practical biotechnology. Currently, MPRA is used for validation of clinically significant mutations, identification of tissue-specific regulatory elements, identification of the favorable loci for transgene integration, as well as represents an essential tool for creating highly efficient expression systems, with a wide range of applications from protein production and design of novel therapeutic antibody super-producers to gene therapy. In this review, the basic principles and areas of practical application of high-throughput reporter assays will be discussed.

Summary: Genome-wide association studies (GWASs) have successfully identified 145 genomic regions that contribute to schizophrenia risk, but linkage disequilibrium makes it challenging to discern causal variants. We performed a massively parallel reporter assay (MPRA) on 5,173 fine-mapped schizophrenia GWAS variants in primary human neural progenitors and identified 439 variants with allelic regulatory effects (MPRA-positive variants). Transcription factor binding had modest predictive power, while fine-map posterior probability, enhancer overlap, and evolutionary conservation failed to predict MPRA-positive variants. Furthermore, 64% of MPRA-positive variants did not exhibit expressive quantitative trait loci signature, suggesting that MPRA could identify yet unexplored variants with regulatory potentials. To predict the combinatorial effect of MPRA-positive variants on gene regulation, we propose an accessibility-by-contact model that combines MPRA-measured allelic activity with neuronal chromatin architecture.

Genome-wide association studies (GWAS) have localized variants to diverse classes of genomic elements, including coding sequences, introns, promoters, enhancers, 5′ and 3′-untranslated regions (UTRs), long noncoding RNA (lncRNA), and miRNAs. Annotation of diverse GWAS-identified loci from precise variant to biologic function to pathogenic impact requires understanding how different classes of genomic elements are affected by genetic variants and appropriately adapting a wide range of genomic tools to uncover variant function and effect. A variety of high-throughput screening methods and informatics tools have been adapted to capture the specific ways in which different variant classes affect gene expression and/or function. Precision gene-editing approaches that produce isogenic cells and tissues that differ only at the variant of interest comprise the gold standard for characterizing the effects of a GWAS variant in its native context. The burden of human disease lies predominantly in polygenic diseases. Since the early 2000s, genome-wide association studies (GWAS) have identified genetic variants and loci associated with complex traits. These have ranged from variants in coding sequences to mutations in regulatory regions, such as promoters and enhancers, as well as mutations affecting mediators of mRNA stability and other downstream regulators, such as 5′ and 3′-untranslated regions (UTRs), long noncoding RNA (lncRNA), and miRNA. Recent research advances in genetics have utilized a combination of computational techniques, high-throughput in vitro and in vivo screening modalities, and precise genome editing to impute the function of diverse classes of genetic variants identified through GWAS. In this review, we highlight the vastness of genomic variants associated with polygenic disease risk and address recent advances in how genetic tools can be used to functionally characterize them. [ABSTRACT FROM AUTHOR]

The integrative analysis of high-throughput reporter assays, machine learning, and profiles of epigenomic chromatin state in a broad array of cells and tissues has the potential to significantly improve our understanding of noncoding regulatory element function and its contribution to human disease. Here, we report results from the CAGI 5 regulation saturation challenge where participants were asked to predict the impact of nucleotide substitution at every base pair within five disease-associated human enhancers and nine disease-associated promoters. A library of mutations covering all bases was generated by saturation mutagenesis and altered activity was assessed in a massively parallel reporter assay (MPRA) in relevant cell lines. Reporter expression was measured relative to plasmid DNA to determine the impact of variants. The challenge was to predict the functional effects of variants on reporter expression. Comparative analysis of the full range of submitted prediction results identifies the most successful models of transcription factor binding sites, machine learning algorithms, and ways to choose among or incorporate diverse datatypes and cell-types for training computational models. These results have the potential to improve the design of future studies on more diverse sets of regulatory elements and aid the interpretation of disease-associated genetic variation.

Abstract A growing number of variants associated with risk for neurodevelopmental disorders have been identified by genome-wide association and whole genome sequencing studies. As common risk variants often fall within large haplotype blocks covering long stretches of the noncoding genome, the causal variants within an associated locus are often unknown. Similarly, the effect of rare noncoding risk variants identified by whole genome sequencing on molecular traits is seldom known without functional assays. A massively parallel reporter assay (MPRA) is an assay that can functionally validate thousands of regulatory elements simultaneously using high-throughput sequencing and barcode technology. MPRA has been adapted to various experimental designs that measure gene regulatory effects of genetic variants within cis- and trans-regulatory elements as well as posttranscriptional processes. This review discusses different MPRA designs that have been or could be used in the future to experimentally validate genetic variants associated with neurodevelopmental disorders. Though MPRA has limitations such as it does not model genomic context, this assay can help narrow down the underlying genetic causes of neurodevelopmental disorders by screening thousands of sequences in one experiment. We conclude by describing future directions of this technique such as applications of MPRA for gene-by-environment interactions and pharmacogenetics.

The enteric nervous system (ENS) is an intrinsic network of neuronal ganglia in the intestinal tube with about 100 million neurons located in the myenteric plexus and submucosal plexus. These neurons being affected in neurodegenerative diseases, such as Parkinson's disease, before pathological changes in the central nervous system (CNS) become detectable is currently a subject of discussion. Understanding how to protect these neurons is, therefore, particularly important. Since it has already been shown that the neurosteroid progesterone mediates neuroprotective effects in the CNS and PNS, it is now equally important to see whether progesterone has similar effects in the ENS. For this purpose, the RT-qPCR analyses of laser microdissected ENS neurons were performed, showing for the first time the expression of the different progesterone receptors (PR-A/B; mPRa, mPRb, PGRMC1) in rats at different developmental stages. This was also confirmed in ENS ganglia using immunofluorescence techniques and confocal laser scanning microscopy. To analyze the potential neuroprotective effects of progesterone in the ENS, we stressed dissociated ENS cells with rotenone to induce damage typical of Parkinson's disease. The potential neuroprotective effects of progesterone were then analyzed in this system. Treatment of cultured ENS neurons with progesterone reduced cell death by 45%, underscoring the tremendous neuroprotective potential of progesterone in the ENS. The additional administration of the PGRMC1 antagonist AG205 abolished the observed effect, indicating the crucial role of PGRMC1 with regard to the neuroprotective effect of progesterone. [ABSTRACT FROM AUTHOR]

Compared to transcription initiation, much less is known about transcription termination. In particular, large-scale mutagenesis studies have, so far, primarily concentrated on promoter and enhancer, but not terminator sequences. Here, we used a massively parallel reporter assay (MPRA) to systematically analyze the influence of short (8 bp) sequence variants (mutations) located downstream of the polyadenylation signal (PAS) on the steady-state mRNA level of the upstream gene, employing an eGFP reporter and human HEK293T cells as a model system. In total, we evaluated 227,755 mutations located at different overlapping positions within +17..+56 bp downstream of the PAS for their ability to regulate the reporter gene expression. We found that the positions +17..+44 bp downstream of the PAS are more essential for gene upregulation than those located more distal to the PAS, and that the mutation sequences ensuring high levels of eGFP mRNA expression are extremely T-rich. Next, we validated the positive effect of a couple of mutations identified in the MPRA screening on the eGFP and luciferase protein expression. The most promising mutation increased the expression of the reporter proteins 13-fold and sevenfold on average in HEK293T and CHO cells, respectively. Overall, these findings might be useful for further improving the efficiency of production of therapeutic products, e.g., recombinant antibodies. [ABSTRACT FROM AUTHOR]

The most recent genome-wide association study (GWAS) of cutaneous melanoma identified 54 risk-associated loci, but functional variants and their target genes for most have not been established. Here, we performed massively parallel reporter assays (MPRAs) by using malignant melanoma and normal melanocyte cells and further integrated multi-layer annotation to systematically prioritize functional variants and susceptibility genes from these GWAS loci. Of 1,992 risk-associated variants tested in MPRAs, we identified 285 from 42 loci (78% of the known loci) displaying significant allelic transcriptional activities in either cell type (FDR < 1%). We further characterized MPRA-significant variants by motif prediction, epigenomic annotation, and statistical/functional fine-mapping to create integrative variant scores, which prioritized one to six plausible candidate variants per locus for the 42 loci and nominated a single variant for 43% of these loci. Overlaying the MPRA-significant variants with genome-wide significant expression or methylation quantitative trait loci (eQTLs or meQTLs, respectively) from melanocytes or melanomas identified candidate susceptibility genes for 60% of variants (172 of 285 variants). CRISPRi of top-scoring variants validated their cis -regulatory effect on the eQTL target genes, MAFF (22q13.1) and GPRC5A (12p13.1). Finally, we identified 36 melanoma-specific and 45 melanocyte-specific MPRA-significant variants, a subset of which are linked to cell-type-specific target genes. Analyses of transcription factor availability in MPRA datasets and variant-transcription-factor interaction in eQTL datasets highlighted the roles of transcription factors in cell-type-specific variant functionality. In conclusion, MPRAs along with variant scoring effectively prioritized plausible candidates for most melanoma GWAS loci and highlighted cellular contexts where the susceptibility variants are functional. Long et al. used massively parallel reporter assays combined with variant scoring to identify functional variants from 78% of known melanoma GWAS loci, including those specific to cell of origin versus cancer contexts. Linking prioritized functional variants to eQTLs identified target genes as validated by CRISPRi. [ABSTRACT FROM AUTHOR]

In any given cell type, dozens of transcription factors (TFs) act in concert to control the activity of the genome by binding to specific DNA sequences in regulatory elements. Despite their considerable importance, we currently lack simple tools to directly measure the activity of many TFs in parallel. Massively parallel reporter assays (MPRAs) allow the detection of TF activities in a multiplexed fashion; however, we lack basic understanding to rationally design sensitive reporters for many TFs. Here, we use an MPRA to systematically optimize transcriptional reporters for 86 TFs and evaluate the specificity of all reporters across a wide array of TF perturbation conditions. We thus identified critical TF reporter design features and obtained highly sensitive and specific reporters for 62 TFs, many of which outperform available reporters. The resulting collection of "prime" TF reporters can be used to uncover TF regulatory networks and to illuminate signaling pathways. A record of this paper's transparent peer review process is included in the supplemental information., Competing Interests: Declaration of interests The authors declare that they have filed a patent application to secure intellectual property rights for the designed TF reporters. C.R. is a co-founder and shareholder of Metric Biotechnologies, Inc., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Individuals infected with the SARS-CoV-2 virus present with a wide variety of symptoms ranging from asymptomatic to severe and even lethal outcomes. Past research has revealed a genetic haplotype on chromosome 3 that entered the human population via introgression from Neanderthals as the strongest genetic risk factor for the severe response to COVID-19. However, the specific variants along this introgressed haplotype that contribute to this risk and the biological mechanisms that are involved remain unclear. Here, we assess the variants present on the risk haplotype for their likelihood of driving the genetic predisposition to severe COVID-19 outcomes. We do this by first exploring their impact on the regulation of genes involved in COVID-19 infection using a variety of population genetics and functional genomics tools. We then perform a locus-specific massively parallel reporter assay to individually assess the regulatory potential of each allele on the haplotype in a multipotent immune-related cell line. We ultimately reduce the set of over 600 linked genetic variants to identify four introgressed alleles that are strong functional candidates for driving the association between this locus and severe COVID-19. Using reporter assays in the presence/absence of SARS-CoV-2, we find evidence that these variants respond to viral infection. These variants likely drive the locus’ impact on severity by modulating the regulation of two critical chemokine receptor genes: CCR1 and CCR5. These alleles are ideal targets for future functional investigations into the interaction between host genomics and COVID-19 outcomes.

Summary: Both upregulation and downregulation by cis-regulatory elements help modulate precise gene expression. However, our understanding of repressive elements is far more limited than activating elements. To address this gap, we characterized RE1, a group of transcriptional silencers bound by REST, at genome-wide scale using a modified massively parallel reporter assay (MPRAduo). MPRAduo empirically defined a minimal binding strength of REST (REST motif-intrinsic value [m-value]), above which cofactors colocalize and silence transcription. We identified 1,500 human variants that alter RE1 silencing and found that their effect sizes are predictable when they overlap with REST-binding sites above the m-value. Additionally, we demonstrate that non-canonical REST-binding motifs exhibit silencer function only if they precisely align half sites with specific spacer lengths. Our results show mechanistic insights into RE1, which allow us to predict its activity and effect of variants on RE1, providing a paradigm for performing genome-wide functional characterization of transcription-factor-binding sites.

A growing number of variants associated with risk for neurodevelopmental disorders have been identified by genome-wide association and whole genome sequencing studies. As common risk variants often fall within large haplotype blocks covering long stretches of the noncoding genome, the causal variants within an associated locus are often unknown. Similarly, the effect of rare noncoding risk variants identified by whole genome sequencing on molecular traits is seldom known without functional assays. A massively parallel reporter assay (MPRA) is an assay that can functionally validate thousands of regulatory elements simultaneously using high-throughput sequencing and barcode technology. MPRA has been adapted to various experimental designs that measure gene regulatory effects of genetic variants within cis- and trans-regulatory elements as well as posttranscriptional processes. This review discusses different MPRA designs that have been or could be used in the future to experimentally validate genetic variants associated with neurodevelopmental disorders. Though MPRA has limitations such as it does not model genomic context, this assay can help narrow down the underlying genetic causes of neurodevelopmental disorders by screening thousands of sequences in one experiment. We conclude by describing future directions of this technique such as applications of MPRA for gene-by-environment interactions and pharmacogenetics. [ABSTRACT FROM AUTHOR]

A common goal in the convolutional neural network (CNN) modeling of genomic data is to discover specific sequence motifs. Post hoc analysis methods aid in this task but are dependent on parameters whose optimal values are unclear and applying the discovered motifs to new genomic data is not straightforward. As an alternative, we propose to learn convolutions as multinomial distributions, thus streamlining interpretable motif discovery with CNN model fitting. We developed MuSeAM (Multinomial CNNs for Sequence Activity Modeling) by implementing multinomial convolutions in a CNN model. Through benchmarking, we demonstrate the efficacy of MuSeAM in accurately modeling genomic data while fitting multinomial convolutions that recapitulate known transcription factor motifs. [ABSTRACT FROM AUTHOR]

Hematopoietic stem cell gene therapy is a promising approach for treating disorders of the hematopoietic system. Identifying combinations of cis-regulatory elements that do not impede packaging or transduction efficiency when included in lentiviral vectors has proven challenging. In this study, we deploy LV-MPRA (lentiviral vector-based, massively parallel reporter assay), an approach that simultaneously analyzes thousands of synthetic DNA fragments in parallel to identify sequence-intrinsic and lineage-specific enhancer function at near-base-pair resolution. We demonstrate the power of LV-MPRA in elucidating the boundaries of previously unknown intrinsic enhancer sequences of the human β-globin locus control region. Our approach facilitated the rapid assembly of novel therapeutic βAS3-globin lentiviral vectors harboring strong lineage-specific recombinant control elements capable of correcting a mouse model of sickle cell disease. LV-MPRA can be used to map any genomic locus for enhancer activity and facilitates the rapid development of therapeutic vectors for treating disorders of the hematopoietic system or other specific tissues and cell types.

Abstract Deriving mechanisms of immune‐mediated disease from GWAS data remains a formidable challenge, with attempts to identify causal variants being frequently hampered by strong linkage disequilibrium. To determine whether causal variants could be identified from their functional effects, we adapted a massively parallel reporter assay for use in primary CD4 T cells, the cell type whose regulatory DNA is most enriched for immune‐mediated disease SNPs. This enabled the effects of candidate SNPs to be examined in a relevant cellular context and generated testable hypotheses into disease mechanisms. To illustrate the power of this approach, we investigated a locus that has been linked to six immune‐mediated diseases but cannot be fine‐mapped. By studying the lead expression‐modulating SNP, we uncovered an NF‐κB‐driven regulatory circuit which constrains T‐cell activation through the dynamic formation of a super‐enhancer that upregulates TNFAIP3 (A20), a key NF‐κB inhibitor. In activated T cells, this feedback circuit is disrupted—and super‐enhancer formation prevented—by the risk variant at the lead SNP, leading to unrestrained T‐cell activation via a molecular mechanism that appears to broadly predispose to human autoimmunity.

Summary: Alternative DNA conformations, termed non-B DNA structures, can affect transcription, but the underlying mechanisms and their functional impact have not been systematically characterized. Here, we used computational genomic analyses coupled with massively parallel reporter assays (MPRAs) to show that certain non-B DNA structures have a substantial effect on gene expression. Genomic analyses found that non-B DNA structures at promoters harbor an excess of germline variants. Analysis of multiple MPRAs, including a promoter library specifically designed to perturb non-B DNA structures, functionally validated that Z-DNA can significantly affect promoter activity. We also observed that biophysical properties of non-B DNA motifs, such as the length of Z-DNA motifs and the orientation of G-quadruplex structures relative to transcriptional direction, have a significant effect on promoter activity. Combined, their higher mutation rate and functional effect on transcription implicate a subset of non-B DNA motifs as major drivers of human gene-expression-associated phenotypes.

The rapid improvement of descriptive genomic technologies has fueled a dramatic increase in hypothesized connections between cardiovascular gene expression and phenotypes. However, in vivo testing of these hypotheses has predominantly been relegated to slow, expensive, and linear generation of genetically modified mice. In the study of genomic cis-regulatory elements, generation of mice featuring transgenic reporters or cis-regulatory element knockout remains the standard approach. While the data obtained is of high quality, the approach is insufficient to keep pace with candidate identification and therefore results in biases introduced during the selection of candidates for validation. However, recent advances across a range of disciplines are converging to enable functional genomic assays that can be conducted in a high-throughput manner. Here, we review one such method, massively parallel reporter assays (MPRAs), in which the activities of thousands of candidate genomic regulatory elements are simultaneously assessed via the next-generation sequencing of a barcoded reporter transcript. We discuss best practices for MPRA design and use, with a focus on practical considerations, and review how this emerging technology has been successfully deployed in vivo. Finally, we discuss how MPRAs are likely to evolve and be used in future cardiovascular research.

AU-rich elements (AREs) are 30 UTR cis-regulatory elements that regulate the stability of mRNAs. Consensus ARE motifs have been determined, but little is known about how differences in 30 UTR sequences that conform to these motifs affect their function. Here, we use functional annotation of sequences from 30 UTRs (fast-UTR), a massively parallel reporter assay (MPRA), to investigate the effects of 41,288 30 UTR sequence fragments from 4653 transcripts on gene expression and mRNA stability in Jurkat and Beas2B cells. Our analyses demonstrate that the length of an ARE and its registration (the first and last nucleotides of the repeating ARE motif) have significant effects on gene expression and stability. Based on this finding, we propose improved ARE classification and concomitant methods to categorize and predict the effect of AREs on gene expression and stability. Finally, to investigate the advantages of our general experimental design we examine other motifs including constitutive decay elements (CDEs), where we show that the length of the CDE stem-loop has a significant impact on steady-state expression and mRNA stability. We conclude that fast-UTR, in conjunction with our analytical approach, can produce improved yet simple sequence-based rules for predicting the activity of human 30 UTRs. [ABSTRACT FROM AUTHOR]

Genetic studies, including genome-wide association studies, have identified many common variants that are associated with autoimmune diseases. Strikingly, in addition to being frequently observed in healthy individuals, a number of these variants are shared across diseases with diverse clinical presentations. This highlights the potential for improved autoimmune disease understanding which could be achieved by characterising the mechanism by which variants lead to increased risk of disease. Of particular interest is the potential for identifying novel drug targets or of repositioning drugs currently used in other diseases. The majority of autoimmune disease variants do not alter coding regions and it is often difficult to generate a plausible hypothetical mechanism by which variants affect disease-relevant genes and pathways. Given the interest in this area, considerable effort has been invested in developing and applying appropriate methodologies. Two of the most important technologies in this space include both low- and high-throughput genomic perturbation using the CRISPR/Cas9 system and massively parallel reporter assays. In this review, we introduce the field of autoimmune disease functional genomics and use numerous examples to demonstrate the recent and potential future impact of these technologies. [ABSTRACT FROM AUTHOR]

Understanding how enhancers drive cell-type specificity and efficiently identifying them is essential for the development of innovative therapeutic strategies. In melanoma, the melanocytic (MEL) and the mesenchymal-like (MES) states present themselves with different responses to therapy, making the identification of specific enhancers highly relevant. Using massively parallel reporter assays (MPRAs) in a panel of patient-derived melanoma lines (MM lines), we set to identify and decipher melanoma enhancers by first focusing on regions with state-specific H3K27 acetylation close to differentially expressed genes. An in-depth evaluation of those regions was then pursued by investigating the activity of overlapping ATAC-seq peaks along with a full tiling of the acetylated regions with 190 bp sequences. Activity was observed in more than 60% of the selected regions, and we were able to precisely locate the active enhancers within ATAC-seq peaks. Comparison of sequence content with activity, using the deep learning model DeepMEL2, revealed that AP-1 alone is responsible for the MES enhancer activity. In contrast, SOX10 and MITF both influence MEL enhancer function with SOX10 being required to achieve high levels of activity. Overall, our MPRAs shed light on the relationship between long and short sequences in terms of their sequence content, enhancer activity, and specificity across melanoma cell states.

Enhancers are cis-regulatory elements that play critical regulatory roles in modulating developmental transcription programs and driving cell-type-specific and context-dependent gene expression in the brain. The development of massively parallel reporter assays (MPRAs) has enabled high-throughput functional screening of candidate DNA sequences for enhancer activity. Tissue-specific screening of in vivo enhancer function at scale has the potential to greatly expand our understanding of the role of non-coding sequences in development, evolution, and disease. Here, we adapted a self-transcribing regulatory element MPRA strategy for delivery to early postnatal mouse brain via recombinant adeno-associated virus (rAAV). We identified and validated putative enhancers capable of driving reporter gene expression in mouse forebrain, including regulatory elements within an intronic CACNA1C linkage disequilibrium block associated with risk in neuropsychiatric disorder genetic studies. Paired screening and single enhancer in vivo functional testing, as we show here, represents a powerful approach towards characterizing regulatory activity of enhancers and understanding how enhancer sequences organize gene expression in the brain.

Our study investigated the underlying mechanism for the 14q24 renal cell carcinoma (RCC) susceptibility risk locus identified by a genome-wide association study (GWAS). The sentinel single-nucleotide polymorphism (SNP), rs4903064, at 14q24 confers an allele-specific effect on expression of the double PHD fingers 3 (DPF3) of the BAF SWI/SNF complex as assessed by massively parallel reporter assay, confirmatory luciferase assays, and eQTL analyses. Overexpression of DPF3 in renal cell lines increases growth rates and alters chromatin accessibility and gene expression, leading to inhibition of apoptosis and activation of oncogenic pathways. siRNA interference of multiple DPF3-deregulated genes reduces growth. Our results indicate that germline variation in DPF3 , a component of the BAF complex, part of the SWI/SNF complexes, can lead to reduced apoptosis and activation of the STAT3 pathway, both critical in RCC carcinogenesis. In addition, we show that altered DPF3 expression in the 14q24 RCC locus could influence the effectiveness of immunotherapy treatment for RCC by regulating tumor cytokine secretion and immune cell activation. [ABSTRACT FROM AUTHOR]

Massively parallel reporter assays (MPRAs) enable high-throughput functional evaluation of numerous DNA regulatory elements and/or their mutant variants. The assays are based on the construction of reporter plasmid libraries containing two variable parts, a region of interest (ROI) and a barcode (BC), located outside and within the transcription unit, respectively. Importantly, each plasmid molecule in a such a highly diverse library is characterized by a unique BC–ROI association. The reporter constructs are delivered to target cells and expression of BCs at the transcript level is assayed by RT-PCR followed by next-generation sequencing (NGS). The obtained values are normalized to the abundance of BCs in the plasmid DNA sample. Altogether, this allows evaluating the regulatory potential of the associated ROI sequences. However, depending on the MPRA library construction design, the BC and ROI sequences as well as their associations can be a priori unknown. In such a case, the BC and ROI sequences, their possible mutant variants, and unambiguous BC–ROI associations have to be identified, whereas all uncertain cases have to be excluded from the analysis. Besides the preparation of additional “mapping” samples for NGS, this also requires specific bioinformatics tools. Here, we present a pipeline for processing raw MPRA data obtained by NGS for reporter construct libraries with a priori unknown sequences of BCs and ROIs. The pipeline robustly identifies unambiguous (so-called genuine) BCs and ROIs associated with them, calculates the normalized expression level for each BC and the averaged values for each ROI, and provides a graphical visualization of the processed data.

The Neanderthal and Denisovan genomes enabled the discovery of sequences that differ between modern and archaic humans, the majority of which are noncoding. However, our understanding of the regulatory consequences of these differences remains limited, in part due to the decay of regulatory marks in ancient samples. Here, we used a massively parallel reporter assay in embryonic stem cells, neural progenitor cells, and bone osteoblasts to investigate the regulatory effects of the 14,042 single-nucleotide modern human-specific variants. Overall, 1791 (13%) of sequences containing these variants showed active regulatory activity, and 407 (23%) of these drove differential expression between human groups. Differentially active sequences were associated with divergent transcription factor binding motifs, and with genes enriched for vocal tract and brain anatomy and function. This work provides insight into the regulatory function of variants that emerged along the modern human lineage and the recent evolution of human gene expression.

Massively parallel reporter assays (MPRAs) enable high-throughput functional evaluation of numerous DNA regulatory elements and/or their mutant variants. The assays are based on the construction of reporter plasmid libraries containing two variable parts, a region of interest (ROI) and a barcode (BC), located outside and within the transcription unit, respectively. Importantly, each plasmid molecule in a such a highly diverse library is characterized by a unique BC–ROI association. The reporter constructs are delivered to target cells and expression of BCs at the transcript level is assayed by RT-PCR followed by next-generation sequencing (NGS). The obtained values are normalized to the abundance of BCs in the plasmid DNA sample. Altogether, this allows evaluating the regulatory potential of the associated ROI sequences. However, depending on the MPRA library construction design, the BC and ROI sequences as well as their associations can be a priori unknown. In such a case, the BC and ROI sequences, their possible mutant variants, and unambiguous BC–ROI associations have to be identified, whereas all uncertain cases have to be excluded from the analysis. Besides the preparation of additional "mapping" samples for NGS, this also requires specific bioinformatics tools. Here, we present a pipeline for processing raw MPRA data obtained by NGS for reporter construct libraries with a priori unknown sequences of BCs and ROIs. The pipeline robustly identifies unambiguous (so-called genuine) BCs and ROIs associated with them, calculates the normalized expression level for each BC and the averaged values for each ROI, and provides a graphical visualization of the processed data. [ABSTRACT FROM AUTHOR]

The prenatal environment can alter neurodevelopmental and clinical trajectories, markedly increasing risk for psychiatric disorders in childhood and adolescence. To understand if and how fetal exposures to stress and inflammation exacerbate manifestation of genetic risk for complex brain disorders, we report a large-scale context-dependent massively parallel reporter assay (MPRA) in human neurons designed to catalogue genotype x environment (GxE) interactions. Across 240 genome-wide association study (GWAS) loci linked to ten brain traits/disorders, the impact of hydrocortisone, interleukin 6, and interferon alpha on transcriptional activity is empirically evaluated in human induced pluripotent stem cell (hiPSC)-derived glutamatergic neurons. Of ~3,500 candidate regulatory risk elements (CREs), 11% of variants are active at baseline, whereas cue-specific CRE regulatory activity range from a high of 23% (hydrocortisone) to a low of 6% (IL-6). Cue-specific regulatory activity is driven, at least in part, by differences in transcription factor binding activity, the gene targets of which show unique enrichments for brain disorders as well as co-morbid metabolic and immune syndromes. The dynamic nature of genetic regulation informs the influence of environmental factors, reveals a mechanism underlying pleiotropy and variable penetrance, and identifies specific risk variants that confer greater disorder susceptibility after exposure to stress or inflammation. Understanding neurodevelopmental GxE interactions will inform mental health trajectories and uncover novel targets for therapeutic intervention., Competing Interests: CONFLICT OF INTEREST STATEMENT K.J.B is a scientific advisor to Rumi Scientific Inc. and Neuro Pharmaka Inc. All other authors declare no conflicts of interest

Background: STARR-seq and other massively-parallel reporter assays are widely used to discover functional enhancers in transfected cell models, which can be confounded by plasmid vector-induced type-I interferon immune responses and lack the multicellular environment and endogenous chromatin state of complex mammalian tissues., Results: Here, we describe HDI-STARR-seq, which combines STARR-seq plasmid library delivery to the liver, by hydrodynamic tail vein injection (HDI), with reporter RNA transcriptional initiation driven by a minimal Albumin promoter, which we show is essential for mouse liver STARR-seq enhancer activity assayed 7 days after HDI. Importantly, little or no vector-induced innate type-I interferon responses were observed. Comparisons of HDI-STARR-seq activity between male and female mouse livers and in livers from males treated with an activating ligand of the transcription factor CAR ( Nr1i3 ) identified many condition-dependent enhancers linked to condition-specific gene expression. Further, thousands of active liver enhancers were identified using a high complexity STARR-seq library comprised of ~ 50,000 genomic regions released by DNase-I digestion of mouse liver nuclei. When compared to stringently inactive library sequences, the active enhancer sequences identified were highly enriched for liver open chromatin regions with activating histone marks (H3K27ac, H3K4me1, H3K4me3), were significantly closer to gene transcriptional start sites, and were significantly depleted of repressive (H3K27me3, H3K9me3) and transcribed region histone marks (H3K36me3)., Conclusions: HDI-STARR-seq offers substantial improvements over current methodologies for large scale, functional profiling of enhancers, including condition-dependent enhancers, in liver tissue in vivo, and can be adapted to characterize enhancer activities in a variety of species and tissues by selecting suitable tissue- and species-specific promoter sequences., Competing Interests: Competing interests The authors declare that they have no competing interests.

Abstract Background Recently, it has become clear that some promoters function as long-range regulators of gene expression. However, direct and quantitative assessment of enhancer activity at long intergenic noncoding RNA (lincRNA) or mRNA gene bodies has not been performed. To unbiasedly assess the enhancer capacity across lincRNA and mRNA loci, we performed a massively parallel reporter assay (MPRA) on six lincRNA loci and their closest protein-coding neighbors. Results For both gene classes, we find significantly more MPRA activity in promoter regions than in gene bodies. However, three lincRNA loci, Lincp21, LincEnc1, and Peril, and one mRNA locus, Morc2a, display significant enhancer activity within their gene bodies. We hypothesize that such peaks may mark long-range enhancers, and test this in vivo using RNA sequencing from a knockout mouse model and high-throughput chromosome conformation capture (Hi-C). We find that ablation of a high-activity MPRA peak in the Peril gene body leads to consistent dysregulation of Mccc1 and Exosc9 in the neighboring topologically associated domain (TAD). This occurs irrespective of Peril lincRNA expression, demonstrating this regulation is DNA-dependent. Hi-C confirms long-range contacts with the neighboring TAD, and these interactions are altered upon Peril knockout. Surprisingly, we do not observe consistent regulation of genes within the local TAD. Together, these data suggest a long-range enhancer-like function for the Peril gene body. Conclusions A multi-faceted approach combining high-throughput enhancer discovery with genetic models can connect enhancers to their gene targets and provides evidence of inter-TAD gene regulation.

Deriving mechanisms of immune‐mediated disease from GWAS data remains a formidable challenge, with attempts to identify causal variants being frequently hampered by strong linkage disequilibrium. To determine whether causal variants could be identified from their functional effects, we adapted a massively parallel reporter assay for use in primary CD4 T cells, the cell type whose regulatory DNA is most enriched for immune‐mediated disease SNPs. This enabled the effects of candidate SNPs to be examined in a relevant cellular context and generated testable hypotheses into disease mechanisms. To illustrate the power of this approach, we investigated a locus that has been linked to six immune‐mediated diseases but cannot be fine‐mapped. By studying the lead expression‐modulating SNP, we uncovered an NF‐κB‐driven regulatory circuit which constrains T‐cell activation through the dynamic formation of a super‐enhancer that upregulates TNFAIP3 (A20), a key NF‐κB inhibitor. In activated T cells, this feedback circuit is disrupted—and super‐enhancer formation prevented—by the risk variant at the lead SNP, leading to unrestrained T‐cell activation via a molecular mechanism that appears to broadly predispose to human autoimmunity. Synopsis: Little progress has been made in resolving causal SNPs, genes and disease mechanisms at GWAS loci. An adapted massively‐parallel reporter assay (MPRA) allows to study immune‐mediated disease loci in CD4 T cells, the cell‐type whose regulatory DNA is most highly enriched for disease‐associated SNPs. Adapted MPRA identifies putative causal SNPs based on their functional effects within primary CD4 T cells—key effectors of immune‐mediated disease.These effects differ from those detected in the Jurkat cell‐line, reinforcing the importance of an appropriate cellular context in disease‐related studies.The results provide a focus for mechanistic studies to resolve the downstream consequences of expression‐modulating variants at multiple loci.At a gene‐desert linked to multiple diseases, the lead MPRA SNP is shown to abrogate NF‐κB binding, disrupt super‐enhancer formation, and reduce TNFAIP3 expression, leading to unrestrained T cell‐driven inflammation.This provides mechanistic insights into disease biology at a locus that cannot be fine‐mapped and illustrates the potential of this method to uncover genetic mechanisms of immune‐mediated disease. [ABSTRACT FROM AUTHOR]