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Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay

ABSTRACT: We apply a massively parallel reporter assay (MPRA) that relies on mRNA and plasmid tag sequencing (Tag-Seq) to compare the regulatory activities of more than 27,000 distinct variants of two inducible enhancers in human cells: a synthetic cAMP-regulated enhancer and the virus-inducible interferon beta enhancer. The resulting data define accurate maps of functional transcription factor binding sites in both enhancers at single-nucleotide resolution and can be used the to train quantitative sequence-activity models (QSAMs).

ORGANISM(S): Escherichia coli Homo sapiens

PROVIDER: GSE31982 | GEO | 2012/02/26

SECONDARY ACCESSION(S): PRJNA145181

REPOSITORIES: GEO

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Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay

Project description:We apply a massively parallel reporter assay (MPRA) that relies on mRNA and plasmid tag sequencing (Tag-Seq) to compare the regulatory activities of more than 27,000 distinct variants of two inducible enhancers in human cells: a synthetic cAMP-regulated enhancer and the virus-inducible interferon beta enhancer. The resulting data define accurate maps of functional transcription factor binding sites in both enhancers at single-nucleotide resolution and can be used the to train quantitative sequence-activity models (QSAMs). Reporter Tag-Seq from HEK293 cells transfected with each of six MPRA plasmid pools, with and without stimulation (forskolin or Sendai virus). The reporter mRNAs contain unique 10 nucleotide tags that facilitates quantitation of their abundances. The same tags were also sequenced from each ransfected plasmid pool to facilitate normalization to plasmid copy numbers. The reporter constructs were designed according to two different mutagenesis strategies: 'single-hit scanning' and 'multi-hit sampling'. The specific variants are included in the processed data files.

2012-02-26 | E-GEOD-31982 | biostudies-arrayexpress

Systematic dissection of regulatory motifs in 2,000 predicted human enhancers using a massively parallel reporter assay

Project description:We employ a massively parallel reporter assay (MPRA) to measure the ex vivo activities of hundreds of K562 and HepG2 enhancers with known transcription factor motif instances. For seven selected motifs that correspond to known or predicted activators and repressors in the two cell types, we make directed modifications of the bases corresponding to these motifs and observe the changes in enhancer activity. Reporter mRNA-seq from HepG2 and K562 cells transfected with a ~55,000-plex MPRA plasmid pool containing 5,418 mutated human enhancer sequences, each linked to 10 distinct 10-nt tags. The reporter mRNA tags facilitate quantitation of their abundances. The same tags were also sequenced from the transfected MPRA plasmid pool to facilitate normalization to plasmid copy numbers.

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Massively Parallel Reporter Assay of human-specific substitutions in neurodevelopmental enhancers

Project description:We performed a Massively Parallel Reporter Assay (MPRA) to screen >30,000 human-specific substitutions in ChIP-seq-identified Human Gain Enhancers (HGEs) and Human Accelerated Regions (HARs), highly conserved non-coding regions that show accelerated sequence evolution in humans. After comparing human and chimpanzee reference alleles, we used a second MPRA to deconvolute individual substitutions within differentially active enhancers from substitutions in the same fragment and from other variants (human segregating variants or chimpanzee-specific variants) to isolate their specific effects on enhancer activity.

2019-11-26 | GSE140983 | GEO

Systematic dissection of regulatory motifs in 2,000 predicted human enhancers using a massively parallel reporter assay

2013-03-15 | GSE33367 | GEO

A systematic evaluation of the design, orientation, and sequence context dependencies of massively parallel reporter assays

Project description:Enhancers play important roles in evolution and disease. However, traditional assays to test enhancers are low throughput and not scalable to the >100,000 enhancers in the human genome. To better prioritize variants associated with disease and to study the role of enhancers, our group and others developed massively parallel reporter assays (MPRAs), which functionally screen thousands of sequences for regulatory activity in parallel. Although MPRAs have been applied to address diverse questions in gene regulation, there has been no systematic comparison of how differences in experimental design influence findings, making it difficult to interpret results and compare between groups. Here, we screen a library of 2,440 sequences, representing candidate liver enhancers and controls, in HepG2 cells for regulatory activity using nine different approaches (including conventional episomal, STARR-seq, and lentiviral MPRA designs). We identify subtle but significant differences in the resulting measurements that correlate with epigenetic and sequence-level features. We also test this library in both orientations with respect to the promoter, validating en masse that enhancer activity is robustly independent of orientation. Finally, we develop and apply a novel method to assemble and functionally test libraries of the same putative enhancers as 192-mers, 354-mers, and 678-mers, and observe surprisingly large differences in functional activity. This work provides a framework for the experimental design of high-throughput reporter assays, suggesting that the extended sequence context of tested elements, and to a lesser degree the precise assay, influence MPRA results.

2019-12-30 | GSE142696 | GEO

Massively parallel reporter assays (MPRAs) of 175bp human genomic fragments containing variants in high LD with lead variants from obesity GWAS associations.

Project description:We performed a massively parallel reporter assay on 2,396 genomic regions containing single nucleotide polymorphisms that are in high linkage disequilibrium with 97 lead variants from an obesity GWAS (PMID: 25673413). Regions were transfected into human SGBS preadipocytes, SGBS mature adipocytes, 3T3-L1 preadipocytes, HT22 hippocampal cells, and GT1-7 cells and assessed for enhancer activity. The processed file contains the MPRA barcodes.

2021-05-22 | E-MTAB-10463 | biostudies-arrayexpress

Genome-wide enhancer identification by massively parallel reporter assay in Arabidopsis

Project description:Enhancers are critical cis-regulatory elements controlling gene expression during cell development and differentiation. However, genome-wide enhancer characterization has been challenging due to the lack of a well-defined relationship between enhancers and genes. Here, we applied a massively parallel reporter assay on Arabidopsis to measure enhancer activities across the genome. We identified 4327 enhancers with various combinations of epigenetic modifications distinctively different from animal enhancers. Furthermore, we showed that enhancers differ from promoters in their preference for transcription factors. Although some enhancers are not conserved and overlap with transposable elements forming clusters, enhancers are generally conserved across thousand Arabidopsis species, suggesting they are selected under evolution pressure and could play critical roles in the regulation of important genes. Moreover, comparison analysis reveals that enhancers identified by different strategies do not overlap, suggesting these methods are complementary in nature. In sum, our work provides an additional catalog of enhancers and lays the foundation for further investigation into enhancers’ functional mechanisms in plants.

2020-10-13 | GSE157030 | GEO

Massively Parallel Reporter Assay for pluripotency factors in mESCs

Project description:A massively parallel reporter assay, MPRA, was conducted in mouse embryonic stem cells (mESC). Synthetic cis-regulatory elements comprised of binding sites for pluripotency transcription factors and genomic sequences with comparable binding sites configurations were used in the assay. Transcripts of dsRed were amplified via PCR from the end of the transcript to sequence 3' UTR barcodes.

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Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay

Project description:Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay

| PRJNA145181 | ENA

Massively parallel reporter assays with multi-layer annotation identified cell-type-specific functional variants and genes associated with melanoma (MPRA)

Project description: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 (MPRA) 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 MPRA, 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 (QTLs) 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, MPRA along with variant scoring effectively prioritized plausible candidates for most melanoma GWAS loci and highlighted cellular contexts where the susceptibility variants are functional.

2022-11-28 | GSE210354 | GEO

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