Strand asymmetry in MPRA Signal
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE156857 | GEO | 2020/12/04
REPOSITORIES: GEO
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Project description:Strand asymmetry in MPRA Signal
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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.
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Project description:Gene regulation occurs through trans-acting factors (e.g. transcription factors) acting on cis-regulatory elements (e.g. enhancers). Massively parallel reporter assays (MPRAs) functionally survey large numbers of cis-regulatory elements for regulatory potential, but do not identify the trans-acting factors that mediate any observed effects. Here we include preliminary data from a pilot transMPRA experiment — a reporter assay that efficiently combines multiplex CRISPR-mediated perturbation and MPRAs to identify trans-acting factors that modulate the regulatory activity of specific enhancers.
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Project description:Human Accelerated Regions (HARs) are the fastest-evolving regions of the human genome and many are hypothesized to function as regulatory elements that drive human-specific gene regulatory programs. We interrogate the in vitro enhancer activity of >3,100 HARs in massively parallel reporter assays (MPRAs), demonstrating that many HARs appear to act as neural enhancers and that sequence divergence at HARs has largely augmented their neuronal enhancer activity.
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Project description:CTCF/cohesin play a central role in insulator function and higher-order chromatin organization of mammalian genomes. Recent studies identified a correlation between the orientation of CTCF-binding sites (CBSs) and chromatin loops. To test the functional significance of this observation, we combined CRISPR/Cas9-based genomic-DNA-fragment editing with chromosome-conformation-capture experiments to show that the location and relative orientations of CBSs determine the specificity of long-range chromatin looping in mammalian genomes, using protocadherin (Pcdh) and β-globin as model genes. Inversion of CBS elements within the Pcdh enhancer reconfigures the topology of chromatin loops between the distal enhancer and target promoters, and alters gene-expression patterns. Thus, although enhancers can function in an orientation-independent manner in reporter assays, in the native chromosome context the orientation of at least some enhancers carrying CBSs can determine both the architecture of topological chromatin domains and enhancer/promoter specificity. The findings reveal how 3D chromosome architecture can be encoded by genome sequence.
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Project description:Cis-regulatory elements (CREs, e.g., promoters and enhancers) regulate gene expression, and variants within CREs can modulate disease risk. Next-generation sequencing has enabled the rapid generation of genomic data that predict the locations of CREs, but a bottleneck lies in functionally interpreting these data. To address this issue, massively parallel reporter assays (MPRAs) have emerged, in which barcoded reporter libraries are introduced into cells and the resulting barcoded transcripts are quantified by next-generation sequencing. Thus far, MPRAs have been largely restricted to assaying short CREs in a limited repertoire of cultured cell types. Here, we present two advances that extend the biological relevance and applicability of MPRAs. First, we adapt exome capture technology to instead capture candidate CREs, thereby tiling across the targeted regions and markedly increasing the length of candidate CREs that can be readily assayed. Second, we package the library into adeno-associated virus (AAV), thereby allowing delivery of candidate CREs to target organs in vivo. As a proof-of-concept, we introduce a capture library of ~46,000 constructs, corresponding to ~3,500 DNase I hypersensitive (DHS) sites, into the mouse retina by ex vivo plasmid electroporation and into the mouse cerebral cortex by in vivo AAV injection. We demonstrate tissue-specific cis-regulatory activity of DHSs and provide examples of high-resolution truncation mutation analysis for multiplex parsing of CREs. Our approach should enable massively parallel functional analysis of a wide range of CREs in any organ or species that can be infected by AAV, such as non-human primates and human stem cell-derived organoids.
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Project description:Alu elements are major contributors to lineage-specific new exons in primate and human genomes. Recent studies indicate that some Alu exons have high transcript inclusion levels or tissue-specific splicing profiles, and may play important regulatory roles in modulating mRNA degradation or translational efficiency. However, the contribution of Alu exons to the human proteome remains unclear and controversial. The prevailing view is that exons derived from young repetitive elements, such as Alu elements, are restricted to regulatory functions and have not had adequate evolutionary time to be incorporated into stable, functional proteins. We adopt a proteotranscriptomics approach to systematically assess the contribution of Alu exons to the human proteome. Using RNA sequencing, ribosome profiling and proteomics data from human tissues and cell lines, we provide evidence for the translational activities of Alu exons and the presence of Alu exon derived peptides in human proteins. These Alu exon peptides represent species-specific protein differences between primates and other mammals, and in certain instances between humans and closely related primates. In the case of the RNA editing enzyme ADARB1, which contains an Alu exon peptide in its catalytic domain, RNA sequencing analyses of A-to-I editing demonstrate that both the Alu exon skipping and inclusion isoforms encode active enzymes. The Alu exon derived peptide may fine tune the overall editing activity and, in limited cases, the site selectivity of ADARB1 protein products. Our data indicate that Alu elements have contributed to the acquisition of novel protein sequences during primate and human evolution.
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Project description:Alu element is a major contributor to lineage-specific new exons in the primate and human genomes. Recent studies indicate that some Alu exons have high transcript inclusion levels or tissue-specific splicing profiles, and may play important regulatory roles in modulating mRNA degradation or translational efficiency. However, the contribution of Alu exons to the human proteome remains unclear and controversial. The prevailing view is that exons derived from young repetitive elements (such as Alu) are restricted to regulatory functions but do not have adequate evolutionary time to be incorporated into stable, functional proteins. In this work, we adopt a proteotranscriptomics approach to systematically assess the contribution of Alu exons to the human proteome. Using RNA sequencing, ribosome profiling, and mass spectrometry data of diverse human tissues and cell lines, we provide evidence for the translational activities of Alu exons and the presence of Alu exon derived peptides in human proteins. These Alu exon peptides represent species-specific protein differences between primates and other mammals, and in certain instances even between humans and closely related nonhuman primates. In the RNA editing enzyme ADARB1, which contains an Alu exon peptide in its catalytic domain, RNA editing analyses of RNA-sequencing data demonstrate that both the Alu exon skipping and inclusion isoforms encode active RNA editing enzymes, while the Alu exon peptide may fine tune the editing activities of the ADARB1 protein products . Together, our data indicate that Alu elements have contributed to the acquisition of novel protein sequences during primate and human evolution. Comparing the A-I RNA editing levels during HEK293 (control), ADARB1 long isoform (with Alu exon) transfected, and short isoform (without Alu exon) transfected cells, each group has 3 replicates.
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