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Massively parallel identification of cis-regulatory variants in yeast promoters

ABSTRACT: Massively parallel identification of cis-regulatory variants in yeast promoters

PROVIDER: PRJNA656111 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR12417822_1.fastq.gz	Fastqsanger.gz
	SRR12417822_2.fastq.gz	Fastqsanger.gz
	SRR12417823_1.fastq.gz	Fastqsanger.gz
	SRR12417823_2.fastq.gz	Fastqsanger.gz
	SRR12417824_1.fastq.gz	Fastqsanger.gz

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Massively parallel identification of cis-regulatory variants in yeast promoters - Experimental measurements

Project description:Sequence variation in regulatory DNA alters gene expression and shapes genetically complex traits. However, the identification of individual, causal regulatory variants is challenging. Here, we used a massively parallel reporter assay to measure the cis-regulatory consequences of 5,832 natural DNA variants in the promoters of 2,503 genes in the yeast Saccharomyces cerevisiae. We identified 451 causal variants, which underlie genetic loci known to affect gene expression. Several promoters harbored multiple causal variants. In five promoters, pairs of variants showed non-additive, epistatic interactions. Causal variants were enriched at conserved nucleotides, tended to have low derived allele frequency, and were depleted from promoters of essential genes, consistent with the action of negative selection. Causal variants were enriched for alterations in transcription factor binding sites. Models integrating these features provided modest, but statistically significant, ability to predict causal variants. This work revealed a complex molecular basis for cis-acting regulatory variation.

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Massively parallel identification of cis-regulatory variants in yeast promoters - Annotation runs

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Massively parallel identification of cis-regulatory variants in yeast promoters - Annotation runs

Project description:Massively parallel identification of cis-regulatory variants in yeast promoters - Annotation runs

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Massively parallel identification of cis-regulatory variants in yeast promoters - Experimental measurements

Project description:Massively parallel identification of cis-regulatory variants in yeast promoters - Experimental measurements

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Massively parallel identification of cis-regulatory variants in yeast promoters

Project description:This SuperSeries is composed of the SubSeries listed below.

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Project description:We developed a single-cell massively parallel reporter assay (scMPRA) to measure the activity of libraries of cis-regulatory sequences (CRSs) across multiple cell-types simultaneously. As a proof of concept, we assayed a library of core promoters in a mixture of HEK293 and K562 cells and showed that scMPRA is a reproducible, highly parallel, single-cell reporter gene assay. Our results show that housekeeping promoters and CpG island promoters have lower activity in K562 cells relative to HEK293, which likely reflects developmental differences between the cell lines. Within K562 cells, scMPRA identified a subset of developmental promoters that are upregulated in the CD34+/CD38- sub-state, confirming this state as more “stem-like.” Finally, we deconvolved the intrinsic and extrinsic components of cell-to-cell variability and found that developmental promoters have a higher proportion of extrinsic noise compared to housekeeping promoters. We anticipate scMPRA will be widely applicable for studying the role of CRSs across diverse cell types.

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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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