Locus-Scale Massively Parallel Reporter Assays

Area of Science:

• Genomics
• Molecular Biology
• Gene Regulation

Background:

• Cis-regulatory elements (CREs) interactions are crucial for mammalian gene regulation.
• Understanding how the arrangement and combination of CREs influence gene expression is essential.

Purpose of the Study:

• To introduce and validate Long Assays for Massively Parallel Reporter Assays (LAMPRAs) for scalable measurement of CRE interactions.
• To investigate how CRE identity, number, spacing, order, orientation, and interactions affect gene regulatory output at large scales.

Main Methods:

• LAMPRAs integrate combinatorial cloning, molecular barcoding, and long- and short-read sequencing.
• Assayed 36,000 synthetic cis-regulatory loci (sCRLs), each a 5kb combination of enhancers, insulators, and spacers.
• Modeled how locus composition drives gene expression.

Main Results:

• Demonstrated the scalability of LAMPRAs for analyzing complex CRE arrangements.
• Quantified the impact of varying CRE combinations and spatial arrangements on gene expression.
• Provided a proof-of-concept for using LAMPRAs to dissect multi-kilobase scale regulatory mechanisms.

Conclusions:

• LAMPRAs offer a powerful, scalable approach to study CRE interactions and their role in gene regulation.
• Locus composition significantly influences gene expression output.
• This methodology advances the understanding of complex gene regulatory architectures.