Understanding how cis-regulatory function is encoded in DNA sequence using massively parallel reporter assays and...

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• Genome-scale methods have identified numerous candidate cis-regulatory elements (CREs).
• Lack of high-throughput methods to functionally validate these CREs has limited understanding of sequence-function relationships.
• Previous limitations hindered the discovery of how cis-regulatory function is encoded in genomic sequences.

Purpose of the Study:

• To introduce and validate massively parallel reporter gene assays (MPRAs) for large-scale functional analysis of CREs.
• To demonstrate the capacity of MPRAs to test and mutationally perturb thousands of cis-regulatory sequences simultaneously.
• To address previously intractable questions regarding cis-regulatory function using high-throughput experimental approaches.

Main Methods:

• Development and application of massively parallel reporter gene assays (MPRAs).
• Integration of next-generation sequencing with high-throughput oligonucleotide synthesis.
• Testing of large libraries of designed and naturally occurring cis-regulatory sequences, including mutational analyses.

Main Results:

• MPRAs provide the capacity to test thousands of cis-regulatory sequences in a single experiment.
• Demonstrated large-scale functional validation of genomic CREs.
• Enabled exhaustive mutational analyses and testing of synthetic CRE libraries, revealing insights into sequence-function relationships.

Conclusions:

• Massively parallel reporter assays are a powerful tool for high-throughput functional genomics.
• These assays provide the statistical power to address complex questions in cis-regulatory biology.
• MPRAs significantly advance the discovery of how cis-regulatory function is encoded in DNA sequences.