# Functional interrogation of candidate cis-regulatory elements at the LDLR locus

## TL;DR

This study identifies 25 regulatory regions that influence LDLR gene expression, a key factor in heart disease, using CRISPR screening and reporter assays.

## Contribution

The study introduces a high-throughput CRISPR approach to validate functional noncoding elements at the LDLR locus.

## Key findings

- 25 candidate cis-regulatory elements significantly impact LDLR expression.
- A 129 bp enhancer region in the first intron was validated and linked to human LDL cholesterol variation.
- The enhancer is conserved, motif-rich, and shows biochemical enhancer features.

## Abstract

Regulation of LDLR gene expression plays an important role in the development of atherosclerotic diseases including heart attack and stroke. Although LDLR regulation by sterol response elements has been well characterized, the functional significance of other noncoding regions at the LDLR locus remains poorly defined. In this study, we developed and applied a high throughput CRISPR screen to test the functional importance of candidate LDLR cis-regulatory elements (CREs) in their native genomic context. In total, we found 25 discrete regions to exhibit a significant impact on LDLR expression. For one of these regions with particularly strong activity in the first intron, we validated the presence of an enhancer by confirming that its disruption reduced endogenous LDLR expression while its insertion upstream of a minimal promoter augmented reporter gene expression. We then applied a massively parallel reporter assay to fine map enhancer activity within this region to a 129 bp interval that is highly conserved among vertebrates, exhibits biochemical hallmarks of enhancer activity, is enriched for transcription factor binding motifs, and contains a common genetic variant (rs57217136) that has been associated with human LDL cholesterol levels by genome-wide association studies. Overall, these findings demonstrate the power of CRISPR screening to interrogate candidate CREs and clarify the functional landscape of noncoding sequences at the LDLR locus.

Expression of the LDLR gene influences a person’s risk of cardiovascular disease due to its impact on the concentration of cholesterol-carrying lipoproteins in the bloodstream. While some of the transcription factors and their corresponding binding sites that control LDLR gene expression have been well characterized, several noncoding regions exhibit features predictive of gene regulatory activity and have not been previously tested. In this study, we leveraged advances in high-throughput CRISPR technology to systematically interrogate the functional significance of these candidate regions with a library of 12,375 gRNAs. We found evidence of activity for 25 distinct CREs, including 1 in the early first intron whose functional significance we then validated and whose precise boundaries we defined using a massively parallel reporter assay. Our findings deepen our basic understanding of LDLR gene regulation, suggest novel targets for therapeutic development, and illustrate the potential of high throughput genomic tools to define the functional landscape of the noncoding genome.