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

Area of Science:

• Genetics
• Molecular Biology
• Cardiovascular Disease Research

Background:

• Low-density lipoprotein receptor (LDLR) gene expression is vital for preventing atherosclerotic diseases like heart attack and stroke.
• While sterol response elements are known regulators, other noncoding regions of the LDLR locus are poorly understood.

Purpose of the Study:

• To functionally interrogate candidate cis-regulatory elements (CREs) at the LDLR locus using a high-throughput CRISPR screen.
• To identify noncoding regions that significantly influence LDLR gene expression in its native genomic context.

Main Methods:

• High-throughput CRISPR screening to assess the function of candidate LDLR CREs.
• Reporter gene assays to validate enhancer activity.
• Massively parallel reporter assays (MPRA) for fine-mapping enhancer function.

Main Results:

• Identified 25 discrete genomic regions significantly impacting LDLR expression.
• Validated an intronic region as a functional enhancer that reduces LDLR expression upon disruption.
• Fine-mapped the enhancer to a conserved 129 bp interval containing transcription factor motifs and a GWAS-implicated variant (rs57217136).

Conclusions:

• CRISPR screening is a powerful tool for dissecting the functional landscape of noncoding sequences.
• Discovered novel CREs regulating LDLR, contributing to understanding atherosclerotic disease mechanisms.
• Identified a specific enhancer linked to human LDL cholesterol levels, offering potential therapeutic targets.