Cell-type-specific long-range looping interactions identify distant regulatory elements of the CFTR gene

Area of Science:

• Genomics
• Molecular Biology
• Epigenetics

Background:

• Identifying gene regulatory elements is challenging due to their potential to act over large genomic distances.
• Long-range regulatory elements are critical for human disease genes, as mutations within them can cause disease.
• Chromatin looping interactions are increasingly recognized as a mechanism facilitating long-range gene expression control.

Purpose of the Study:

• To utilize chromosome conformation capture (3C) as a discovery tool for identifying long-range regulatory elements.
• To identify novel regulatory elements controlling the cystic fibrosis transmembrane conductance regulator (CFTR) gene.
• To investigate the role of these elements in CFTR gene expression.

Main Methods:

• Employed chromosome conformation capture (3C) to detect chromatin looping interactions.
• Identified regulatory elements within a 460-kb region around the CFTR locus.
• Characterized identified elements using DNase I hypersensitive sites and histone modification patterns.
• Performed reporter assays to assess the functional activity of identified elements.

Main Results:

• Discovered four novel long-range acting elements that specifically loop to the CFTR promoter in CFTR-expressing cells.
• Located these elements at 20 kb and 80 kb upstream, and 109 kb and 203 kb downstream of the CFTR promoter.
• Confirmed these elements possess enhancer-like characteristics and interact with each other.
• Demonstrated synergistic activation of the CFTR promoter by the downstream elements in reporter assays.

Conclusions:

• Revealed novel long-range acting elements that regulate CFTR gene expression.
• Established the utility of 3C-based approaches for discovering novel regulatory elements.
• Highlighted the importance of chromatin looping in controlling gene expression, particularly for disease-associated genes like CFTR.