A promoter-dependent upstream activator augments CFTR expression in diverse epithelial cell types

Area of Science:

• Genetics
• Molecular Biology
• Epigenetics

Background:

• Cystic fibrosis transmembrane conductance regulator (CFTR) mutations cause cystic fibrosis (CF), impacting epithelial function and male infertility.
• CFTR is an anion channel crucial for epithelial cell function.

Purpose of the Study:

• To investigate a novel epididymis-selective cis-regulatory element (CRE) located 5' to the CFTR gene promoter.
• To explore a new mechanism for activating or augmenting CFTR gene expression.

Main Methods:

• CRISPR activation (CRISPRa) was used to co-activate the -9.5 kb CRE and the CFTR gene promoter in epididymis cells.
• Chromatin accessibility was assessed using assays.
• CFTR mRNA and protein expression levels were measured.
• Transient reporter gene assays were performed.

Main Results:

• Co-activation of the -9.5 kb CRE and CFTR promoter significantly augmented CFTR mRNA and protein expression in epididymis cells.
• This combined strategy enhanced chromatin accessibility at both the CRE and promoter sites.
• The approach successfully activated CFTR expression in other epithelial cells where the locus is normally inactive.
• The -9.5 kb CRE did not function as a classical enhancer in reporter gene assays.

Conclusions:

• A novel mechanism for activating/augmenting CFTR expression was identified, involving synergistic action of a CRE and promoter activation.
• This finding may hold therapeutic potential for CF mutations that disrupt CFTR transcription.
• The study highlights the importance of cis-regulatory elements in controlling gene expression in specific tissues.