A G-Quadruplex Structure in the Promoter Region of

Area of Science:

• Molecular Biology
• Genetics
• Cancer Research

Background:

• CLIC4's differential expression in cancer suggests a tumor-promoting role.
• Transcriptional regulation of CLIC4 remains poorly understood.

Purpose of the Study:

• To investigate the transcriptional regulation of the CLIC4 gene.
• To analyze the CLIC4 promoter region for regulatory elements.

Main Methods:

• Analysis of the CLIC4 promoter for high GC content and potential G-quadruplex (G4) structures.
• In vitro G4 structure confirmation using Nuclear Magnetic Resonance (NMR) spectroscopy.
• Reporter assays to assess G4 structure's regulatory role in transcription.
• CRISPR/Cas9 gene editing to disrupt the G4-forming sequence in the CLIC4 promoter within A375 cells.
• Pop-in/pop-out strategy for isolating precisely edited cell clones.

Main Results:

• A high GC content near the transcriptional start site (TSS) of CLIC4 was identified, capable of forming G4 structures.
• NMR confirmed the in vitro formation of G4 structures.
• Reporter assays demonstrated that a specific G4 structure regulates gene transcription; mutation disrupting the G4 structure decreased transcription activity.
• CRISPR/Cas9-mediated disruption of the G4-forming sequence in the CLIC4 promoter led to decreased endogenous CLIC4 mRNA expression in edited A375 cell clones.

Conclusions:

• The G-quadruplex (G4) structure within the CLIC4 promoter plays a significant role in regulating the gene's transcriptional level.
• This finding provides insights into the molecular mechanisms controlling CLIC4 expression in cancer.