A G-Quadruplex Structure in the Promoter Region of CLIC4 Functions as a Regulatory Element for Gene Expression.

Mu-Ching Huang,Zi-Fu Wang,Chin-Tin Chen,Ta-Chau Chang,I-Te Chu,Steven Lin

doi:10.3390/ijms19092678

Abstract

The differential transcriptional expression of CLIC4 between tumor cells and the surrounding stroma during cancer progression has been suggested to have a tumor-promoting effect. However, little is known about the transcriptional regulation of CLIC4. To better understand how this gene is regulated, the promoter region of CLIC4 was analyzed. We found that a high GC content near the transcriptional start site (TSS) might form an alternative G-quadruplex (G4) structure. Nuclear magnetic resonance spectroscopy (NMR) confirmed their formation in vitro. The reporter assay showed that one of the G4 structures exerted a regulatory role in gene transcription. When the G4-forming sequence was mutated to disrupt the G4 structure, the transcription activity dropped. To examine whether this G4 structure actually has an influence on gene transcription in the chromosome, we utilized the CRISPR/Cas9 system to edit the G4-forming sequence within the CLIC4 promoter in the cell genome. The pop-in/pop-out strategy was adopted to isolate the precisely-edited A375 cell clone. In CRISPR-modified A375 cell clones whose G4 was disrupted, there was a decrease in the endogenous CLIC4 messenger RNA (mRNA) expression level. In conclusion, we found that the G4 structure in the CLIC4 promoter might play an important role in regulating the level of transcription.

Highlights

Chloride intracellular channel 4, CLIC4, is a multifunctional protein
The deletion of −1700 to −1340 at the promoter region resulted in a significant decrease in the secreted embryonic alkaline phosphatase (SEAP) activity
Since high GC contents were found in the CLIC4 promoter region, we argued that DNA secondary structure might exist to interfere with the PCR and the sequencing process

Summary

Introduction

In addition to its diverse physiological functions [1,2,3,4], the differential expression of CLIC4 between cancer cells and the surrounding stroma has been reported in various tumor types [5]. CLIC4 has been identified for nearly 30 years [10,11], only NANOG, p53, c-Myc, and SP-1 transcription factors have been reported to regulate CLIC4 expression. G4 structures in promoter regions may influence transcription in both positive and negative ways. This depends on the strand that G4 locates in, and the function of the proteins that are bound to the G4 structures. We further evaluated the effect of the G4 structure in regulating CLIC4 gene expression

CLIC4 Promoter Analysis

PG4-3 Is Involved in Regulating CLIC4 Transcription

Cell Culturing

Reporter Assay

Real-Time PCR Analysis