Identification of G-quadruplex structures that possess transcriptional regulating functions in the Dele and Cdc6 CpG islands

Daniyah H Bay,Keisuke Iida,Kazunori Ikebukuro,Annika Busch,Isao Karube,Wataru Yoshida,Fred Lisdat,Kazuo Nagasawa

doi:10.1186/s12867-017-0094-z

Abstract

BackgroundG-quadruplex is a DNA secondary structure that has been shown to play an important role in biological systems. In a previous study, we identified 1998 G-quadruplex-forming sequences using a mouse CpG islands DNA microarray with a fluorescent-labeled G-quadruplex ligand. Among these putative G-quadruplex-forming sequences, G-quadruplex formation was verified for 10 randomly selected sequences by CD spectroscopy and DMS footprinting analysis. In this study, the biological function of the 10 G-quadruplex-forming sequences in the transcriptional regulation has been analyzed using a reporter assay.ResultsWhen G-quadruplex-forming sequences from the Dele and Cdc6 genes have been cloned in reporter vectors carrying a minimal promoter and the luciferase gene, luciferase expression is activated. This has also been detected in experiments applying a promoterless reporter vector. Mutational analysis reveals that guanine bases, which form the G-tetrads, are important in the activation. In addition, the activation has been found to decrease by the telomestatin derivative L1H1-7OTD which can bind to the G-quadruplex DNA. When Dele and Cdc6 CpG islands, containing the G-quadruplex-forming sequence, have been cloned in the promoterless reporter vector, the luciferase expression is activated. Mutational analysis reveals that the expression level is decreased by mutation on Dele G-quadruplex; however, increased by mutation on Cdc6 G-quadruplex.ConclusionDele and Cdc6 G-quadruplex formation is significant in the transcriptional regulation. Dele and Cdc6 G-quadruplex DNA alone possess enhancer and promotor function. When studied in more complex CpG islands Dele G-quadruplex also demonstrates promotor activity, whereas Cdc6 G-quadruplex may possess a dual function of transcriptional regulation.

Highlights

G-quadruplex is a DNA secondary structure that has been shown to play an important role in biological systems
The activation of protein expression is clearly decreased by thymine mutations in the G4 region (Fig. 2; Additional file 3). These results indicate that death ligand signal enhancer (Dele) and celldivision-cycle 6 (Cdc6) G4 DNAs have a role in transcriptional activation, both as a promoter and enhancer
The reporter assay for G4-forming DNA sequences has demonstrated that Dele and Cdc6 G4 DNA may have the role of promoter and enhancer i.e., activating the transcription

Summary

Introduction

G-quadruplex is a DNA secondary structure that has been shown to play an important role in biological systems. We identified 1998 G-quadruplex-forming sequences using a mouse CpG islands DNA microarray with a fluorescent-labeled G-quadruplex ligand. Among these putative G-quadruplex-forming sequences, G-quadruplex formation was verified for 10 randomly selected sequences by CD spectroscopy and DMS footprinting analysis. The biological function of the 10 G-quadruplex-forming sequences in the transcriptional regulation has been analyzed using a reporter assay. G4 ligands, such as TMPyP4 and telomestatin, have been shown to stabilize the G4 structure of the RET protooncogene promoter and lead to the repression of gene expression [21]

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