A G-quadruplex nanoswitch in the SGK1 promoter regulates isoform expression by K+/Na+ balance and resveratrol binding

https://doi.org/10.1016/j.bbagen.2020.129778Get rights and content

Highlights

  • A G-quadruplex in the SGK1 promoter is a molecular switch for isoform expression.

  • The G-quadruplex is stabilized by K+ or Resveratrol, but destabilized by Na+.

  • Potassium supplementation inhibits the transcription of SGK1 isoforms.

Abstract

Background

High sodium intake can up-regulate the level of renal serum- and glucocorticoid-inducible kinase-1 (SGK1), which plays a pivotal role in controlling blood pressure via activation of the epithelial sodium channel (ENaC), which can lead to salt-sensitive hypertension. Increased potassium intake, or a vegetarian diet, counteracts salt-sensitive hypertension, but the underlying mechanisms are not fully understood.

Methods

Bioinformatics and molecular modeling were used to identify G-quadruplex (G4) and their conformations in the SGK1 promoter. CD spectra and UV melting dynamics were measured to study the stability of G4 as influenced by potassium/sodium balance and resveratrol. RT-PCR and Western blot were employed to study the effects of potassium and resveratrol on the SGK1 isoform expression.

Results

The SGK1 gene encodes a G4 structure in the proximal upstream of promoter-2; the G4 structure is stabilized by potassium or resveratrol, but destabilized by sodium. Super-physiological levels of sodium stimulate the transcription of all SGK1 isoforms, whereas resveratrol or potassium supplementation inhibits the transcription of iso-2 and iso-3, but not iso-1.

Conclusions

Stabilizing the G4 by potassium or resveratrol induces alternative promoter usage and/or pre-mRNA splicing in the transcription of SGK1.

General significance

Potassium/sodium ion balance or resveratrol binding can act to regulate G4 molecular switches for controlling SGK1 gene expression, thereby presenting a new avenue for drug development.

Introduction

Potassium/sodium balance helps maintain vital body functions. High sodium intake is responsible for salt-sensitive hypertension. While the effectiveness of the current dietary sodium guidelines in reducing the risk of cardiovascular morbidity and mortality is in question [1], increasing potassium intake or adopting a vegetarian diet is believed to be beneficial in helping to control elevated blood pressure and risk of stroke [2,3]. Possible mechanisms for the antihypertensive effects of potassium or resveratrol, a natural polyphenolic molecule found in fruits and vegetables, are suggested to involve alterations in the activity of the rennin-angiotensin aldosterone axis, or reduced oxidative stress [[4], [5], [6], [7]]. Recent studies have identified serum- and glucocorticoid-inducible kinase-1 (SGK1) as a pivotal player in sodium and potassium homeostasis and blood pressure regulation, via activation of the epithelial sodium channel (ENaC) [[8], [9], [10], [11], [12]]. Furthermore, dietary sodium can regulate the abundance of SGK1 in renal cells and T lymphocytes [9,13]. A better understanding of the mechanism for antihypertensive effects of potassium or resveratrol is therefore crucial for the improvement of dietary guidelines and drug development. Since potassium and sodium are the major monovalent cations that influence the formation of G-quadruplex (G4) [14], which are widely distributed in the genome and recognized as important regulators of gene expression [15], we asked if the SGK1 gene encodes any G4(s) that might be responsible for the regulation of SGK1 gene expression by sodium, potassium or resveratrol.

Section snippets

Identification of potential G4 structures in the SGK1 gene

QGRS Mapper (https://bioinformatics.ramapo.edu/QGRS/index.php) is a web-based server for predicting G-quadruplexes in nucleotide sequences [16]. We used this server to screen the promoter regions of the SGK1 gene.

DNA preparation for the biophysical studies

Three potential G4 structures in the SGK1 gene that were identified by QGRS mapper were synthesized as follows (the quadruplex-forming nucleotides are underlined):

G4-1: GGGCCGGGGTCCCGGCGGCGGGAACGGGA

G4-2: GGGTTGGGGAGGAGGGTGGGA

G4-3: GGGGCGGGGCGAGGGGCGAGGCGAAGGGCGGGA

As previously reported

The SGK1 gene contains three G4 structures in the promoter regions

We first searched for potential G4 in the promoter region of SGK1, because G4 structures are over-represented in gene promoters [22,23]. SGK1 has multiple promoters which drive the transcription of at least three isoforms [24]. By using a web-based program for predicting G4 structure [16], three G4s were identified in the SGK1(iso-1) promoter-2 region, namely G4-1, G4-2 and G4-3 (Fig. 1A). The G4-1 quadruplex sequence is conserved in human, Chimp, Gorilla, and Orangutan (Fig. 1B).

The effects of sodium, potassium and resveratrol on SGK1 G4 conformation depend on specific G4 structural features

In the

The G-quadruplex in SGK1 promoter presents a novel molecular switch in the regulation of gene expression

Potassium and sodium are the major ions that regulate the formation and folding topology of G4 [14]. G4 motifs have been identified in telomeres and promoters and implicated in variety of biological processes such as pre-mRNA splicing, transcription pausing, translation initiation and repression [15,36]. The stability and topology of G4 in the promoter may have an impact on gene expression [37]. G4 structures can be classified into three topologies, i.e., parallel, anti-parallel, and hybrid [38

Conclusion

This study provides evidence for the role of a G-quadruplex-based molecular switch for potassium or resveratrol in counteracting the effects of dietary salt, through altering the transcription of SGK1 isoforms. On the basis of this mechanism, operating on a genetic level, supplementation of potassium and a diet rich in resveratrol or possibly other DNA-binding polyphenols may be an ideal dietary salt guideline. The capacity of resveratrol to bind to G4 structures to regulate gene expression may

Declaration of Competing Interest

None.

Acknowledgments

This work was supported by grants from National Natural Science Foundation of China (NSFC) (No. 81272257 and No.31170855) to Lijun Zhao, and by an unrestricted gift to Ethan W. Taylor from the Dr. Arthur and Bonnie Ennis Foundation, Decatur, IL, USA.

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    1

    These authors contributed equally.

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