# i-Motif, not G-quadruplex, stability regulates insulin expression

## TL;DR

This study shows that i-motif structures, not G-quadruplexes, regulate insulin gene expression in response to glucose levels.

## Contribution

The study reveals that i-motif stability, not G-quadruplex stability, correlates with insulin gene activation.

## Key findings

- Transcriptional activation occurs only when both G-quadruplex and i-motif structures can form.
- Promoter activity is positively correlated with i-motif stability, not G-quadruplex stability.
- G-quadruplexes may act as initiation sites while i-motifs modulate insulin gene expression.

## Abstract

The insulin-linked polymorphic region (ILPR) is a variable number tandem repeat located in the promoter of the human insulin gene. This G-rich sequence can fold into four-stranded G-quadruplex DNA structures, while its complementary C-rich strand forms i-motifs. The ILPR varies in repeat number and sequence composition, but the relationship between sequence diversity, DNA structure, and insulin gene regulation remains poorly understood. Although both G-quadruplexes and i-motifs have been implicated in transcriptional control, their relative contributions, particularly when formed on complementary strands of the same locus, are unclear. Here, we characterized the structure and stability of nine ILPR-based sequences using biophysical techniques and luciferase reporter assays. We demonstrate that transcriptional activation in response to high glucose occurs only when both G-quadruplex and i-motif structures can form. Other combinations of structures do not induce transcription. Moreover, promoter activity correlated positively with i-motif stability, but not with G-quadruplex stability. These results suggest a model in which G-quadruplexes may act as an initiation site, while i-motifs act as modulators of insulin gene expression. Our findings underscore the importance of treating G-quadruplexes and i-motifs as a dynamic, interdependent system in both the regulation of gene expression and also the potential of these structures as therapeutic targets.

Graphical Abstract