Remodeling Ca2+ dynamics by targeting a promising E-box containing G-quadruplex at ORAI1 promoter in triple-negative
Oishika Chatterjee1, Jagannath Jana2, Suman Panda1
1Department of Biological Sciences, Bose Institute, EN-80 Sector V, Salt Lake, Unified Campus, Kolkata 700091, India.
Abstract:
ORAI1 is an intrinsic component of store-operated calcium entry (SOCE) that strictly regulates Ca2+ influx in most non-excitable cells. ORAI1 is overexpressed in a wide variety of cancers, and its signal transduction has been associated with chemotherapy resistance. There is extensive proteomic interaction of ORAI1 with other channels and effectors, resulting in various altered phenotypes. However, the transcription regulation of ORAI1 is not well understood. We have found a putative G-quadruplex (G4) motif, ORAI1-Pu, in the upstream promoter region of the gene, having regulatory functions. High-resolution 3-D NMR structure elucidation suggests that ORAI1-Pu is a stable parallel-stranded G4, having a long 8-nt loop imparting dynamics without affecting the structural stability. The protruded loop further houses an E-box motif that provides a docking site for transcription factors like Zeb1. The G4 structure was also endogenously observed using Chromatin Immunoprecipitation (ChIP) with anti-G4 antibody (BG4) in the MDA-MB-231 cell line overexpressing ORAI1. Ligand-mediated stabilization suggested that the stabilized G4 represses transcription in cancer cell line MDA-MB-231. Downregulation of transcription further led to decreased Ca2+ entry by the SOCE pathway, as observed by live-cell Fura-2 Ca2+ imaging.
Insights
Researchers discovered a G-quadruplex (G4) motif in the ORAI1 gene promoter. Stabilizing this G4 structure represses ORAI1 transcription, reducing calcium (Ca2+) entry in cancer cells.
Area of Science:
- Molecular Biology
- Cancer Biology
- Biophysics
Background:
- ORAI1 regulates calcium (Ca2+) influx via store-operated calcium entry (SOCE) in non-excitable cells.
- ORAI1 overexpression is linked to various cancers and chemotherapy resistance.
- The transcriptional regulation of ORAI1 remains largely uncharacterized.
Purpose of the Study:
- To investigate the transcriptional regulation of the ORAI1 gene.
- To identify and characterize regulatory elements within the ORAI1 promoter region.
- To explore the role of G-quadruplex structures in ORAI1 gene expression.
Main Methods:
- 3-D NMR structure elucidation of the ORAI1-Pu G4 motif.
- Chromatin Immunoprecipitation (ChIP) using an anti-G4 antibody (BG4).
- Live-cell Fura-2 Ca2+ imaging to assess SOCE pathway activity.
Main Results:
- A stable parallel-stranded G4 motif, ORAI1-Pu, was identified in the ORAI1 promoter, featuring a dynamic loop with an E-box.
- The ORAI1-Pu G4 structure was detected endogenously in MDA-MB-231 cells.
- Ligand-mediated stabilization of ORAI1-Pu repressed ORAI1 transcription and decreased Ca2+ influx via SOCE.
Conclusions:
- The ORAI1 promoter contains a functional G4 motif (ORAI1-Pu) that influences gene transcription.
- G4 structure stabilization can serve as a mechanism to downregulate ORAI1 expression in cancer cells.
- Targeting ORAI1-Pu offers a potential strategy to modulate Ca2+ signaling and chemosensitivity in cancer therapy.
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