Promoter G-quadruplex favours epigenetic reprogramming-induced atypical expression of ZEB1 in cancer cells

Background: Aberrant expression of Zinc-finger E-box binding homeobox 1 (ZEB1), which remains repressed in normal cells, is frequently associated with cancer aggressiveness. However, transcriptional mechanism underlying such atypical ZEB1 expression in cancer is not yet well-understood. Methods: ZEB1 promoter G-quadruplexes were studied and modeled extensively using circular dichroism, fluorescence spectroscopy, ITC and DMS protection assay. Luciferase assay, qPCR, FAIRE, ChIP, western blotting, confocal microscopy was used to access the regulation of ZEB1 transcription. Results: Our study unravels the occupancy of nucleolin to ZEB1 promoter as a crucial determinant which facilitates the binding of SP1 transcription factor to chromatin, by locally remodelling the region. SP1, subsequently, recruits P300 acetyl transferase leading to enriched acetyl-histone H3 at promoter and activates ZEB1 transcription. ZEB1 promoter analysis identifies presence of four putative G-quadruplex (G4) forming motifs within 700 bp of TSS; each quadruplex is characterized structurally in details with an array of biophysical techniques. Surprisingly, stabilization of G4 with cationic porphyrin TMPyP4 represses its transcription and eventually impedes cell invasiveness. Conclusions: TMPyP4 binding to a selected G4 motif (5' -534/-511-3' from TSS), where nucleolin/SP1/P300 co-occupies, prevents the association of nucleolin which consequently hinders SP1 binding, leading to chromatin compactness and transcriptional repression. General significance: Our findings demonstrate an epigenetic mechanism of ZEB1 reactivation where dynamic occupancy of transcription regulators encompassing a G4 motif is crucial and thus, small molecule induced G-quadruplex stabilization may act as a potential molecular switch to turn-off gene expression.

Automated summary

Our study identified the crucial role of nucleolin in facilitating SP1 transcription factor binding to the ZEB1 promoter, leading to the activation of ZEB1 transcription. Stabilization of G-quadruplex with TMPyP4 represses ZEB1 transcription and inhibits cell invasiveness. These findings suggest that small molecule induced G-quadruplex stabilization may serve as a potential molecular switch to regulate gene expression.