Vimentin binds to G-quadruplex repeats found at telomeres and gene promoters

Abstract

G-quadruplex (G4) structures that can form at guanine-rich genomic sites, including telomeres and gene promoters, are actively involved in genome maintenance, replication, and transcription, through finely tuned interactions with protein networks. In the present study, we identified the intermediate filament protein Vimentin as a binder with nanomolar affinity for those G-rich sequences that give rise to at least two adjacent G4 units, named G4 repeats. This interaction is supported by the N-terminal domains of soluble Vimentin tetramers. The selectivity of Vimentin for G4 repeats versus individual G4s provides an unprecedented result. Based on GO enrichment analysis performed on genes having putative G4 repeats within their core promoters, we suggest that Vimentin recruitment at these sites may contribute to the regulation of gene expression during cell development and migration, possibly by reshaping the local higher-order genome topology, as already reported for lamin B.

Figure 1.

SDS-PAGE of the last eluted fraction (GLB) obtained from pull-down assays performed with KIT2KIT* folded into (A) G-quadruplex and (B) duplex. Bands corresponding to Streptavidin monomer (S1) and dimer (S2) and Vimentin (V) are highlighted, M indicated the molecular weight markers.

Figure 2.

EMSA of 500 nM (A) KIT2KIT*, KIT2 and KIT*; (B) ILPR and hTERT; (C) TEL, 2TEL, 3TEL and 4TEL folded into G-quadruplexes and incubated in the presence (+) or absence (–) of 8 μM Vimentin in 5 mM Tris-HCl (pH 8.4), 150 mM KCl. Gels were stained with Sybr Green II (panels on the left) and with colloidal Coomassie Brilliant Blue G250 (panels on the right).

Figure 3.

Job plots derived from EMSA performed with Vimentin and (A) 2TEL, (B) 4TEL, in 5 mM Tris–HCl (pH 8.4), 150 mM KCl, at constant sum of oligonucleotide and protein concentrations (10 μM), and by varying their molar fraction. (C) Fluorescence anisotropy variation upon binding of Vimentin to the 2TEL telomeric G4 repeat (5 nM 5′-6-FAM-2TEL) in 5 mM Tris–HCl (pH 8.4), 150 mM KCl at 25°C. The data represent mean ± S.D. from three independent experiments. Vimentin concentration is calculated as tetramers concentration. (D) EMSA of 8 μM Vimentin with 2 μM (+) and 8 μM (++) 2TEL performed in 5 mM Tris–HCl (pH 7.4), 150 mM KCl, stained with Coomassie Brilliant Blue G250 and Sybr Green II.

Figure 4.

SDS-PAGE of the limited proteolysis reactions performed with trypsin on tetrameric Vimentin, in 5 mM Tris–HCl (pH 8.4), 150 mM KCl, in the absence (VIM) or in the presence of stoichiometric amounts of 2TEL (VIM + DNA). The reaction was quenched after 0, 1, 5 and 30 min from trypsin addition. Labelled bands were cut and subjected to in-gel trypsin digestion and LC–MS^E analyses for peptide sequencing. M indicated the molecular weight markers.

Figure 5.

Results of GO enrichment analysis performed with David tool on human genes presenting putative G4 repeats within their promoters.