Insights into telomeric G-quadruplex DNA recognition by HMGB1 protein

Abstract

HMGB1 is a ubiquitous non-histone protein, which biological effects depend on its expression and subcellular location. Inside the nucleus, HMGB1 is engaged in many DNA events such as DNA repair, transcription and telomere maintenance. HMGB1 has been reported to bind preferentially to bent DNA as well as to noncanonical DNA structures like 4-way junctions and, more recently, to G-quadruplexes. These are four-stranded conformations of nucleic acids involved in important cellular processes, including telomere maintenance. In this frame, G-quadruplex recognition by specific proteins represents a key event to modulate physiological or pathological pathways. Herein, to get insights into the telomeric G-quadruplex DNA recognition by HMGB1, we performed detailed biophysical studies complemented with biological analyses. The obtained results provided information about the molecular determinants for the interaction and showed that the structural variability of human telomeric G-quadruplex DNA may have significant implications in HMGB1 recognition. The biological data identified HMGB1 as a telomere-associated protein in both telomerase-positive and -negative tumor cells and showed that HMGB1 gene silencing in such cells induces telomere DNA damage foci. Altogether, these findings provide a deeper understanding of telomeric G-quadruplex recognition by HMGB1 and suggest that this protein could actually represent a new target for cancer therapy.

Figure 1.

Representation of (A) unimolecular parallel (TelG4-up), (B) unimolecular hybrid (TelG4-uh) and (C) tetramolecular parallel (TelG4-tp) G4 structures formed by truncations of human telomeric DNA sequence.

Figure 2.

CD spectra of (A) TelG4-up, (B) TelG4-uh and (C) TelG4-tp G4s (1 μM) (black lines), HMGB1_1–166 (1 μM) (blue lines), and 1:1 G4/HMGB1_1–166 mixtures (gray lines). The spectra derived from the arithmetical sum of the individual spectra of G4s and HMGB1_1-166 are reported as well (cyan lines).

Figure 3.

(A) Per residues intensity changes and (B) chemical shift perturbation of HMGB1_1-166 protein (25 μM) in the presence of 1 μM TelG4-up DNA. The residues exhibiting the largest effects have been colored in red (A) and green (B) on the structure of free HMGB1 (pdb: 2YRQ).

Figure 4.

(A) Per residues intensity changes and (B) chemical shift perturbation of HMGB1_1-166 protein (25 μM) in the presence of 1 μM TelG4-up DNA and Braco-19 (2.5 mM). The residues exhibiting the largest effects have been colored in red (A) and green (B) on the structure of free HMGB1 (pdb: 2YRQ).

Figure 5.

(A) Per residues intensity changes and (B) chemical shift perturbation of box-A domain of HMGB1 (25 μM) in the presence of 1 μM TelG4-up DNA. The residues exhibiting the largest effects have been colored in red (A) and green (B) on the structure of free HMGB1 (pdb: 2YRQ).

Figure 6.

Per residues intensity changes of box-B after the addition of box-A (25 μM) to the complex box-B/TelG4-up (25 μM). The residues exhibiting the largest effects have been colored in red on the structure of free HMGB1 (pdb: 2YRQ).

Figure 7.

(A) Per residues intensity changes and (B) chemical shift perturbation of box-A domain protein (25 μM) in the presence of 1 μM TelG4-up DNA and Braco-19 (2.5 mM). The residues exhibiting the largest effects have been colored in red (A) and green (B) on the structure of free HMGB1 (pdb: 2YRQ).

Figure 8.

(A–C) Fluorescence emission spectra of HMGB1_1–166 (3 μM) in the absence and presence of stepwise addition (5 μl) of (A) TelG4-up, (B) TelG4-uh, and (C) TelG4-tp G4s at 25°C. (D) Titration curves obtained by plotting the fraction of bound protein (α) versus the DNA concentration for TelG4-up (black), TelG4-uh (red), and TelG4-tp (green).The circles represent the experimental data, the lines are the best fit obtained with the theoretical model. (E, F) Time evolution SPR sensorgrams obtained at 25°C by injections of (E) TelG4-up and (F) TelG4-uh at various concentrations on the chip-immobilized HMGB1_1-166 with a contact time of 80 s, a dissociation time of 600 s, and a flow rate of 30 μl/min. The sensorgrams are shown as colored lines and their respective fits, based on the 1:1 kinetic interaction model, as black lines.

Figure 9.

Best first models of HMGB1_1-166 and TelG4-up obtained performing multi-body docking calculations, with the software HADDOCK (see main text), and considering in one case the interaction of box-A with the G-tetrad plane (model A) and in the other case the interaction with the loop (model B). The interaction of box-B is with the G-tetrad plane in both cases. Box-A is in blue, box-B in marine, TelG4-up in yellow, and the ‘active’ residues in the two domains as magenta spheres.

Figure 10.

HMGB1 localizes at telomeres. (A) To establish whether HMGB1 colocalizes with telomeres, telomerase-positive HeLa cells and telomerase-negative/ALT-positive U2OS cells were processed for immunofluorescence using antibodies against the telomeric protein TRF1 (green) and HMGB1 (red). Nuclei are indicated by dotted lines. The images were acquired with a Leica Deconvolution microscope (magnification 63×). Enlarged views of co-localization spots are reported. (B) Formaldehyde-cross-linked chromatin fragments, obtained from HeLa cells, were immunoprecipitated with antibodies against HMGB1 and TRF1, as positive control for telomeric sequences. Chromatin immunoprecipitation with Rabbit immunoglobulins (IgG) and without antibody were used as negative controls. To verify that an equivalent amount of chromatin was used in the immunoprecipitates, 0.5 and 0.05 μg of the total chromatin (Input) were included in the blot. Specific (Telo) and nonspecific (Alu) probes were used. (C) U2OS cells were treated with Braco-19 at the indicated doses for 24 h and processed for IF analysis as in (A). Left panel: Representative immunofluorescence images. Right panel: Quantitative analysis of HMGB1/TRF1 colocalization, expressed as the number of HMGB1/TRF1 colocalizations for cell. Histograms show the mean values ± S.D. of three independent experiments.

Figure 11.

Knocking-down of HMGB1 induces telomere instability. HeLa and U2OS cells were transfected with a siRNA against HMGB1 (siHMGB1) or a non-targeting siRNA (siControl) and assayed. (A) Representative western blot showing the expression levels of HMGB1 evaluated in HeLa and U2OS cells, silenced or not (siControl) for HMGB1. Analysis of β-actin was reported as loading control. (B–E) The indicated cell lines were processed for immunofluorescence (IF) using antibodies against γH2AX and TRF1 to visualize the DNA damage and telomeres, respectively. (B) Histogram showing the percentage of γH2AX-positive cells. (C) Representative images of IF microscopy experiments. γH2AX spots are visualized in green, TRF1 in red and nuclei are stained in blue. Enlarged views of Telomere Induced Foci (TIFs) are reported. The images were acquired with a Leica Deconvolution microscope (magnification 63x). (D, E) Quantitative analysis of TIFs. The graphs represent the percentages of (D) TIF-positive cells and (E) the mean number of TIFs per cell in the indicated samples. Cells with at least four γH2AX/TRF1 foci were scored as TIF positive. Histograms show the mean values ± S.D.