Interferon-inducible protein 16 (IFI16) is a member of the HIN-200 protein family, containing two HIN domains and one PYRIN domain. IFI16 acts as a sensor of viral and bacterial DNA and is important for innate immune responses. IFI16 binds DNA and binding has been described to be DNA length-dependent, but a preference for supercoiled DNA has also been demonstrated. Here we report a specific preference of IFI16 for binding to quadruplex DNA compared to other DNA structures. IFI16 binds to quadruplex DNA with significantly higher affinity than to the same sequence in double stranded DNA. By circular dichroism (CD) spectroscopy we also demonstrated the ability of IFI16 to stabilize quadruplex structures with quadruplex-forming oligonucleotides derived from human telomere (HTEL) sequences and the MYC promotor. A novel H/D exchange mass spectrometry approach was developed to assess protein interactions with quadruplex DNA. Quadruplex DNA changed the IFI16 deuteration profile in parts of the PYRIN domain (aa 0–80) and in structurally identical parts of both HIN domains (aa 271–302 and aa 586–617) compared to single stranded or double stranded DNAs, supporting the preferential affinity of IFI16 for structured DNA. Our results reveal the importance of quadruplex DNA structure in IFI16 binding and improve our understanding of how IFI16 senses DNA. IFI16 selectivity for quadruplex structure provides a mechanistic framework for IFI16 in immunity and cellular processes including DNA damage responses and cell proliferation.

Since the description of double-stranded B-DNA, the knowledge of variability of DNA structures has greatly expanded. The presence of cruciform, triplex and quadruplex structures was demonstrated by many techniques in vitro. Nowadays, there is substantial evidence for the presence of these unusual structures in vivo and their significance is being uncovered [ 27 , 28 ]. Large numbers of potential quadruplex sequences were predicted by in silico analysis [ 29 ]. To date, many quadruplex DNA sequences in the human genome were characterized, for example in repetitive G-rich sequences such as telomeres [ 30 ] and in the promoters of oncogenes such as MYC [ 31 ], KIT [ 32 ], BCL2 [ 33 ] and TERT [ 34 ]. The transition from double stranded DNA to quadruplex structure influences processes related to cancer through expression of target genes [ 35 – 37 ] or through inhibition of telomerase processivity [ 38 ]. Recently, quadruplex DNA and RNA structures have been detected in viral genomes, notably Epstein-Barr virus [ 39 ], HIV-1 [ 40 , 41 ] and human papillomaviruses [ 42 ]. Contemporary results show the importance of quadruplex structure in maintaining chromosome integrity, replication, regulation of transcription and translation [ 43 ].

IFI16 was first identified as a DNA binding protein by Dawson and Trapani in 1995 [ 21 ]. In 2008, IFI16 HIN-A was described as an RPA-like protein, sharing the same oligonucleotide / oligosaccharide domain and preference for single stranded DNA over double stranded DNA [ 22 ]. According to Unterholzner et al., IFI16 binding to DNA is not sequence-specific or AT content-dependent, but is strongly DNA length-dependent [ 9 ]. Based on crystallographic studies, the IFI16 HIN-B—double stranded DNA interface is accomplished through electrostatic interactions between the negatively charged sugar-phosphate backbone and positively charged protein residues [ 23 ]. Based on structural analysis and binding experiments of the HIN-A and HIN-B domains with double stranded DNA, a model of non-interacting beads on a string was proposed [ 23 , 24 ]. In a recent study, the single stranded DNA preference was questioned for the full length wild type protein and DNA-length dependence was characterized in more detail, revealing cooperative assembly of IFI16 filaments on double stranded DNA [ 25 ]. IFI16 binding to long plasmid DNA was studied and preferences for supercoiled over linear forms and for cruciform structure over double stranded DNA was observed [ 26 ].

IFI16 cooperates with other proteins in transcriptional regulation and DNA repair. Binding of IFI16 HIN-A domain to the C-terminus of p53 results in enhanced DNA binding of p53 and increased transcriptional activation of p21 [ 14 ]. Moreover, IFI16 is involved in the p53-mediated pathway and DNA damage recognition through breast cancer-associated protein-1 (BRCA1) interaction, where BRCA1 relocates IFI16 from the cytoplasm to the nucleus and IFI16 is necessary for full activation of DNA repair after ionizing radiation [ 15 – 17 ]. As a DNA sensor, IFI16 stimulates the formation of inflammasomes in certain cell types during infection with Kaposi Sarcoma-associated herpesvirus [ 1 , 2 ], Herpes simplex virus 1 [ 18 ], Epstein-Barr virus [ 19 ] and Human immunodeficiency virus (HIV-1) [ 20 ]. The DNA sensing ability of IFI16 is also related to the activation of interferon β expression through interaction with stimulator of interferon genes [ 9 ], and interferon α expression [ 4 ].

IFI16 (interferon-inducible protein 16) has multiple biological functions; it is a DNA sensor important in inflammasome activation [ 1 , 2 ], but it also plays roles in transcriptional regulation [ 3 , 4 ] and cell proliferation [ 5 ]. IFI16 belongs to the highly homologous HIN-200 (hemopoietic expression—interferon-inducibility—nuclear localization) protein family characterized by a 200 amino acid motif containing a DNA binding domain at the C-terminus and a PYRIN domain at the N-terminus, involved mainly in protein-protein interactions. The human HIN-200 family is composed of four characterized members; absent in melanoma 2 (AIM2), interferon-inducible protein X (IFIX), myeloid cell nuclear differentiation antigen (MNDA) and IFI16 [ 6 , 7 ]. IFI16 differs from other members by the presence of two HIN domains [ 7 ] and was detected not only in the nucleus, but also in the cytoplasm [ 8 , 9 ]. IFI16 subcellular localization is influenced by the cell type [ 10 ], post-translational modification [ 11 , 12 ] and cell treatment. For example, pathogen invasion causes the formation of IFI16 foci in the cytoplasm and induces interferon β (IFNB) gene expression [ 9 ] and UV-light causes the transfer of IFI16 from the nucleus to the cytoplasm [ 13 ].

Each sample was thawed and injected onto an immobilized pepsin column (66 μl bed volume, flow rate 20 μl/min, 2% acetonitrile / 0.05% trifluoroacetic acid). Peptides were trapped and desalted on-line on a peptide microtrap (Michrom Bioresources, Auburn, CA, USA) for 2 min at flow rate 20 μl/min. Next, the peptides were eluted onto an analytical column (Jupiter C18, 1.0 x 50 mm, 5 μm, 300Å, Phenomenex, Torrance, CA, USA) and separated using a linear gradient elution of 10% B in 2 min, followed by 31 min isocratic elution at 40% B. Solvents were: A– 0.1% formic acid in water, B– 80% acetonitrile / 0.08% formic acid. The immobilized pepsin column, trap cartridge and the analytical column were kept at 1°C. Mass spectrometric analysis was carried out using an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, Whaltan, MA, USA) with ESI ionization on-line connected with a robotic system based on the HTS-XT platform (CTC Analytics company, Zwingen, Switzerland). The instrument was operated in a data-dependent mode for peptide mapping (LC-MS/MS). Each MS scan was followed by MS/MS scans of the top three most intensive ions from both CID and HCD fragmentation spectra. Tandem mass spectra were searched using SequestHT against the cRap protein database ( ftp://ftp.thegpm.org/fasta/cRAP ) containing the IFI16 protein sequence. Sequence coverage was analysed with Proteome Discoverer 1.4 software (Thermo Fisher Scientific). Analysis of deuterated samples was performed in LC-MS mode with ion detection in the orbital ion trap and the data were processed using HD Examiner (Sierra Analytics, Modesto, CA, USA).

At first a sample for peptide mapping was prepared to obtain the protein coverage. IFI16 protein (1 μM) was dissolved in 1% DMSO and the pH adjusted using 0.88 M HCl in 1 M glycine. The next step was preparation of deuterated samples. IFI16 protein was incubated with DNA (single stranded, double stranded NHEIII, quadruplex NHEIII) for 5 min, then the protein-DNA complex was initiated for deuteration by dilution into 1% DMSO in deuterated water containing 5 mM Tris/HCl, 0.1 mM EDTA, 50 mM KCl, pH 7.6 to stabilize DNA. The H/D exchange was carried out at 4°C and was quenched by the addition of 0.88 M HCl in 1 M glycine after 15 min. Then 3 μg of pepsin was added and the protein was digested at 4°C. After 2 min the sample was placed on a strong anionic exchange column (Q-sepharose). Peptides were spun through the column (1 min, 8000 rcf) while DNA was captured on the column. Finally, the sample was rapidly frozen in liquid nitrogen. Simultaneously a control sample was prepared, where the protein was incubated with 1% DMSO (instead of DNA).

CD measurements were carried out in a Jasco 815 (Jasco International Co., Ltd.,Tokyo, Japan) dichrograph in 1 cm path-length quartz Hellma microcells placed in a thermostatically regulated cell holder at 23°C. A set of four scans was averaged for each sample with a data pitch of 0.5 nm and 100 nm.min -1 scan speed. CD signal was expressed as the difference in the molar absorption, Δε of the left- and right-handed circularly polarized light, molarity being related to DNA strands. Precise DNA strand concentrations were determined on the basis of UV absorption at 260 nm measured in TE buffer pH 8, using molar extinction coefficients of 539,600, and 541,400 M -1 cm -1 calculated according Gray et al [ 45 ] for HTEL and NHEIII sequences respectively. Experimental conditions were changed directly in the cells by adding solution (KCl protein buffer: 50 mM KCl, 5 mM Tris/HCl pH 7.6, 10% glycerol, 2 mM DTT, 0.1 mM EDTA; NaCl protein buffer: 20 mM HEPES, pH 7.6, 500 mM NaCl, 10% glycerol, 2 mM DTT) with or without the protein and the final DNA strand concentration was corrected according to the increase in volume.

Synthetic oligonucleotides with and without FAM-3’-end labeling were purchased from Integrated DNA Technologies, Inc., Coralville, IA, USA. The oligonucleotide sequences of single stranded, double stranded, cruciform and quadruplex DNA are shown in Table 1 . Complementary oligonucleotides for double stranded and cruciform structure were annealed by incubation at 95°C for 5 min with subsequent cooling to room temperature. Oligonucleotide for quadruplex formation was heated to 95°C in TE buffer and then incubated with 50 mM KCl at room temperature for 16 h.

Supercoiled plasmid DNAs of pBluescript II SK (-) and the derived plasmid pCMYC were isolated from DH5α as described in the QIAGEN protocol (QIAGEN GmbH, Hilden, Germany). pCMYC plasmid (containing 141 bp of nuclease hypersensitive element III1a (NHEIII) region of the human MYC promoter forming G-quadruplex) was constructed by cloning the 141 bp EcoRI/HindIII restriction fragment of pNHE III 1a plasmid [ 44 ] into the EcoRI/HindIII site of pBluescript and was kindly provided by Dr. Marie Brazdova. Plasmids were linearized by EcoRI restriction enzyme (New England Biolabs, Ipswich, MA, USA).

Results

Recognition of quadruplex structures by IFI16 in plasmid DNA To compare IFI16 binding to quadruplex and double stranded DNAs derived from the MYC promoter we used electrophoretic mobility shift assay with DNA plasmids on agarose gel. It was previously demonstrated that IFI16 binds preferentially to supercoiled DNA compared to the linear form of the same plasmid DNA [29]. Moreover, IFI16 was described as a length-dependent DNA binding protein [22, 28]. Considering these observations, we were interested in whether IFI16 is capable of recognizing quadruplex structures stabilized as a local structure in large negatively supercoiled DNA molecules (where they represent only a small portion of a DNA substrate which per se is relatively strongly bound by the protein). In this study we used two supercoiled plasmids: pBluescript was used as a model of supercoiled DNA without quadruplex structure and pCMYC containing 141 bp from the NHEIII region of MYC promoter that includes G:C-rich sequence was used as a model for binding to quadruplex DNA. Formation of the quadruplex structure in the plasmid was induced by negative supercoiling (due to destabilization of duplex DNA, thus favoring separation of strands and folding of the G-rich strand into the quadruplex). Presence of the quadruplex, featuring an open non-B structure in the plasmid, was confirmed by S1 nuclease cleavage as described earlier for plasmids with cruciform structure [46]. In addition, probabilities of quadruplex formation in both plasmids were analyzed by the free software QGRS Mapper [47] and only pCMYC (but not pBluescript) showed possible formation of one predicted G-quadruplex for 4 minimal G-group size, in agreement with expectations (Table 1, underlined G-quartets in the quadruplex NHEIII sequence are predicted to form quadruplex structure in pCMYC). Various amounts of IFI16 protein were incubated with 100 ng of plasmid DNAs and the resulting complexes were then separated on 1% agarose gels. In Fig 1A, lanes 1 and 7, free DNAs without IFI16 protein were loaded. After addition of IFI16 (molar ratio protein: DNA 1.25:1 (lane 2 and 8), 2.5:1 (lane 3 and 9), 5:1 (lane 4 and 10), 10:1 (lane 5 and 11) 20:1 (lane 7 and 12)) we observed different band patterns due to IFI16 DNA binding. The binding of IFI16 to DNA was visible as shifted (retarded) band(s) and/or as a decrease of the free DNA band intensity (decreasing to total loss caused by saturation of protein binding). While we observed retarded band(s) of pBluescript from molar ratio 2.5:1 (Fig 1A, lane 3), pCMYC was evidently bound from the lowest protein concentration tested (molar ratio1.25:1, Fig 1A, lane 8). At higher protein concentration (lane 6) pBluescript-IFI16 complexes formed multiple shifted bands but the free form of pBluescript was still visible even at a protein:DNA ratio of 20:1, whereas pCMYC (lane 12) was completely bound with IFI16 protein under the latter conditions and free pCMYC was not observed. Strikingly, for protein:DNA ratios 1:1.25–1:5 pCMYC formed a single strong retarded band (compared to pBluescript forming multiple weak bands), suggesting a single strongly preferred protein-DNA complex formed at a specific site. Similar behavior was previously observed with a plasmid containing a single target site for p53 upon formation of specific p53-DNA complexes [48]. In pCMYC, such a preferred site for IFI16 binding can be the quadruplex structure. In contrast, pBluescript contains no preferentially bound site and at higher protein-DNA ratios it forms IFI16-DNA complexes with various stoichiometries (again, in analogy with earlier observed p53-DNA binding -[48]). Hence, our results strongly suggest selective binding of IFI16 to a quadruplex existing as a local supercoil-stabilized structure in plasmid DNA i.e., with structural arrangement more complex than represented by short oligonucleotide targets. In Fig 1B we compared the binding of IFI16 to the linear forms of both tested plasmids. In contrast to the above described results with scDNAs, structurally unconstrained linearized forms of the same plasmid did not apparently bind IFI16 at protein-DNA ratios between 1.25:1 and 20:1 (Fig 1B). PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Fig 1. Binding of IFI16 protein to supercoiled DNAs. (A) 100 ng sc pBluescript (lane 1–6) and sc pCMYC (lane 7–12) were incubated with increasing concentrations of IFI16 (molar ratio DNA:protein 1:0 / 1:1.25 / 1:2.5 / 1:5/ 1:10 / 1:20) in binding buffer (5 mM Tris-HCl, pH 7.0; 1 mM EDTA, 50 mM KCl and 0.01% Triton X-100) on ice for 15 min. The electrophoresis ran for 3 h at 100 V at 4°C. (B) 100 ng linear pBluescript (lane 1–6) and linear pCMYC (lane 7–12) were incubated with increasing concentrations of IFI16 (molar ratio DNA: protein 1:0 / 1:1.25 / 1:2.5 / 1:5/ 1:10 / 1:20) in binding buffer (5 mM Tris-HCl, pH 7.0; 1 mM EDTA, 50 mM KCl and 0.01% Triton X-100) on ice for 15 min. The electrophoresis ran for 3 h at 100 V at 4°C. https://doi.org/10.1371/journal.pone.0157156.g001

Stabilization of quadruplex structure by IFI16 Many quadruplex binding proteins were described recently [28]. Some quadruplex binding proteins resolve these structures, while others induce and enhance quadruplex formation. Having provided initial evidence that IFI16 recognizes quadruplex structure in plasmid DNA, we validated binding to two short quadruplexes and further investigated the effect of IFI16 protein on the formation and stabilization of quadruplex structures by CD spectroscopy. We used the HTEL oligonucleotide which forms an antiparallel (2+2) quadruplex structure and the NHEIII oligonucleotide which folds into a parallel quadruplex [49–51]. The HTEL oligonucleotide CD spectra in the presence of 50 mM KCl and 50 mM NaCl are shown at Fig 2A—the unstructured oligonucleotide HTEL (blue line) is represented by the peak at 255 nm, the quadruplex structure is demonstrated by formation of a peak at 296 nm typical for antiparallel (2+2) quadruplex structure. The NHEIII oligonucleotide folds into a parallel quadruplex in the presence of 50 mM KCl more efficiently than in the presence of 50 mM NaCl [52] (Fig 2B). The quadruplex structure is demonstrated by peak shift to 260 nm and an increase in height. The differences in structure of unfolded oligonucleotides, oligonucleotides folded to quadruplex parallel or antiparallel structure and double helical oligonucleotides by CD spectroscopy are summarized in Vorlickova et al. [50]. PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Fig 2. CD spectroscopy of quadruplexes and their stabilization by IFI16. (A) CD spectra of oligonucleotide HTEL in TE buffer after denaturation (blue line), in TE buffer + 50 mM NaCl (red line) and in TE buffer + 50 mM KCl (green line). (B) CD spectra of oligonucleotide NHEIII in TE buffer after denaturation (blue line), in TE buffer + 50 mM NaCl (red line) and in TE buffer + 50 mM KCl (green line). The schematic drawings represent quadruplex structures of HTEL and NHEIII sequences. (C) The effect of recombinant IFI16 on HTEL quadruplex formation in potassium ions. CD spectra description: HTEL oligonucleotide in TE buffer (blue line), HTEL in TE buffer with 50 mM KCl (red line), HTEL in TE buffer + protein buffer with final concentration 3.4 mM KCl (green line), HTEL in TE buffer + IFI16 in protein buffer at molar ratio 1:1 and final concentration 3.4 mM KCl (violet line), IFI16 protein in protein buffer with final concentration 3.4 mM KCl in TE buffer (black line). (D) The effect of recombinant IFI16 on NHEIII quadruplex formation in potassium ions. The same description of curves as in C (NHEIII instead of HTEL). (E) The effect of recombinant IFI16 on HTEL quadruplex formation in sodium ions. CD spectra description: HTEL oligonucleotide in TE buffer (blue line), HTEL in TE buffer with 50 mM NaCl (red line), HTEL in TE buffer + protein buffer with final concentration 3.2 mM NaCl (green line), HTEL in TE buffer + IFI16 in protein buffer at molar ratio 1:2 and final concentration 3.2 mM NaCl (violet line), IFI16 protein in protein buffer with final concentration 3.2 mM NaCl in TE buffer (black line). (F) The effect of recombinant IFI16 on NHEIII quadruplex formation in sodium ions. The same description of curves as in E (NHEIII instead of HTEL). https://doi.org/10.1371/journal.pone.0157156.g002 The effect of IFI16 on quadruplex stability was studied in the presence of either of the salts for both quadruplex forming oligonucleotides. First, we measured the CD spectra of the oligonucleotides in TE buffer after denaturation where the CD spectra suggest their unfolded state. Then we added the protein in buffer containing KCl or NaCl to the oligonucleotide in TE buffer. The same volume of protein buffer was added to the unfolded oligonucleotide as a control to see the effect of protein buffer itself on DNA structure. CD spectra indicating the IFI16 stabilization effect are shown in Fig 2C–2F. The unstructured oligonucleotide HTEL (Fig 2C) in TE buffer (blue line) is represented by the peak at 255 nm. After addition of the protein-free buffer (3.4 mM KCl in final volume) (green line), the initiation of formation of the quadruplex structure is visible as peaks appearing at 296 nm and 264 nm, and a decrease of the 255 nm peak. The addition of IFI16 (in molar ratio IFI16:oligonucleotide 1:1) to the unfolded oligonucleotide causes stronger quadruplex formation (magenta line), surprisingly even stronger than in the presence of 50 mM KCl (red line). Hence, IFI16 stimulates and stabilizes quadruplex structure formation. The short wavelength part of the spectrum is influenced by absorption of protein (black line for IFI16 without oligonucleotide). The same experiment was performed with the protein dissolved in buffer containing NaCl (Fig 2E). The CD spectra are colored as in Fig 2C. CD spectrum of HTEL in 50 mM NaCl is characterized by a maximum at 296 nm, similar to that observed in the presence of potassium ions (for comparison of the spectra see Fig 2A) and a minimum at 264 nm. At low NaCl concentrations (3.2 mM NaCl corresponding to the salt concentration after protein addition) there is only a small increase at 296 nm in the CD spectrum. IFI16 addition induced a larger change in the CD spectrum shape compared to the effect of 50 mM NaCl in the absence of the protein. Quadruplex formation in the presence of sodium ions (without IFI16) required higher salt concentration than observed for potassium ions. For this reason, molar ratio 1:2 (DNA:protein) was used (ratio 1:1 was too low to induce quadruplex formation in 1.6 mM NaCl, not shown). In the presence of protein, synergic effects of potassium or sodium ions and IFI16 on quadruplex formation were observed. Similarly, the unfolded oligonucleotide NHEIII spectrum (Fig 2D, blue line) showed a characteristic peak around 253 nm. Addition of protein-free buffer containing 3.4 mM KCl (green line) caused a shift to 260 nm and an increase in height, suggesting formation of the parallel quadruplex structure. The addition of IFI16 in the same buffer containing 3.4 mM KCl in the final volume (magenta line) increased the peak height more than addition of buffer alone (green line). The stabilization effect of IFI16 was even stronger than that of 50 mM KCl (red line). The stabilization effect of IFI16 on NHEIII quadruplex structure was less visible because the presence of 3.4 mM KCl (without protein) induced quadruplex formation. NHEIII quadruplex formation in sodium ions is less effective (the signature quadruplex peak was at 260 nm and was smaller in 50 mM NaCl than in 50 mM KCl, for comparison see Fig 2B). Therefore, the differences in CD spectra with and without protein are considerably larger (Fig 2F). The CD spectrum of NHEIII containing 3.2 mM NaCl (green line) exhibited a maximum at 253 nm, similar to unfolded NHEIII (blue line), and initial quadruplex formation was visible as an increase of the band. IFI16 addition caused a considerable increase in peak height and a shift to 260 nm, comparable to the effect of 50 mM NaCl alone. Again, higher protein amount was used (1:2 NHEIII:IFI16 molar ratio) because low sodium ion concentration was insufficient to support quadruplex formation at the 1:1 protein:DNA ratio. Thus, the synergic effect of ions and protein on quadruplex formation is predicted for all experimental conditions and it appears that IFI16 binds and stabilizes both quadruplexes to a comparable extent No preference for parallel/antiparallel conformation was observed by either CD spectroscopy or EMSA.