Study of the Interaction Between Zinc Finger Protein ZNF191(243-368) and DNA by Fluorescence Quenching

C₂H₂ zinc finger proteins are the predominant class of human transcription factors, mediating sequence-specific DNA recognition. ZNF191(243–368), the C-terminal domain of ZNF191 containing four C₂H₂ motifs, is implicated in hepatocellular carcinomaproliferation. To investigate its DNA-binding properties, a GST-ZNF191(243–368) fusion protein was used in fluorescence quenching studies with two DNA sequences (GGAGGGTGGTTA and GAAATAATGTTA). Results indicate static quenching due to protein-DNA complex formation, with quenching constants >100 L/mol. Slight differences in binding affinities suggest multiple factors influence DNA recognition. These findings highlight the complexity of zinc finger–DNA interactions and the need for caution in predicting sequence specificity solely from motif analysis.

Krüppel-type (C₂H₂) zinc finger is a ubiquitous motif that mediates the sequence-specific recognition of DNA and widely exists in eukaryotes, which can bind DNA, RNA, or other proteins and assume critical roles in various biological functions, including cell differentiation and embryo development(1–3). Previous studies explored the crystal/NMR structures and DNA binding sites of zinc finger proteins, and some works reported the possibility of previewing the recognition site of a novel C₂H₂ zinc finger protein by sequence analysis (4–8). These observations are insufficient to affirm the recognition code of zinc finger protein for the synergistic impact of multiple factors on its DNA-binding potential.Therefore, the obtaining of specific DNA-binding sequences of zinc finger proteins and understanding their functions remains challenging (9–11).

The zinc finger transcription factor 191 (ZNF191), a Krüppel-typezinc finger protein, was reported to bind to β-catenin promoter, promoting the cell proliferation of hepatocellular carcinoma (HCC), and regulatea wingless-type family member 8B (Wnt8B); this plays a pivotal role in HCC proliferation by serving as a new prognostic biomarker and a potential therapeutic target for HCC patients (12–14). ZNF191(243-368), the zinc finger region protein of ZNF191, presumably binds to a specific DNA sequence, which acts as the main functional region of ZNF191. Though several studies reported on the AT-binding inclination of ZNF191(243-368), the consensus binding site of ZNF191(243-368) had an AGGG core also reported(14–16). The actual function and DNA-binding site of ZNF191(243-368), therefore, still need to be further investigated. In this study, the fusion protein of ZNF191(243-368) with glutathione-S-transferase (GST)—that is, GST-ZNF191(243-368)—is used to elucidate the interaction of ZNF191(243-368) and DNA by fluorescence quenching.

Materials and Methods

Expression and Purification of Fusion Protein

The zinc finger fusion protein GST-ZNF191(243-368) was expressed from the recombinant vector pGEX-ZF and purified through Glutathione Sepharose 4B slurry (Amersham Pharmacia Biotech), Amicon YM-10 (Millipore) and Sephadex G75 columns. The detailed processes were as showninthe literature (16).

Synthesis of DNA

Two DNA duplexes (DNA1: GGAGGGTGGTTA, DNA2: GAAATAATGTTA) were used to study the DNA binding activity of ZNF191(243-368) (15,16). The four single-strand DNAs were synthesized (Sangon Biotech) with the following sequences: DNAa: ‘5-GGAGGGTGGTTA-3’; DNAb: ‘5-TAACCACCCTCC-3’; DNAc: ‘5-GAAATAATGTTA-3’; DNAd: ‘5-TAACATTATTTC-3’. Each single-strand DNA was prepared into a 200 μM solution according to its molecular weight, and mixed the complementary DNA solutions in equal volumes, boiled in a water bath for 10 min, cooled at room temperature, and purified by nondenaturing polyacrylamide gel electrophoresis. The DNA concentration was then determined using ultraviolet (UV) spectroscopy with an absorption at 260 nm, resulting in double stranded DNA1 (a-b) and DNA2 (c-d).

DNA Binding Experiment

DNA binding studies were performed in a buffer (10 mmol/L Tris-HCl, pH 7.5). Titrations are conducted by adding the DNA stock solution (50 μmol/L) to 2 mL of the protein solution of GST-ZNF191(243-368) or GST (1 μmol/L).Fluorescence emission spectra are obtained on a Cary Eclipse fluorescence spectrometer (Varian Company) within 300-400 nm at an excitation wavelength of 280 nm in a 1 cm fluorescence cuvette at 20 °C. The entrance and exit slits for all fluorescence measurements are maintained at 10 nm.

Results and Discussion

Fluorescence spectroscopy is widely used to probe protein-DNA interactions. Using tyrosine in the protein molecule as the endogenous fluorescence probe, the slight alteration in the conformation of protein, the micro-environment round Tyr, and the interaction between protein and DNA based on the fluorescence quenching effect can be analyzed (17–20).

The fluorescence spectra of GST-ZNF191(243-368) and GST proteins with two synthesized DNA sequences were then screened. The different fluorescence spectra of GST-ZNF191(243-368) fusion protein and GST were obtained by subtracting the fluorescence spectra of GST from those of GST-ZNF191(243-368) to eliminate the effect of GST on ZNF191(243-368), as shown in Figure 1.

Figure 1: Difference fluorescence emission spectra of GST-ZNF191(243-368) and GST titrated by DNA. (a) and (b) separately belong to DNA1 and DNA2.C_protein = 1 μmol/L, from up to down: C_DNA = 0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 μmol/L, respectively.

The fluorescence difference spectra show that the endogenous fluorescence intensity of ZNF191(243-368) is decreased regularly with the increase of double-strand DNA concentration. DNA is the fluorescence quenching agent for GST and ZNF191(243-368) proteins. The fluorescence quench of DNA to ZNF191(243-368)protein is accordance with the Stern-Volmer equation:

in which F₀ and F are the fluorescence intensities of the protein without and with DNA respectively, [Q] is theconcentration of double-strand DNA, and K_sv is the quenching rate constant of DNA to protein (21–23). Based on equation 1, the quenching rate constant can be calculated by the fitting curves of the quenching of DNA to protein.

Figure 2: Stern-Volmer plot of the fluorescence quenching of DNA to ZNF191 (243-368).

The quenching rate constants of DNA1 or DNA2 and ZNF191(243-368) obtained from Figure 2 are 8.52×10⁵ L/mol and 5.68×10⁵ L/mol, respectively. Because the maximum rate constant of dynamic quenching process of biological macromolecules is less than 100 L/mol (24), the fluorescence quenching of DNA to ZNF191 (243-368) is a static quenching process—that is, the fluorescence quenching of DNA to ZNF191(243-368) is caused for the formation of protein-DNA complex.

Figure 3: Plots of lg[(F₀-F)/F] vs. lg[Q] of ZNF191(243-368) by DNA.

For the static quenching process, the changes of fluorescence intensity are associated with the DNA concentration as the following equation:

in which F₀, F, and [Q] are the same as those of equation 1, K_a is the binding constant of DNA to protein, and n is the binding number of DNA in the ZNF191(243-368)protein(25,26). The K_a and n can then beobtained from the intercept and slope of the curves of lg[(F₀‒F)/F] vs. lg[Q] in Figure 3, and the values are shown in Table Ⅰ.

The experiment results indicate that ZNF191(243-368) and the DNA molecule forma 1:1 type complex. The binding constant of DNA1 to ZNF191(243-368) is only 1.35 times greater than that of DNA2 with protein. This difference is not as significant as the difference in the three-component system, but this result is consistent with our previous research; using three-component systems proves that fluorescence spectroscopy is a relatively reliable method to study the interaction (16). The interaction between ZNF191(243-368) and DNA1 is strong, which is inconsistent with the research results of ZNF191 in vivo; this difference may be attributed to the effect of SCAN box located before ZNF191(243-368), the difference in in vitro and in vivo experiments, or synergistic effects of many factors on the recognition of zinc finger protein to DNA (27–30). Also, it may be the great interference of the GST protein or the low sensitivity of the endogenous fluorescence.

Conclusions

This study elucidates that the fluorescence quenching of DNA1 or DNA2 to ZNF191(243-368) is a static quenching process, and a complex of protein-DNA is formed in the titration process. The slight difference in two quenching constants also indicates that the sequence-specific recognition of zinc finger protein to DNA should be studied cautiously, because the zinc finger domain of a protein is not the only factor that affects the DNA-binding properties of the protein. The property, structure, and function of zinc finger proteinZNF191 and ZNF191(243-368) still need further investigations, and the sequence-specific recognition of zinc finger protein to DNA should be studied cautiously.

Conflicts of Interest

There are no conflicts of interest among the authors.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 21301050) and the Horizontal Scientific Research Project of Henan University of Technology (No. 51001089).

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Wenbo Jia, Libo Yuan, and Dongxin Zhao are with the School of Chemistry and Chemical Engineering at Henan University of Technology, in Zhengzhou, China. Direct correspondence to: zhaodx798@163.com