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Original Paper	Pdf file on Synergy OPEN omments
Acta Biochim Biophys Sin 2008, 40: 704-710
doi:10.1111/j.1745-7270.2008.00450.x

Characterization of the target DNA sequence for the DNA-binding domain of zinc finger protein 191

Haoyue Wang^1#, Ruilin Sun^3,4#, Guoxiang Liu^3,4, Minghui Yao^3,4, Jian Fei²*, and Hebai Shen¹*

1 Department of Chemistry, Life and Environmental Science College, Shanghai Normal University, Shanghai 200234, China

2 School of Life Science and Technology, Tongji University, Shanghai 200092, China

3 Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China

4 Graduate School of Chinese Academy of Sciences, Beijing 100049, China

Received: March 29, 2008�������

Accepted: May 30, 2008

This work was supported by grants from the National Basic Research Program of China (No. 2002CB713803), the National Natural Science Foundation of China (No. 20573075) and the Science and Technology Commission of Shanghai Municipality (Nos. 06XD14014, 0752nm028, 07DZ22303, 06DZ05137 and 54319939)

#These authors contributed equally to this work

*Corresponding authors:

Jian Fei: Tel, 86-21-65982429; Fax, 86-21-65982429; E-mail, [email protected]

Hebai Shen: Tel, 86-21-64321800; Fax, 86-21-54242640; E-mail, [email protected]

Studies on the DNA-binding properties of transcription factors are important in searching for the downstream genes regulated by these factors. In the present study, we report on the DNA-binding property of a Cys2His2-type transcription factor, zinc finger protein 191 (Zfp191), which has been newly found to play a significant role in mice. By constructing a fusion protein containing the DNA-binding domain of Zfp191, we characterized target DNA by determining the protein's binding specificity and dependence on zinc. The data showed that the DNA-binding domain of Zfp191 can specifically bind to the TCAT repeat motif and that there is a cooperative effect among the target DNA's multiple binding sites. Furthermore, the binding reaction is dependent on zinc. This work provides a foundation for further studies on the role of Zfp191 in gene regulation and development.

Keywords������ zinc finger protein 191; DNA-binding protein; microsatellite

As an important class of transcription factors in eukaryotes, zinc finger proteins play a very important role in the regulation of gene expression, signal transduction [1,2], cell growth, differentiation and development [3-5] by binding to their DNA recognition sites. Zinc finger protein 191(Zfp 191), a transcription factor recently found in mice, is widely expressed in multiple tissues and organs of mice, such as heart, brain, liver, kidney and skeletal muscles. According to data from a complementary DNA (cDNA) subtraction library and Northern blot analysis, Zfp191 was developmentally regulated, and was related to cartilage differentiation and basic cellular processes [6]. In recent years, Zfp191 gene knockout mice were found, and the embryos of homozygous (Zfp191-/-) were retarded in development and died at approximately 7.5 d post-fertilization, indicating Zfp191抯 invaluable role in early embryonic development [7]. Moreover, the Zfp191 expression pattern during early embryogenesis, particularly in areas of proliferation in the development of the central nervous system, also provided strong support of its important role and possibly accounted for the phenotypic features of Zfp191-/- embryos [8]. However, the exact role of Zfp191 in development was unknown. Therefore, it was necessary to ascertain its DNA-binding properties and investigate how Zfp191 regulates the expression of its downstream genes during development.

Zfp191 shows 94% identity in amino acid sequence and 88% identity in cDNA sequence to its human homolog, zinc finger protein 24 (ZNF24), a Cys2His2 (C2H2)-type zinc finger protein [9]. Zfp191 has four C2H2-type zinc fingers at its C-terminal, which constitute the potential DNA-binding domain; between the fingers, there is a highly conserved seven-amino acid inter-finger spacer, TGEKP(Y/F)X, a member of a Kr�ppel-like factor family [10]. We have designed a fusion protein, glutathione S-transferase-Zfp191 binding domain (GST-Zfp191BD), in which the N-terminal part of Zfp191 was replaced by the GST sequence (Fig. 1). Furthermore, the DNA-binding properties of this fusion protein were investigated by electrophoretic mobility shift assay (EMSA) and microwell colorimetric assay. Similar to its homolog ZNF24, Zfp191 can specifically interact with an intronic polymorphic TCAT motif in the first intron of human tyrosine hydroxylase (TH) gene [11-13]. The detection of some mutants by microwell colorimetric assay suggests that the fourth nucleotide in tetranucleotide core sequence is relatively flexible to its binding specificity, while the others are conserved. The binding of the fusion protein to its target sequence is cooperative. Finally, the DNA-binding activity of Zfp191 is shown to be zinc dependent.

Materials and Methods

Materials

Our laboratory derived the vector pX-C from pGEX-3. We used Escherichia coli BL21(DE3) which was conserved by our laboratory as the expression strain. Glutathione-affinity resin was purchased from Yeli Bioscience Company (Shanghai, China). Our laboratory produced anti-GST antibodies. General reagents were of analytical grade.

Plasmid construction

The Zfp191BD fragment, which encodes Zfp191 residues 229-368, was amplified by polymerase chain reaction from pZfp cDNA with the following primers: 5-GGGGCGG�C�C�GCATATGGAGAAACTTGTTTTC-3 (forward) and 5-G�G�GGGGTACCGTG�CT�GATTGT�TTCCTCCT-3 (reverse). The fragment was inserted into the vector pX-C and digested with NotI and KpnI to construct the pX-C-Zfp191BD plasmid. The constructed plasmid was identified by polymerase chain reaction and confirmed by DNA sequencing.

Expression and purification of the fusion protein

A 3 ml Luria broth culture, supplemented with 100 mg/ml ampicillin, was inoculated from monoclonal host strain E. coli BL21(DE3) containing recombinant plasmid, and was incubated overnight at 37 �C. The overnight culture was diluted 1:100 into 100 ml Terrific broth medium with 100 mg/ml ampicillin. When an absorbance of about 1.5 at 600 nm was reached, the bacterial culture was induced by 0.05 mM isopropyl b-D-thiogalactopyranoside (IPTG). After 4 h IPTG induction at 30 �C, the bacteria were harvested by centrifugation (7500 g at 4 �C for 10 min). The bacteria were resuspended in 20 ml lysis buffer [phosphate-buffered saline (PBS) (pH 7.4), 1 % Triton X-100, 1 mM phenylmethylsulphonyl fluoride (PMSF)] and lysed by ultrasonication in an ice bath. The lysate was centrifuged at 13,400 g for 20 min at 4 �C. The supernatant, containing mainly the fusion protein of GST-Zfp191BD, was collected and filtrated with microfiltration membranes (0.22 mm pore size).

The clear solution containing GST-Zfp191BD was applied to a pre-equilibrated glutathione-affinity resin column at a flow rate of 0.75 ml/min. The column was washed with 20 bed volumes PBS immediately after all the protein solution had passed through the column. Then the fusion protein was eluted with 10-15 bed volumes of freshly made elution buffer [10 mM glutathione in 50 mM Tris-HCl (pH 8.0)], and the elution was monitored using Coomassie brilliant blue G250 stain. Fractions containing purified protein were added with 50% (W/V) glycerol and stored at -80 �C. All the samples were analyzed by SDS-PAGE.

Electrophoretic mobility shift assay (EMSA)

The complementary oligonucleotides encoding the (TCAT)n or (CCAC)n motifs were annealed and labeled by incubating them in T4 polynucleotide kinase and [g-32P]ATP (250 Ci/mmol). Each binding reaction mixture contained 0.6 mg purified GST-Zfp191BD protein and appropriately labeled DNA in 10 ml buffer [15 mM HEPES (pH 7.6), 60 mM KCl, 6 mM EDTA, 7.5 mM MgCl2, 7.5 mM ZnCl2, 6% glycerol, 0.6% Nonidet P-40, 0.3 mM PMSF, 0.6 mM dithiothreitol, 1 mg poly(dI)/poly(dC)]. Protein, poly(dI)/poly(dC) and unlabeled competitors were first incubated for 15 min at room temperature. A radiolabeled probe was then added and the samples were incubated for another 15 min. The DNA-protein complexes were analyzed by electrophoresis on a 6% polyacrylamide gel, which was then dried for autoradiography [13].

Microwell colorimetric assay

The 96-well enzyme immunoassay/radioimmunoassay plate (Corning, New York, USA) was coated with diluted streptavidin [1 mg/ml in 50 mM carbonate buffer (pH 9.6)] and incubate overnight at 4 �C. The plate was then washed three times with PBS containing 0.1% Tween-20. Non-specific binding sites were blocked using 8% non-fat milk/PBS and incubated for 120 min at room temperature. After being washed three times with PBS containing 0.1% Tween-20, the plate was incubated with 10 pmol of biotin-dsDNA (Sangon, Shanghai, China) for 1 h at 37 �C in 100 ml PBS per well. The wells were then washed three times with PBS containing 0.1% Tween-20 and 75 mM Zn(Ac)2. The GST-Zfp191BD was diluted in the binding buffer [PBS containing 0.2 mg/ml bovine serum albumin, 2% non-fat milk, 0.05 mg/ml salmon sperm DNA (Genebase, Guangzhou, China), 1 mM PMSF, 50 mM ZnSO4, 0.6 mM dithiothreitol] to an appropriate concentration [15], added to the wells and incubated for 1 h at 25 �C. The plate was then washed three times with PBS containing 0.1% Tween-20. Mouse anti-GST antibodies, diluted 4000 times in 10 mM phosphate buffer (pH 7.4) containing 50 mM NaCl and 1% non-fat milk, were added and incubated for 1 h at 25 �C. The plate was washed three times with PBS containing 0.1% Tween-20 and 75 mM Zn(Ac)2. Then the appropriate dilution of horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G antibody was added to the wells and incubated for 1 h at room temperature. The plate was washed four times with PBS containing 0.1% Tween-20 and 75 mM Zn(Ac)2 and then once again with PBS. Next, 100 ml colorimetric buffer [0.1 mg/ml tetramethylbenzidin, 0.1 M NaH2PO4, 0.05 M citric acid, 0.0024% H2O2 (pH5.5)] was added and incubated for 5-10 min at 37 �C before adding 25 ml stopping solution (2 M H2SO4). Optical density was then read at 450 nm, using a 630 nm reference wavelength, on an enzyme-linked immunosorbent assay microplate reader (Bio-Rad, Vienna, Austria) [14].

Data processing

The data form the microwell colorimetric assay was processed using OriginPro 7.0 (OriginLab, Northampton, USA). The apparent dissociation constant (K_d^app) was calculated by fitting a sigmoid binding curve and determined as the concentration achieved half-maximal A450 [16]. Because the binding reactions were washed, the K_d^app represents non-equilibrium binding constants. Binding saturation of the DNA ligands could only be achieved with the P10 oligomer (Sangon) due to non-specific background signals when using GST-Zfp191BD at concentrations above 300 nM. The P value was obtained by comparing the plot of P3, P10, M2G, M3T, M4A or M5, respectively, to the plot of M1A as a reference. Sigmoidal kinetics can be described by the Hill equation. Thus, the Hill coefficient was calculated by fitting a Hill curve to the data until the Chi-sqr was no longer reduced.

Atomic absorption spectrometry

A 1.5-mg GST-Zfp191BD in 150 ml of 50 mM Tris-HCl (pH 8.0) containing 10 mM glutathione and 50% glycerol was digested using mixed acid (HNO3:HClO4=4:1) at approximately 200 �C. It was subsequently dissolved in 25 ml 1% HCl and then the Zn2+ ion concentration was accessed with flame atomic absorption spectroscopy on a Varian AA240FS spectrometer (Varian, Palo Alto, USA) (wavelength 213.9 nm for Zn).

Results

Expression and purification of the GST-Zfp191BD fusion proteins

GST-Zfp191BD was expressed in its recombinant forms in E. coli BL21(DE3), with an IPTG induction at 30 �C. The entire expression and purification process was monitored by SDS-PAGE (Fig. 2). Comparison of lane 2 [lysate of BL21-pX-C-Zfp191BD (non-induced by IPTG)] with lane 3 [lysate of BL21-pX-C-Zfp191BD (induced for 4 h by IPTG)] indicates fusion protein GST-Zfp191BD (approximately 42.7 kDa) was expressed successfully. The fusion protein was both in the precipitate and supernatant of bacteria lysate (lane 4 and lane 5) and only the one in the supernatant was used for further purification. The fusion protein bound to the glutathione-affinity resin column successfully (comparing lane 5 and lane 6), and was easily obtained by eluting the column using 10 mM glutathione, which showed the only one visual band in the correct position in SDS-PAGE (lane 8).

Sequence dependency for binding GST-Zfp191BD to DNA

ZNF24, the transcription factor identified by the yeast one-hybrid-system from a human brain cDNA library, can interact with the intronic polymorphic TCAT repeat in the TH gene [12]. We presumed that the binding domain of Zfp191 has the similar properties to its homolog ZNF24, so the eP3[(TCAT)3] was chosen as the positive probe for EMSA (Table 1). The data suggested that only the positive protein GST-Zfp191BD and the positive probe eP3 could specifically form the complex in EMSA, while the others could not (Fig. 3).

In order to know more about the specificity of the binding reaction, we analyzed GST-Zfp191's binding to different mutations of TCAT motif by microwell colorimetric assay. The positive controls P3 and P10, which are the variants of repeat sequence in HUMTH01, as well as the mutates including four transversional mutants M1A, M2G, M3T, M4A and the recommended negative control M5 were detected by microwell colorimetric assay using PBS buffer as the reference [12,17]. As shown in Fig. 4, there was no significant difference in the binding abilities of M1A, M2G and PBS; M3T, M4A and M5 showed significant binding ability to GST-Zfp191BD (P<0.05), especially M4A (P<0.01). The weak binding observed with the mutated probes M1A, M2G, and M3T suggests that positions 1-3 are critical and that Zfp191 may interact with a 3-bp binding site. The fourth nucleotides in the tetranucleotide core sequence were more flexible than others.

To further investigate the binding properties of GST-Zfp191BD, we also detected the dynamic parameters of the binding reaction. The combination curve was analyzed (Fig. 5), and the apparent dissociation constant () and the Hill coefficient were calculated to evaluate the binding affinity and cooperation (Table 2). The� values indicated that P10, P3 and M4A provide the best binding sequences for GST-Zfp191BD. It was also observed that the Hill coefficient for GST-Zfp191BD binding to P10 is greater than 1 (nH=1.42740.1865), to P3 is less than 1 (nH=0.48650.1088), and to M4A is not significantly different from 1 (nH=0.72390.3185).

As there are four zinc finger motifs in the GST-Zfp191BD based on the Conserved Domain Database (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi?seqinput=NP_067534.2), we expected to verify the effect of zinc ion upon the binding property through experimentation. By microwell colorimetric assay, we were able to detect whether GST-Zfp191BD could binding to P10 or P3 in the binding buffer with 50 mM Zn2+, without Zn2+, or without Zn2+ but with 10 mM EDTA (Fig. 6). Our data showed that the GST-Zfp191BD binding to P3 and P10 is significantly stronger than its binding to M5 with 50 mM Zn2+ or without Zn2+ binding buffer. However, when 10 mM EDTA was added to the binding buffer, the GST-Zfp191BD binding to P3 and P10 was as weak as its binding to M5. So the binding of GST-Zfp191BD to its target DNA is ion-dependent.

Discussion

The data from the Reviewed Computational Analysis (http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=Retrieve&dopt=full_report&list_uids=59057&log$=databasead&dbfrom=protein) and gene knockout experiments indicated that Zfp191 is an important transcriptional factor in C2H2 zinc-finger protein family [7]. Identifying its target DNA sequences is a key step to discover its downstream genes. The fusion protein GST-Zfp191BD was designed and purified from E. coli to investigate the DNA-binding properties of the conserved DNA-binding domain at Zfp191's-terminal. The GST domain in the N-terminal region not only facilitates the purification of the fusion protein, but also acts as a reporter tag for functional detection. Both qualitative and quantitative data of DNA-protein interactions were obtained from EMSA and microwell colorimetric assays. GST-Zfp191BD is an artificial protein in which the SCAN domain of Zfp191 has been replaced by the GST domain, so our data only show the properties of GST-Zfp191BD, but not native Zfp191. GST-Zfp191BD has similar properties to ZNF24 in qualitative analysis in that it can specifically interact with the TCAT repeat motif in the TH gene. The different binding affinity between GST-Zfp191BD and the modified TCAT target DNA sequences shows the specificity of the interaction. The results indicated that the position 1-3 in the TCAT motif are important to the binding and cannot be substituted, while the fourth is more tolerant.

According to the Conserved Domain Database, Zfp191 has four consecutive C2H2 zinc-finger conserved sequences (residues 251-273, 279-301, 307-329, and 335-357), indicating that there are potential zinc finger domains in the protein. Based on evidence from Inferred from Electronic Annotation (http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=Retrieve&dopt=full_report&list_uids=59057&log$=databasead&dbfrom=protein), Zfp191 was inferred to have the function of DNA binding, and we gave the solid evidence from experiment. The necessity of Zn2+ was observed in the microwell colorimetric assay, and we assumed that GST-Zfp191BD could not bind to P10 or P3 without Zn2+ in the binding buffer (Fig. 6). To explain this result we quantified the concentration of zinc ion by atomic absorption spectrometry. The data showed that the purified GST-Zfp191BD sample (containing 5.0 mg/ml GST-Zfp191BD) contained 28.6 ppm Zn2+, and the GST-Zfp191BD/Zn2+ molar ratio was 1:3.76, which is close to the theoretical value 1:4. Therefore, we concluded that Cys2His2 tetrahedral complexes assisted by Zn2+ which stabilize the supersecondary structure of zinc fingers were already formed during the expression in E. coli.

It is notable that the specific GST-Zfp191BD binding sequence (TCAT)n is the microsatellite in the first intron of the TH gene. Due to its polymorphism in different individuals, microsatellites have been regarded as markers related to mental disorders and some diseases, such as schizophrenia and pancreaticobiliary malignancies [18-20]. In recent years, microsatellite has been reported to regulate a gene promoter through direct interaction with a transcription factor. For example, ZNF24 can specifically silence the TH gene by binding to the TCAT repeat motif in HUMTH01 [12,13], and p53 can activate the PIG3 gene by binding the pentanucleotide (TGYCC)n in its promoter [21]. The specificity of GST-Zfp191BD binding to the microsatellite in the first intron of the TH gene implies that Zfp191 might regulate the downstream gene by the novel way of directly binding to the microsatellite near or in the genes. The quantitative difference in affinity between GST-Zfp191BD and its various target DNA sites also gives some hints as to the genetic traits associated with the polymorphism of microsatellites.

Acknowledgements

We would like to thank Ms. Jiajuan Shen (School of Life Science and Technology, Tongji University, Shanghai, China) for her help in ordering reagents and experiment materials.

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