α-Fetoprotein gene DNA-binding proteins

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 243, No. 1, November 15, pp. 320-324,1985

COMMUNICATION a-Fetoprotein

Gene DNA-Binding

GILBERT J. COTE, ZHI WANG, Lkpartnznt of Bio&emist?y, University

AND

Proteins

JEN-FU CHIU’

of Vermon t CoUege of Medicine,

Burlington,

Vermm t 05405

Received June 13, 1985, and in revised form July 30, 1985

The technique of protein blotting was used to study nuclear protein interaction with the cY-fetoprotein (AFP) gene. The DNA-binding specificity was optimized by varying the amount of competitor DNA and the ionic strength. The specific binding of AFP gene DNA was observed for a set of Morris hepatoma 7777 nuclear proteins. Similar specificity was not seen for these same proteins in liver. In normal rat livers, however, two unique proteins were observed which displayed specific binding. The speculated involvement of these proteins in AFP gene regulation is discussed. o 1% Academic PM, I~~.

A mechanism of gene regulation in procaryotes is the sequence-specific binding of proteins to DNA regulatory regions. Evidence is accumulating that sequence-specific DNA-binding proteins may play an important role in eucaryotic gene regulation as well. Proteins such as the Xenopus 5 S RNA transcription factor (l), the papova virus T-antigens (2), and the glucocorticoid receptor (3) are examples of proteins whose binding to specific DNA sequences is required for gene regulation. Our laboratory is interested in determining the existence of specific protein factors which may be involved in the regulation of the a-fetoprotein (AFP)2 gene. During embryonic development AFP is synthesized by the fetal liver and yolk sac (4, 5). Although AFP is a prominent protein in fetal blood its level decreases rapidly after birth (5). The decrease in AFP synthesis is the result of direct transcriptional regulation (6). Transcriptional activity can reappear during liver regeneration (7) or in certain hepatocellular carcinomas (8). In this study we employ protein blotting as a technique for the identification of DNA-binding proteins. This technique has been successfully used in the identification of sequence-specific binding proteins of a Drosophilia heat shock gene (9). The use of protein blotting allows the direct comparison of fractionated

proteins from different sources. The present study uses total nuclear proteins isolated from the AFP-producing Morris hepatoma 7777, as well as from normal adult rat liver. The conditions required for the binding of these proteins to DNA encoding the 5’ region of the AFP gene and the specificity of this binding were studied for these proteins. MATERIALS

1 To whom correspondence should be addressed. ’ Abbreviations used: AFP, a-fetoprotein; SDS, sodium dodecyl sulfate. 0003-9861/85 $3.00 Copyright All rights

8 1995 by Academic Press. Inc. of reproduction in any form reserved.

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METHODS

Animals and tumors. The rapidly growing, AFPproducing Morris hepatoma 7777 was maintained as a transplantable tumor in the hind legs of Buffalo rats. Normal liver tissue was obtained from Buffalo rats. Preparation of nuclear extra,& Nuclei were prepared from liver or tumor tissue by homogenization of freshly obtained tissue in 10 vol of 10 mM Tris-HCl, pH 7.6,0.25 M sucrose, 25 mM NaCI, 5 mM MgClz, 0.1% Triton X-100, and 0.5 mM phenylmethylsulfonyl fluoride. Crude nuclei were pelleted by centrifugation at 8OOg for 10 min at 4°C. Nuclear pellets were resuspended in homogenization buffer containing 2.2 M sucrose, and purified nuclei were isolated by centrifugation at 45,000g for 1 h at 4°C. This nuclear pellet was washed once to remove the heavy sucrose and was resuspended at 100 A&ml in DNase 1 digestion buffer (10 mM Tris-HCI, pH 7.4, 10 mM NaCI, 3 mM MgCl, 10% glycerol). DNA was digested by the addition of 50 pg/ml DNase 1 and incubation for 1 to 2 h on ice. Following this incubation an equal volume of 0.125 M Tris-HCl, pH 6.8,4% sodium dodecyl sulfate

a-FETOPROTEIN

GENE

(SDS), 10% glycerol, and 10% @-mercaptoethanol was added to prepare samples for electrophoresis. Samples prepared in this way can be stored at -70°C for extended periods until use. Gel electrophoresti and transfer. SDS-polyacrylamide gel electrophoresis was performed as previously described (lo), with minor modifications. The stacking and separating gels contained 0.1% SDS and 1% glycerol. The upper electrode buffer was 0.1 M Tris-HCl, 0.77 M glyeine, and 1% SDS, 2X the Laemmli concentration. Samples were applied to a 3% polyacrylamide stacking gel and separated on a 7.5% running gel. Electrophoresis was performed at a constant current of 10 mA per 30-ml gel. Pyronin Y was used as a tracking dye. Following electrophoresis, proteins were transferred to nitrocellulose paper as previously described (11). Transfer of proteins from untreated polyacrylamide gels was performed in 24 mM Tris, pH 8.3,192 mM glycine, 20% methanol, for 3-5 h at 60 V. DNA binding and detection Following electrophoretie transfer, the nitrocellulose filters were preincubated in 10 mM Tris-HCl, pH 7.4, 50 mM NaCI, 1 mM EDTA and 5X Denhardt’s solution (0.1% Ficoll, 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone) (binding buffer), at room temperature with gentle agitation or stored at 4°C until use. After 1 h, the preincubation buffer was removed and replaced with fresh binding buffer containing sheared Escherichia coli DNA and [*rP]end-labeled pXRAF 6/R6.2. The plasmid pXRAF 6/R6.2 was end-labeled as previously described by the addition of [82PjdATP to restricted DNA (12). Labeled DNA was allowed to bind for 1 h at room temperature at a concentration of 0.1 pg/ml binding buffer. Following binding, the filters were washed 3X in 10 mM Tris-HCI, pH 7.4, 1 rnrd EDTA, 0.2 M NaCl, and 1X Denhardt’s at room temperature. DNA binding was determined by autoradiography using Kodak XRP X-ray film. Elutim of DNA from dried&filters. Following autoradiography the radiolabeled bands were localized to their corresponding positions on the nitrocellulose filters. The bands were cut from the filter and the strip was shaken in 0.75 ml of 0.1% SDS, 1 mM EDTA for 1 h. The filter was removed and the DNA was concentrated by lyophilization. Lyophilized DNA was dissolved in 3% Ficoll containing bromphenol blue as a tracking dye, and was fractionated on 1% agarose gels. Agarose gels were dried and autoradiographed to detect the presence of DNA. RESULTS

AND

DISCUSSION

Several studies suggest that the regulation of class II genes involves sequences 5’ to the mRNA coding region. In this study we employ rat genomic DNA containing 5’ region of the AFP gene. The DNA is a 6.2-kb EcoRl fragment originating from the bacte-

DNA-BINDING

PROTEINS

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riophage XRAF 6 and has been subcloned into the EcoRl site of the vector pUC8 (Fig. 1). This subclone was designated pXRAF 61R6.2. In the experiments presented here the plasmid pXRAF 6/R6.2 was restricted with EcoRl to create insert fragments of rat genomic and vector DNA that exist in equimolar amounts. Fractionated proteins are then exposed to a mixture of labeled DNAs, and the binding specificity of individual proteins determined by the DNA fragment retained by that protein. Initial experiments were performed to optimize the detection of specific binding. Nuclear proteins were obtained from two sources, normal rat liver in which the AFP gene is transcriptionally inactive, and the Morris hepatoma 7777 in which the AFP gene is active. These proteins were fractionated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose paper as shown in Fig. 2A. The binding of radiolabeled DNA was carried out in the presence of unlabeled nonspecific E. ooli DNA which served as a competitor. The use of competitor DNA reduces the amount of probe bound nonspecifically and prevents the possibility of DNA probe exhaustion prior to specific binding. Figure 2A shows the results of varying the amount of competitor DNA on the DNA-binding pattern of the fractionated proteins. When radiolabeled DNA is incubated in the presence of low amounts of competitor DNA the binding pattern reveals many more proteins than are seen at higher DNA competitor concentrations. A point of saturation appears when little change in binding is seen upon addition of greater amounts of competitor (data not shown). To determine the specificity of this binding, DNA fragments were eluted from several hands and electrophoresed on agarose gels to determine the molecular weights of the bound DNAs (Fig. 2B). The histone proteins by definition bind DNA nonspecifically. Electrophoresis of the DNA eluted from histone pro-

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FIG. 2. The effect of competitor DNA on specific AFP gene DNA binding. (A) Amido black-stained nitrocellulose of transferred hepatoma and normal liver proteins, and autoradiographs of identical transfers probed in the presence of varying amounts of E. coli DNA. Filters were washed in the presence of 0.2 M NaCl. (B) Autoradiograph of agarose gel containing DNA eluted from proteins in (A). Numbers correspond to bands in (A). LH, Liver histone; HH, hepatoma histone.

teins (LH and HH) reveals two DNA bands of molecular weights 6.2 kb (AFP gene insert) and 2.7 kb (vector DNA). The autoradiographic intensity is equal for the two bands, indicating that histones do not show preferential or specific binding. The nonhistone proteins vary in their ability to bind DNA. At low concentrations of competitor DNA several proteins show the ability to bind radiolabeled DNA. Figure 2B reveals that this binding is largely nonspecific with regard to the AFP gene insert. As the concentration of competitor DNA is increased, the detection of proteins which bind the 6.2-kb fragment specifically becomes evident. Two nonhistone (73 and 62 kDa) proteins isolated from normal liver show specific binding to AFP gene DNA (Bands 34 and 35). Proteins of comparable molecular weight and DNA-binding ability do not exist in the hepatoma sample. However, nonhistone proteins of molecular weights 83K, 98K, and 160K show specific binding in hepatoma (bands 41,42, and 43) and no preferential binding in normal liver samples of similar molecular weights. In Figure 2, bands 34,35,41,42, and 43 show pref-

erential binding of the 6.2-kb AFP gene insert. In these bands, however, the relationship to vector DNA binding is not apparent. To determine the degree of binding specificity, these bands were subjected to a longer autoradiographic exposure and band intensity quantitated by densitometric scanning (Shimadzu, Model CS930). As shown in Fig. 3, the binding of the 2.7-kb vector DNA fragment is apparent upon the longer exposure time. Densitometric scanning reveals the intensity of binding to AFP gene to be three to six times that of vector DNA. Band 42, which shows binding to vector DNA (Figure 2B), binds the 6.2-kb band with twice the intensity of the 2.7-kb band (data not shown). Ionic strength is one of several factors which affects protein-DNA interaction and chromatin structure. For this reason protein-DNA complex stability and specificity were measured as a function of the salt concentration in the wash buffer (Fig. 4). The use of low-ionic-strength wash buffer (0.05 M NaCl) promotes the retention of DNA by a majority of nuclear proteins (Fig. 4). This interaction, however, is largely nonspe-

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FIG. 3. Assessment of DNA-binding specificity. to a longer autoradiographic exposure. The ratio kb band was then quantitated by densitometric the ratio (% ) of the individual peak area to total cific in nature, as seen in Fig. 4B. The elution of DNA from these proteins reveals the presence of both DNA fragments bound to the majority of proteins tested. An increase in the ionic strength of the washing buffer results in a decrease in overall binding, as DNA is

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FIG. 4. The effect of ionic strength on DNA binding and specificity. (A) Autoradiographs of filters washed in the presence of 0.05,O.lO and 0.20 M NaCl following DNA binding in the presence of 1500-fold E. wli DNA. (B) Autoradiograph of DNA eluted from corresponding bands in (A).

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The DNA agarose gel from Fig. 2B was subjected of the intensity of the 6.2-kb DNA band to the 2.‘7scanning (Shimadzu, CS-930). Numbers represent peak area. removed from proteins of lower binding affinity. The concentrations of 0.1 and 0.2 M NaCl show a concurrent increase in binding specificity with the loss of loosely bound DNA. This suggests then that the AFP genespecific binding is associated with a protein of higher binding affinity. This however may be an over generalization. Although we found no AFP sequencespecific binding proteins with low DNA-binding affinity, our survey was not extensive, so the possibility of proteins being masked by ones of similar molecular weight exists. In this study, we separated chromosomal proteins with one-dimensional gel electrophoresis. It is possible that each protein band represents a mixture of proteins and only one or some of which are DNA-binding proteins. In this study we have compared DNA-binding proteins isolated from actively and nonactively AFPproducing cells. In the Morris hepatoma 7777, three proteins (160, 98, and 83 kDa) were identified which bind AFP gene DNA preferentially. Proteins of similar molecular weight were identified in normal liver but did not show the same preferential binding seen in tumor. Two AFP gene-binding proteins (73 and 62 kDa) were found specific to normal rat liver, which is inactive in AFP synthesis. The functions of these AFP gene-binding proteins are not known. However, the possibility of participation in the control of AFP gene expression is speculated. Since the DNA-binding assay is carried out on proteins which have been denatured by SDS-polyacrylamide gel electrophoresis, some proteins may have lost their DNA-sequence recognition domains due to becoming irreversibly denatured. Future studies will be directed toward the purification and characterization of these proteins to determine their relationship to AFP gene expression. ACKNOWLEDGMENTS This investigation was supported by USPHS Grant CA 25098 awarded by NCI. G. J. Cote is a trainee of

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COTE,

WANG,

Cancer Biology Training Grant T32-09286. The hacteriophage ARAFG was a generous gift of Dr. Thomas Sargent at NIH.

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