Tohoku

J. Exp.

Med., 1992, 168, 175-182

Complex

Regulation

of a Tumor

Marker

Expression. Enhancer

and

Silencer

of the GST-P

Gene

MASAMIMURAMATSU,MITCHELLB. DICCIANNI, SHIGERU MORIMURA,TOSHIYASUZUKIand MASAYOSHI IMAGAWA Department of Biochemistry, Saitama Saitama 350-04

Medical

School,

MURAMATSU, M., DICCIANNI,MB., MORIMURA, S., SUZUKI,T. and IMAGAWA, M. Complex Regulation of a Tumor Marker Expression. Enhancer and Silencer of the GST-P Gene. Tohoku J. Exp. Med., 1992, 168 (2), 175-182 Glutathione transferase P (GST-P) is expressed at high levels in precancerous lesions and hepatocellular carcinomas from a very early stage of chemically-induced hepatocarcinogenesis in the rat. To explore the molecular mechanisms of its specific activation, we are investigating the regulation mechanisms of the GST-P gene expression. By using gene technology, we have identified a strong enhancer, GPEI, at 2.5 Kb and a silencer region at about 400 by upstream from the transcription start site. GPEI has a palindromic structure composed of two TPAresponsive element (TRE)-like sequences and binds at least three proteins including AP-1 (c-jun/c-fos). The silencer is composed of several sequences resembling each other and binds at least three proteins including SF-B/LAP/LIP. To determine whether the GST-P gene is activated together with a putative hepatooncogene because they are located close to each other (cis-mechanism), or because they share a trans-acting factor that can activate both genes simultaneously (trans-mechanism), transgenic rats were produced with GST-P control region connected to the CAT reporter. The results unequivocally demonstrate that GST-P gene is activated position-independently by a traps-mechanism. AP-1; enhancer ; hepatocarcinogenesis ; glutathione transferase P gene ; silencer

Some antigens or enzymes are specifically produced in certain tumor cells and frequently useful for diagnosis. Therefore, they are sometimes called "tumor markers". Whereas a number of tumor markers are reported, the molecualr mechanisms of their specific expression are yet to be clarified. Glutathione transferase P (GST-P) is highly and specifically expressed during hepatocarcinogenesis of the rat caused by hepatocarcinogens such as diethylnitrosoamine (DEN), acetylaminofluorene (AAF) and 3'-methyl-4-dimethylaminoazobenzene (3'MeDAB). The almost 100% occurrence of this phenotype in the precancerous lesion and early hepatocellular carcinoma suggest the presence of a mechanism by which GST-P gene is activated together with a crucial transforming gene of liver cells. To address this question, we first cloned the GST-P gene from the rat Addressfor reprints : 38 Morohongo,Moroyama,Iruma-gun,Saitama 350-04,Japan. 175

M. Muramatsu

176

genome and characterized the Then, we produced transgenic hooked

to the

carried

out on these

control elements present in the upstream region. rats with the control region of the GST-P gene

E. coli CAT reporter. rats

et al.

The results

of the carcinogenic

experiments

are described.

MATERIALS AND METHODS The cloning of the GST-P gene has been described (Sugioka et al. 1985b ; Okuda et al. 1987). Cell culture and transfection procedures were also described (Sakai et al. 1988; Okuda et al. 1990). Gene constructs for transfection experiments and the methods for constructing mutants were also described (Okuda et al. 1987, 1990; Imagawa et al. 1991a, b). Chemical carcinogenesis has been performed by the Solt-Farber procedure as described previously (Solt and Farber 1976; Sugioka et al. 1985a). Other procedures are described in the literature cited at each section.

RESULTSAND DISCUSSION To determine the control region of the GST-P gene, we examined various upstream fragments is terms of the enhancing activity to the minimal promoter (Sakai et al. 1988). Fig. 1 summarizes the results. Experiment 1 shows that the SA fragment located at about 2.7 Kb upstream has essentially all the enhancing activity, whereas experiment 2 indicates the presence of a silencer activity, in BP fragment located at about 400 by upstream. The SA fragment contained an enchancer, designated GPEI, composed of a palindrome of two TREs (Okuda et al. 1989). CAT assays in Fig. 2 show that the GPEI can act as an enhancer even

Fig .1.

Characterization

of enhancer

GST-P

gene.

A. Each

activity GST-P

was gene.

determined. Positions

construct

and

silencer

was introduced

B. Restriction of the fragments

elements into

in the

dRLh84

upstream

of

cells and CAT

map of the 5' flanking used are shown by bold

region bars.

of

Complex

Regulation

of a Tumor

Fig. 2. Activity of GPEI and collagenase and c-fox co-expression.

Marker,

GST-P,

enhancers

Gene

in F9 cells.

Exp

ression

177

Effects of c-jun

in F9 embryonal carcinoma cells which lack AP1 (c-jun/c-fos heterodimer) and cannot support the collagenase enhancer having an authentic THE (Okuda et al. 1990). Fig. 3 shows the results of mutational analysis which indicate that the downstream THE-Like sequence is the dominant component in GPEI, but the upstream THE-like sequence is also required for the enhancer activity in F9 cells. The requirement of the divergent arrangement of the THE-like sequence is shown in Fig. 4 (Okuda et al. 1990). This holds true in HeLa cells as well as in F9 cells. We also confirmed that the distance between the two THE-like sequence was also important and the 3 by was the optimal spacing (data not shown). Recently, we detected by gel-shift assay at least two proteins that bound to GPEI in F9 cell extract (Diccianni et al. 1992). They are apparently different from AP1 that could be detected in HeLa cells, but their nature is now under study. The silencer region detected in Fig. 1 was further studies by directly connecting to different promoters. As shown in Table 1, the region had a silencing effect independently of the orientation and location to the three different promoters (Imagawa et al. 1991a). Rat liver nuclear extract revealed clear footprints on this region (Fig. 5) and the deletion of each segment showed a significant release from the silencing effect (Fig. 6). At least three proteins appear to bind to this regions. The protein that binds to several sequences resembling each other was designated SF-A and purified to near homogeneity. This purified protein showed almost identical footprints on the silencer region. Another protein designated SF-B that binds to only one element of the silencer region has been cloned by a Southwestern protocol using the polymerized binding element as a probe. Surprisingly, the cDNA was found to have a sequence identical with the previously identified LAP/ NF-1L6 which acts as a liver specific enhancer (Descombes et al. 1990; Imagawa et al. 1991b). The binding of a recombinant SF-B to the silencer element was

M. Muramatsu

178

et al.

Fig . 3.

Two THE-like sequences mediate the activity of GPEI in F9 cells. A. Structure of GPEI mutants used. B. Examination of the downstream TRElike sequence by single point mutants. C. Examination of the upstream THE-like sequence by triple-point mutants. From Okuda (1990) by permission of Oxford University Press.

competed by the NF-1L6 binding sequence of C reactive protein (CRP) at the same efficiency as itself confirming their identity (Fig. 7). These results indicate that SF-B/LAP/NF-1L6 can act as either positively or negatively depending upon the context of regulation, the precise mechanism of which remains to be determined. Apart from the interacting factors, the crucial point of tumor marker expression remains to be answered ; i.e. why and how is the GST-P gene activated together with the putative transforming gene of the liver. To investigate this aspect of the problem, we made a reporter gene in which the bacterial chloramphenicol acetyltransferase (CAT) structural gene was ligated to the 2.9 Kb fragment containing the upstream region of the GST-P gene. It was microinjected to the fertilized eggs and several lines of transgenic rats were produced. Then

Corn plex

Fig. 4. of

Regulation

of a Tumor

Marker,

GST-P,

Gene Ex pression

A certain palindromic orientation is required for the proper the two TREs. Synthetic DNAs shown are connected

thymidine

TABLE 1.

kinase

promoter

and CAT

activity

assayed

cooperativity to the HSV

in respective

Silencing effectsof negative fragment in GST-P activities of heterologous promoters and enhancer hepatoma and hepatoma cells

179

cells.

gene on in non-

180

M. Muramatsu

et al.

Fig. 5. DNase I footprints of rat liver nuclear extract on promoter and silencer of GST-P gene. A. Footprints on coding and non-coding strands. B. Binding sites of nuclear factors in promoter region. Arrow and arrowhead indicate the Pst I site (3' end of BP fragment) and cap site (+ 1), respectively.

Complex

Regulation

Fig. 6.

Fig.

of a Tumor

Internal

deletion

Marker,

analysis

GST-P,

Gene

Ex pression

181

of th e silencer.

7. Competition analysis of SF-B using GPS1 oligonucleotide as a probe. Left panel shows a gel mobility shift assay using f3-galactosidase/SF-B fusion protein. As competitors, 10, 50 and 250 fold molar excess of GPSI and CRPa oligonucleotides were used. The dried gel was cut and counted by liquid scintillation counter, and competition ratio (% of control) is shown in right panel.

these rats were subjected to the Solt-Farber

(1976) procedure of chemical he-

patocarcinogenesis, all the liver tumors tested from three independent lines of transgenic animals expressed CAT activity at high levels, whereas no cat activity was detected in the control liver of these rats (data not shown). Since the probability of all three transgenes being integrated close to the putative hepatooncogene is virtually zero, these data unequivocally establishes that the GSTPgene is activated by a trans-mechanism. If the putative hepato-oncogene shares a trans-acting factor with GST-P gene, identification of this factor via GST-P regulatory elements (possibly GPEI) may become a promising tool for the cloning of the relevant hepato-oncogene.

182

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et al.

References 1) Descombes, P., Chojkier, M., Lichtsteiner, S., Folvey, E. & Schibler, U. (1990) LAP, a novel member of the C/EBP gene family, encodes a liver-enriched transcriptional activator protein. Genes Dev., 4, 1541-1551. 2) Diccianni, M.B., Imagawa, M. & Muramatsu, M. (1992) The dyad palindromic glutathione transferase P enhancer binds multiple factors including AP-1. Nucleic Acids Res., 20, 5153-5158. 3) Imagawa, M., Osada, S., Okuda, A. & Muramatsu, M. (1991a) Silencer binding proteins function of multiple cis-elements in the glutathione transferase P gene. Nucleic Acids Res., 19, 5-10. 4) Imagawa, M., Osada, S., Koyama, Y., Suzuki, S., Hirom, P.C., Diccianni, M.B., Morimura, S. & Muramatsu, M. (1991b) SF-B that binds to a negative element in glutathione transferase P gene is similar or identical to trans-activator LAP/IL6-DBP. Biochem. Biophys. Res. Commun., 179, 293-300. 5) Okuda, A., Sakai, M. & Muramatsu, M. (1987) The structure of the rat glutathione S-transferase P gene and related pseudogenes. J Biol. Chem., 262, 3858-3863. 6) Okuda, A., Imagawa, M., Maeda, Y., Sakai, M. & Muramatsu, M. (1989) Structure and functional analysis of an enhancer GPEI having a phorbol 12-0-tetradecanoate 13-acetate responsive element-like sequence found in the rat glutathione transferase P gene. J. Biol. Chem., 264, 16919-16926. 7) Okuda, A., Imagawa, M., Sakai, M. & Muramatsu, M. (1990) Functional cooperativity between two TPA responsive elements in undifferentiated F9 embryonic stem cells. EMBO J., 9, 1131-1135. 8) Sakai, M., Okuda, A. & Muramatsu, M. (1988) Multiple regulatory elements and phorbol 12-0-tetradecanoate 13-acetate responsiveness of the rat placental glutathione transferase gene. Proc. Natl. Acad. Sci. USA, 85, 9456-9460. 9) Solt, D. & Farber, E. (1976) A new principle for the analysis of chemical carcinogenesis. Nature, 263, 701-703. 10) Sugioka, Y., Fujii-Kuriyama, Y., Kitagawa, T. & Muramatsu, M. (1985a) Changes in polypeptide pattern of rat liver cells during chemical hepatocarcinogenesis. Cancer Res., 45, 365-378. 11) Sugioka, Y., Kano, T., Okuda, A., Sakai, M., Kitagawa, T. & Muramatsu, M. (1985b) Cloning and the nucleotide of sequence of rat glutathione S-transferase P cDNA. Nucleic Acids Res., 13, 6049-6057.

Complex regulation of a tumor marker expression. Enhancer and silencer of the GST-P gene.

Glutathione transferase P (GST-P) is expressed at high levels in precancerous lesions and hepatocellular carcinomas from a very early stage of chemica...
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