Structure of the promoter for the rat Fas antigen gene

Biochimica et Biophysica Acta 1352 Ž1997. 238–242

Short sequence-paper

Structure of the promoter for the rat Fas antigen gene Kotohiko Kimura ) , Kimie Asami, Mikio Yamamoto Department of Biochemistry, National Defense Medical College 3-2, Namiki, Tokorozawa, Saitama 359, Japan Received 3 July 1996; accepted 1 April 1997

Abstract The Fas antigen is a receptor protein transducing cell death signals. Binding of Fas ligands to Fas antigens provokes apoptosis in target cells. Here we report the structure of the promoter of the gene coding for rat Fas antigens. The major transcription start site, identified by the S1 nuclease protection assay, was situated 188 nucleotides upstream of the translational initiation site. The promoter activity was located in the region at the nucleotide position from y142 to y24. In this region we identified a consecutive sequence of NF-k B and NF-IL6 consensus sequences, spanning from y142 to y122. q 1997 Elsevier Science B.V. Keywords: Fas antigen; Promoter; NF-IL6; NF-k B

Fas antigen ŽAg. is a transmembrane receptor protein for an apoptosis-mediating signal molecule termed Fas-ligand. We have previously described the cloning of a cDNA coding for rat Fas antigen ŽrFas Ag. gene w1x. The human and mouse homologues have been reported earlier w2,3x. The expression of Fas Ag was demonstrated to be induced by interferon g and CD40, which indicates that there are some mechanism to control the expression of Fas Ag w1,2x. The promoter analysis is an important first step to elucidate the mechanism of gene expression. Although the sequence of the promoter region of human Fas Ag gene was already reported independently by three laboratories w4–6x, the promoter analysis of murine Fas Ag gene was not yet performed. In this

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Corresponding author. Present address: Kimura Auxological Institute 3-3-63 Chuo, Sayama, Saitama 350-13, Japan. Fax: q81 429 599964.

study we sequenced the promoter region of rFas Ag and examined the promoter activity of the region. A genomic DNA library constructed with the EMBL3 vector and DNA from rat liver cells was purchased from Clonetec Co. Ltd. About one million clones were screened by plaque hybridization using a cDNA fragment of rFas Ag as a probe. By analyzing two different clones isolated by the method, we obtained three EcoRIrBamHI-restricted fragments of the length of 1.8 kb, 1.3 kb, and 0.6 kb. The fragment of 1.3 kb was demonstrated to include 5X terminal region of rFas Ag mRNA. Two other fragments were verified to be located as shown in Fig. 1. By sequencing these fragments, we determined the sequence of 5X untranslated region spanning for 1.5 kb Ž Fig. 2. . The locations of the primers used in the study below are depicted in Fig. 2. Since both human and rat promoters of Fas Ag are lacking distinct sequences of TATA, GC, or CCAAT boxes, Fas Ag gene is supposed to be among housekeeping genes ŽFig. 2, w3,4x..

0167-4781r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 7 8 1 Ž 9 7 . 0 0 0 6 0 - 2

K. Kimura et al.r Biochimica et Biophysica Acta 1352 (1997) 238–242

Fig. 1. The schematic depiction of the structure of promoter region of rat Fas antigen gene. The letters above the bar indicate the restriction sites as follows; B: BamHI; E: EcoRI; Bgl; BglII; P: Pst I. The shaded area indicates the first exon. The 5X terminal of the first exon was determined based upon the result of the S 1 nuclease protection assay shown in Fig. 3.

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The 5X terminal of rFas Ag mRNA was determined by the S 1 nuclease protection assay using polyŽA.q RNA extracted from the liver of rats injected with cycloheximide, based on the method of Sambrook et al. w7x. Cycloheximide was used because it enhanced Fas Ag mRNA expression about 100-fold w8x. Specifically, the PCR product generated by using total liver RNA and a primer pair C and F was cloned as a target sequence into a MV1184 vector. A singlestranded DNA carrying the target sequence was iso-

Fig. 2. The sequence of the promoter region of rat Fas antigen gene. Arrows designated by the letters ‘A’ through ‘F’ indicate the primers used for either constructing Cat vectors or producing a probe for the S1 nuclease protection assay. The nucleotide residues designated by bold underlines correspond to either transcription factor consensus sequences or restriction sites. An underlined ‘ATG’ is a translational start site. A major transcription start site determined by the S1 nuclease protection assay shown in Fig. 3 is indicated by an asterisk. The nucleotide residues corresponding to the first exon are written in uppercase letters.

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lated and used as a template to synthesize the radiolabeled DNA probe by a Klenow fragment and the primer F ŽFig. 2.. The synthesized DNA was digested with EcoRI enzymes that cleaved at the distal end of the target sequence. The obtained DNA fragment was constituted from a radiolabeled probe and an unlabeled complementary sequence. The radiolabeled probe was separated by a polyacrylamide gel electrophoresis. Thirty m g of polyŽ A.q RNA was hybridized with the radiolabeled probe, and the unhybridized probe was digested with an S 1 nuclease ŽTakara Shuzo, Co. Ltd., Kyoto.. Digested samples were electrophoresed on an 8% urea polyacrylamide gel and analyzed by using a bioimaging analyzer, BAS-2000 ŽFuji Photos Co. Ltd., Tokyo.. Although only a faint band could be detected because of the extremely small content of Fas Ag mRNAs, this band was obtained repeatedly by independent experiments ŽFig. 3, an arrowhead.. There were some minor bands shorter in length by one to three nucleotide residues than the major band designated by an arrowhead. These minor bands were probably produced as a result of digestion of terminal nucleotide residues of the DNA fragment corresponding to the major band, considering that these minor bands increased their thickness along with the increase in S 1 nuclease activity in the assay. The major mRNA starting point thus determined is depicted as an asterisk in Fig. 2. The promoter analysis revealed that there was a consecutive sequence of NF-k B and IL6-DBP consensus sequences in the 5X terminal region of an rFas Ag gene, spanning from y142 to y122 ŽFig. 2.. On the other hand, while three laboratories have reported so far the structure of a promoter sequence of human Fas Ag independently, there are some controversies on the positions of both transcription initiation sites and putative transcription factor binding sites w4–6x. Behrmann et al. reported that there were two NF-k B consensus sequences in the promoter region of human Fas Ag, while Wada et al. reported that an NF-IL6 consensus sequence, a human analogue of IL6-DBP, was located at eight different distinct positions in the region prior to the translation initiation site w5,6x. The latter authors claimed that two of these NF-IL6 consensus sequences were probably located in the first exon, although there were some controversies on the positions of transcription initiation sites as mentioned above w6x. In any cases these results sug-

Fig. 3. The determination of a major transcription start site by the S1 nuclease protection assay. The probe was hybridized with liver polyŽA.q RNA. For the control experiment, Escherichia coli total RNA was used instead of liver RNA. The lanes designated by the number 1 through 4 indicate the amount of an S1 nuclease used for the assay as follows; 1: 10 units; 2: 20 units; 3: 50 units; 4: 100 units. The lanes depicted by the letter ‘ACTG’ indicate the sequence ladder obtained by using the primer F as a sequencing primer. Due to the difference of sample buffers, the samples digested by S1 nuclease moved faster by four bases than the DNA samples for the sequence ladder, based upon the comparison of the undigested probe with the sequence ladder. Then the protected band designated by an arrowhead corresponds to the nucleotide residue designated by an asterisk in Fig. 2.

gested a possibility that an NF-k B and an NF-IL6 consensus sequences might play some important roles in the expression of human Fas Ag gene. However, it must be pointed out that, while both rat and human promoter regions contain a consensus sequence of the same group, the overall structure of the promoters is quite different between rat and human ŽFig. 2 w4–6x.. To ascertain whether the region including a consecutive sequence of NF-k B and IL6-DBP in rat Fas

K. Kimura et al.r Biochimica et Biophysica Acta 1352 (1997) 238–242

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Table 1 The Cat activity of rat Fas antigen promoter region Vector Activity

A 1.85

B 2.57

Bgl 2.61

Eco 2.87

C 1.92

D 1.44

E 1.07

pSCo 1.00

pSV2 124

The Cat vectors were constructed by the method described in the text. The 5X and 3X terminal of nucleotide positions of the promoter sequence constructed into a pSV0Cat vector are as follows; A: y653 to 144; B: y489 to 144; Bgl: y348 to 144; Eco: y220 to 144; C: y141 to 144; D: y22 to 144; E: 45 to 144. The pSV0 and pSV2 were used as a negative and a positive control respectively. The Cat activities are depicted as relative values to the value of the pSV0 as 1.00.

Ag gene did indeed show transcription activity, a promoter activity assay using chloramphenicol acetyltransferase ŽCat. vectors was performed based on the method of Sambrook et al. w7x. The PCR products generated from total liver RNA by using the primer F as a reverse primer and each one of the primers A through E as a forward one were cloned into a pSV0Cat vector. These vectors were named after forward primers used for their construction. Further, in order to produce another two kinds of Cat vectors, the PCR fragment generated by the primer pair A and F was restricted with either BglII or EcoRI and the restricted fragments including the sequence corresponded to the primer F were cloned into a pSV0Cat vector. These two vectors were named after restriction enzymes used for their construction. A pSV2Cat vector containing an SV40 early promoter region was used as a positive control. These vectors were transfected into L929 mouse fibroblast cells by the calcium phosphate method. The cell line L929 was obtained from Riken Cell Bank ŽTsukuba, Japan. and cultured in Dulbecco’s modified essential medium ŽGibco-BRL, Gaithersburg. supplemented with 10% foetal calf serum. While a Cat activity was low in the cells transfected with either the vector D or E lacking NF-k B and IL6-DBP consensus sequences, a high Cat activity was obtained in the cells transfected with vectors including these consensus sequences such as the vector B, Eco, Bgl, and C ŽTable 1.. The Cat analysis hence clearly showed that the region includ-

ing a consecutive sequence of NF-k B and IL6-DBP consensus sequences was essential for a transcription activity. Van Bladel et al. demonstrated that tumor necrosis factor Ž TNF.-a receptor expression was induced markedly in hepatocytes by interleukin-6 ŽIL-6. w9x. There is a possibility that Fas Ag expression may be induced by IL-6 in a similar manner, since TNF-a receptors and Fas Ags belong to the same family of membrane proteins and yet both receptors function to transduce death signals to cells Žsee Table 2.. Recently Wada et al. reported that a transcription factor NF-IL6 augmented slightly the Fas Ag expression in a tissue culture system w5x. Since an NF-IL6 is known to function as a transcription factor to transduce signals from stimulus of lipopolysaccharides or IL-6, their report was supposed to be related to the results of Van Bladel et al. In this study we showed that the region including a consecutive NF-k B and NF-IL6 consensus sequence was responsible for the promoter activity of an rFas Ag gene. There were some reports on the synergistic activation of genes by NF-k B and NF-IL6 transcription factors. And in some cases the NF-k B consensus sequence is located adjacent to the NF-IL6 consensus sequence w10–12x. The rFas Ag gene probably belongs to such cases, although direct evidence indicating an essential role of such consecutive sequence for the promoter activity was not given in this study. On the contrary, human Fas Ag promoter does not include such consecutive sequences,

Table 2 The effect of cycloheximide and lipopolysaccharide upon the expression of mouse Fas mRNA in L929 fibroblast cells Žy. 0.072

cyc 0.01 mgrml 0.19 )

cyc 1 mgrml 0.31 )

lps 10 mgrml 0.15

lps q cyc 10 mgrml 0.01 mgrml 0.54 )

lps q cyc 10 mgrml 1 mgrml 1.0 )

The data were expressed as relative values to the value obtained in the presence of both lipopolysaccharide Ž10 m grml. and cycloheximide Ž1 m grml. as 1.0. The asterisks indicate the difference of P - 0.05, when compared with medium alone.

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although there are many NF-IL6 and NF-k B consensus sequences in the promoter region w4–6x. These results suggested a possibility that the cellular mechanism of the regulation of Fas Ag expression might somehow be different between human and rat.

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w5x I. Behrmann, H. Walczak, P.H. Krammer, Eur. J. Immunol. 24 Ž1994. 3057–3062. w6x N. Wada, M. Matsumura, Y. Ohba, N. Kobayashi, T. Takizawa, Y. Nakanishi, J. Biol. Chem. 270 Ž1995. 18007– 18012. w7x J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning Ž2nd Ed.., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989. w8x K. Kimura, M. Yamamoto Ž1996. Cell Biochemistry and Function, in press. w9x S. Van Bladel, C. Libert, W. Fiers, Cytokine 3 Ž1991. 149–154. w10x Y. Zhang, M. Broser, W.N. Rom, Proc. Natl. Acad. Sci. USA 91 Ž1994. 2225–2229. w11x N. Mukaida, Y. Mahe, K. Matsushima, J. Biol. Chem. 265 Ž1990. 21128–21133. w12x A.R. Brasier, D. Ron, J.E. Tate, J.F. Habener, EMBO J. 9 Ž1990. 3933–3944.