- Email: [email protected]
Analysis of the promoter region of the human FcRn gene
Biochimica et Biophysica Acta 1492 (2000) 180^184
www.elsevier.com/locate/bba
Promoter paper
Analysis of the promoter region of the human FcRn gene Joanna E. Mikulska a b
a;
*, Neil E. Simister
b
Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, R. Weigla 12, 53-114 Wroclaw, Poland Rosenstiel Center for Basic Biomedical Sciences, W.M. Keck Institute for Cellular Visualization, and Biology Department, Brandeis University, Waltham, MA 02254-9110, USA Received 11 January 2000; accepted 2 March 2000
Abstract The 5P-flanking region of the human FcRn K-chain gene was analyzed for its ability to directly express the chloramphenicol acetyltransferase (CAT) reporter gene in NIH3T3 and Lu106 cells. Transient transfection of the CAT constructs revealed that there was promoter activity in the region 3660 to +300 of the 5P-flanking sequence. Electrophoretic mobility-shift assays showed that there are functional binding sites for Sp1 or Sp1-like factors, AP1 or a related factor, and additional unidentified proteins in the promoter region. ß 2000 Elsevier Science B.V. All rights reserved. Keywords : FcRn; Promoter; 5P-Flanking region ; Transcription factor; Gene regulation
The neonatal Fc receptor, FcRn, was identi¢ed as the receptor that transports IgG from milk across the intestinal epithelium of the suckling rat [1^3]. The human homologue of FcRn was ¢rst found in the placenta, where it is thought to transport maternal IgG to the fetus [4]. FcRn has since been found in several cell types, including capillary endothelium in some locations [5], where it appears to regulate plasma IgG concentrations [6^8]. FcRn structurally resembles the MHC class I molecules [3,9], but the FcRn K-chain is encoded outside the major histocompatibility complex [10^12]. We previously reported the sequence of the gene encoding human FcRn K-chain (HFGRT, GenBank AF200219 and AF200220) [13]. We mapped the transcription start sites, which were not associated with typical TATA or initiator sequences [13]. In the present work, we investigated the promoter activity of the 5P-£anking sequence of the human FcRn K-chain gene, and the binding of nuclear proteins to this region. These studies provide a starting point for examining the transcriptional regulation of the human FcRn gene. To test for promoter activity, fragments of the 5P-£anking sequence of the hFcRn gene were subcloned into the promoterless chloramphenicol acetyltransferase (CAT) expression vector pCAT-enhancer (Promega), as shown in Fig. 1. The plasmid CAT-1426 contained the 5P-region
* Corresponding author. Fax: +48-71-373-2587; E-mail : [email protected]
of the FcRn gene (bases 3660 to +766) in the HindIII/ AccI sites of pCAT-enhancer. CAT-712 contained the HindIII/AvrII fragment (3660 to +52) of the 5P-region in the HindIII/XbaI sites of pCAT-enhancer. The HindIII/AvrII fragment was also digested with SdaI to release two fragments: SdaI/AvrII (3230 to +52) was cloned into the PstI/XbaI sites of pCAT-enhancer creating CAT-281, and HindIII/SdaI (3660 to 3230) was inserted in the HindIII/PstI sites to make CAT-431. CAT-T2 was made by subcloning a TaqI fragment (373 to +300) into the AccI site of pCAT-enhancer. Additionally, this TaqI fragment was digested with AvrII: the TaqI/AvrII (373 to +52) and AvrII/TaqI (+52 to +300) fragments were subcloned into AccI/XbaI sites of pCAT-enhancer, creating CAT-124 and CAT-248, respectively. Each construct was sequenced to establish the orientation of the insert. The human cell lines HeLa, and Lu106 cells (embryo lung ¢broblast) were cultured in minimal essential medium (MEM), K-modi¢cation, containing L-glutamine (GibcoBRL), 100 U/ml of penicillin, 100 Wg/ml streptomycin, and 10% fetal bovine serum (Gibco-BRL). The mouse ¢broblast-like cell line NIH3T3 was grown in Dulbecco's modi¢ed Eagle's medium supplemented with antibiotics and fetal bovine serum as above. Cells were grown to 50^60% con£uence at 37³C in an atmosphere of 5% CO2 . Cells were washed twice with Opti-MEM I (GibcoBRL) and transfected with plasmids (10 Wg) using Lipofectin Reagent (Gibco-BRL). The pCAT-control vector containing the SV40 promoter and promoterless pCAT-
0167-4781 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 0 ) 0 0 0 6 8 - 3
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Fig. 1. Schematic diagram of the 5P-£anking region of the human FcRn K-chain gene (A) and CAT constructs (B). The restriction sites AvrII (A), BamHI (B), BsaHI (Bs), BstXI (Bx), EcoRI (E), HindIII (H), SmaI (S), SacI (Sa), SdaI (Sd), TaqI (T) are shown. The transcription initiation sites are indicated by arrows. Numbers 1^7 indicate the plasmid constructs. Constructs were generated by a restriction endonucleases digestion of the 5P-£anking region of the human FcRn K-chain gene and inserted to the promoterless CAT expression vector.
enhancer vector with no insert were used as positive and negative controls, respectively. After 5 h, Opti-MEM I was replaced with culture medium supplemented with 10% fetal bovine serum, and cells were grown for 43 h before being harvested. Cell extracts were prepared by three cycles of freeze^thaw in 0.25 M Tris^HCl, pH 8.0. Extracts were incubated at 60^65³C for 10 min to inactivate endogenous acetylases, then assayed for CAT activity [14]. The CAT induction from each plasmid in a given cell line was assayed in duplicate from three independent transfections, and was expressed relative to the CAT activity of the pCAT-control vector (100%). We found that a construct containing most of the cloned 5P-£anking region, CAT-1426 (bases 3660 to 6
Fig. 2. Functional analysis of the 5P-£anking region of the human FcRn K-chain gene. (A) Representative autoradiogram from analysis of promoter activity of the plasmid constructs transiently transfected into Lu106 cells: lane 1, control reaction containing only 1-deoxy [dichloroacetyl-1-14 C]chloramphenicol; lane 2, pCAT-control vector (positive control) ; lane 3, pCAT-enhancer vector (negative control) ; lanes 4^10, plasmid constructs, respectively, CAT-1426, CAT-712, CAT-281, CAT431, CAT-T2, CAT-124, and CAT-248. (B) Relative CAT activity of the promoter constructs transfected into NIH3T3 cells (open columns) and into Lu106 cells (¢lled columns). CAT activity (percentage of the acetylated form of chloramphenicol) of each construct in a given cell line was expressed relative to the CAT activity of the pCAT-control vector (100%). The pCAT-enhancer is the promoterless plasmid vector (negative control) used to prepare CAT constructs. Transfections were carried out three times with all plasmid constructs including positive and negative controls. The results shown here are the means of duplicate assays for three independent transfections.
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+766) supported transcription of the CAT reporter in NIH3T3 and Lu106 cells (Fig. 2). The construct was inactive in HeLa cells (data not shown). CAT-712 (3660 to +52) and CAT-T2 (373 to +300) had similar promoter activities to CAT-1426 (Fig. 2). When the insert in CAT712 was divided into two parts, in CAT-431 (3660 to 3230) and CAT-281 (3230 to +52), both were inactive (Fig. 2). Likewise, neither part of the 5P-£anking sequence from CAT-T2, in CAT-124 (373 to +52) and CAT-248 (+52 to +300) was capable of driving the transcription of the CAT gene (Fig. 2). These data indicate that there is promoter activity in the region 3660 to +300, and suggest that there are multiple positive regulatory elements throughout this region. Regulatory sequences downstream of the transcription start sites have been described for other genes, including osteocalcin [15], angiotensinogen [16], insulin-like growth factor-I [17], and elk-1 [18]. Thus the engagement both upstream and downstream elements in the regulation of expression is not unique to FcRn.
Potential binding sites for transcription factors within the 5P-£anking sequence were identi¢ed using TESS [19] in the string search mode and MatInspector Pro version 3.3 [20] (Genomatix, Munich, Germany) in the matrix search mode, in both cases using version 3.2 of the Transfac database [21]. Possible binding sites in the region 3660 to 3230 included a consensus Sp1 element at 3640 and an Sp1-like sequence at 3625. Sp1 is important in enhancing the expression of some TATA-less genes [22]. There was also a potential AP1 binding site at 3270. In the region +53 to +300, there was a potential Sp1 site at +80, and Ets family consensus sequences at +125 and +190. Ets family members may have a role in the formation of the transcription initiation complex on promoters lacking TATA and Inr [23,24]. Ets family members often require cooperation with Sp1 [25,26] or AP1 transcription factors [27,28] for selective and e¤cient activation of their target genes. To test the possible interactions of the putative sites in the sequence 3660 to 3230 and +52 to +300 with nuclear proteins, electrophoretic mobility shift assays were per-
Fig. 3. Electrophoretic mobility shift assays with: probe A, 197 bp fragment (3660 to 3464) of the 5P-£anking region of the human FcRn gene (A); probe B, 172 bp (3463 to 3292) (B); probe C, 62 bp (3291 to 3230) (C); probe D, 248 bp (+53 to +300) (D). Probes A^D, were end-labeled and incubated with nuclear extracts (12 Wg) from Lu106 cells, then analyzed by electrophoresis in non-denaturing 4% polyacrylamide gels. The binding reactions were carried out in the presence of 6 Wg of poly(dI-dC) and 4.5 Wg of BSA. Lane 1, probe alone (negative control) ; lane 2, labeled probe with nuclear extract. Competitors used were as follows: unlabeled probe (lane 3); AP1 consensus oligonucleotide (lane 4); Sp1 consensus (lane 5, except in B); CREB consensus (lane 6, and lane 5 of B). A 50-fold molar excess of the competitors was used except in lane 3, where a 35-fold excess was used.
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formed. Nuclear extracts of Lu106 cells were prepared as described by Bae et al. [29]. The protein concentrations of the extracts were determined by the Bradford assay [30], and aliquots were snap-frozen in liquid nitrogen and stored at 380³C until use. Gel mobility shift assays were done essentially as described [31], using 12 Wg of nuclear extract. Three DNA probes were used. Probe A is a 197bp DNA fragment (nucleotides 3660 to 3464) obtained by digesting the plasmid CAT-431 (above) with XmaI/HindIII. CAT-431 was also digested with BstXI and then XmaI/PstI to release a 172-bp fragment (3463 to 3292) and a 62-bp fragment (3291 to 3230), respectively probes B and C. Probe D is a 248-bp fragment (+53 to +300) made by cutting CAT-T2 (above) with AvrII and TaqI. After dephosphorylation, probes were end-labeled using [Q-32 P]ATP (NEN) and T4 polynucleotide kinase (Amersham). Double-stranded oligonucleotides containing the recognition sites for the transcription factors AP1, Sp1, and CREB (Promega) and unlabeled DNA probes were used as competitors. Probe A (3660 to 3464) formed ¢ve complexes with nuclear proteins from Lu106 cells (Fig. 3A, lane 2). Unlabeled probe competed with the labeled probe for binding, indicating that the interactions are speci¢c (Fig. 3A, lane 3). A 50-fold molar excess of DNA containing the consensus sequence for Sp1 competed away four complexes (I, II, III and V, Fig. 3A, lane 5), suggesting that these complexes contain Sp1 or Sp1-like factors. AP1 and CREB consensus oligonucleotides did not compete for binding of nuclear protein with probe A (Fig. 3A, lanes 4 and 6). Probe B (3463 to 3292) formed four speci¢c complexes (Fig. 3B). Neither AP1, nor CREB (Fig. 3B, lanes 4 and 5), nor Sp1 (not shown) consensus oligonucleotides competed for binding. Probe C (3291 to 3230) formed three complexes, which were competed by excess unlabeled probe (Fig. 3C, lanes 2 and 3). AP1 and CREB consensus oligonucleotides (Fig. 3C, lanes 4, 6), but not Sp1 competed for binding (Fig. 3C, lane 5). The target sequences for AP1 and CREB are very similar. Both AP1 and CREB oligonucleotides could therefore compete with probe C for binding to the same nuclear factor (complex I in Fig. 3C). Probe D (+53 to +300) formed two speci¢c complexes (Fig. 3D). Complex I was competed by the Sp1 consensus oligonucleotide (Fig. 3D, lane 5), but not AP1 or CREB oligonucleotides (Fig. 3D, lanes 4 and 6). These results were consistent with the binding of Sp1 or Sp1-like factors to the predicted sites at 3640, 3625, and +80, and with the binding of AP1 to the predicted site at 3270. Our results also showed the binding of additional unidenti¢ed proteins to sites in the 5P-£anking region. The presence of binding sites for several transcription factors suggests that expression of the human FcRn K-chain is controlled by a complex array of proteins. Further studies will be needed for a fuller understanding of the regulation of this physiologically important gene. The
183
partial characterization of the promoter region presented in this report will serve as a basis for these studies. We thank Dr. M. Ugorski and Dr. D. Dus for cell lines, M. Banas for technical assistance in cell culture and Dr. T. Niedziela and Dr. W. Jachymek for help in preparing the ¢gures. This work was supported by the State Committee for Scienti¢c Research (KBN, Poland, Grant 4 P05A 033 08) and NIH Grants HD27691 and HD01146.
References [1] R. Rodewald, J.-P. Kraehenbuhl, Receptor-mediated transport of IgG, J. Cell Biol. 99 (1984) 159s^164s. [2] N.E. Simister, A.R. Rees, Isolation and characterization of an Fc receptor from neonatal rat small intestine, Eur. J. Immunol. 15 (1985) 733^738. [3] N.E. Simister, K.E. Mostov, An Fc receptor structurally related to MHC class I antigens, Nature 337 (1989) 184^187. [4] C.M. Story, J.E. Mikulska, N.E. Simister, A major histocompatibility complex class I-like Fc receptor cloned from human placenta: possible role in transfer of immunoglobulin G from mother to fetus, J. Exp. Med. 180 (1994) 2377^2381. [5] J. Borvak, J. Richardson, C. Medesan, F. Antohe, C. Radu, M. Simionescu, V. Ghetie, E.S. Ward, Functional expression of the MHC class I-related receptor, FcRn, in endothelial cells of mice, Int. Immunol. 10 (1998) 1289^1298. [6] V. Ghetie, J.G. Hubbard, J.K. Kim, M.F. Tsen, Y. Lee, E.S. Ward, Abnormally short serum half-lives of IgG in beta 2-microglobulinde¢cient mice, Eur. J. Immunol. 26 (1996) 690^696. [7] R.P. Junghans, C.L. Anderson, The protection receptor for IgG catabolism is the beta 2-microglobulin-containing neonatal intestinal transport receptor, Proc. Natl. Acad. Sci. USA 93 (1996) 5512^5516. [8] E.J. Israel, D.F. Wilsker, K.C. Hayes, D. Schoenfeld, N.E. Simister, Increased clearance of IgG in mice that lack L2-microglobulin possible protective role of FcRn, Immunology 89 (1996) 573^578. [9] W.P. Burmeister, L.N. Gastinel, N.E. Simister, M.L. Blum, P.J. î resolution of the MHC-related Bjorkman, Crystal structure at 2.2 A neonatal Fc receptor, Nature 372 (1994) 336^343. [10] J.J. Ahouse, C.L. Hagerman, P. Mittal, D.J. Gilbert, N.G. Copeland, N.A. Jenkins, N.E. Simister, A mouse MHC class-I-like Fc receptor encoded outside the MHC, J. Immunol. 151 (1993) 6076^6088. [11] E. Kandil, M. Noguchi, T. Ishibashi, M. Kasahara, Structural and phylogenetic analysis of the MHC class I-like Fc receptor gene, J. Immunol. 154 (1995) 5907^5918. [12] E. Kandil, M. Egashira, O. Miyosi, N. Niikawa, T. Ishibashi, M. Kasahara, The human gene encoding the heavy chain of the major histocompatibility complex class I-like Fc receptor (FCGRT) maps to 19q13.3, Cytogenet. Cell Genet. 73 (1996) 97^98. [13] J.E. Mikulska, L. Pablo, J. Canel, N.E. Simister, Cloning and analysis of the gene encoding the human neonatal Fc receptor, Immunogenetics, submitted for publication. [14] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning : A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. [15] B. Frenkel, M. Montecino, J.L. Stein, J.B. Lian, G.S. Stein, A composite intragenic silencer domain exhibits negative and positive transcriptional control of the bone-speci¢c osteocalcin gene: promoter and cell type requirements, Proc. Natl. Acad. Sci. USA 91 (1994) 10923^10927. [16] Y. Nibu, S. Takahashi, K. Tanimoto, K. Murakami, A. Fukamizu, Identi¢cation of cell type-dependent enhancer core element located in the 3P-downstream region of the human angiotensinogen gene, J. Biol. Chem. 269 (1994) 28598^28605.
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[17] C.I. Pao, J.L. Zhu, D.G. Robertson, K.W. Lin, P.K. Farmer, S. Begovic, G.J. Wu, L.S. Phillips, Transcriptional regulation of the rat insulin-like growth factor-I gene involves metabolism-dependent binding of nuclear proteins to a downstream region, J. Biol. Chem. 270 (1995) 24917^24923. [18] U. Lehmann, P. Brocke, J. Dittmer, A. Nordheim, Characterization of the human elk-1 promoter. Potential role of a downstream intronic sequence for elk-1 gene expression in monocytes, J. Biol. Chem. 274 (1999) 1736^1744. [19] J. Schug, G.C. Overton, TESS: Transcription Element Search System on the WWW, Technical Report CBIL-TR-1997-1001-v0.0, Computational Biology and Informatics Laboratory, School of Medicine, University of Pennsylvania, 1997. [20] K. Quandt, K. Frech, H. Karas, E. Wingender, T. Werner, MatInd and MatInspector ^ new fast and versatile tools for detection of consensus matches in nucleotide sequence data, Nucleic Acids Res. 23 (1995) 4878^4884. [21] T. Heinemeyer, X. Chen, H. Karas, A.E. Kel, O.V. Kel, I. Liebich, T. Meinhardt, I. Reuter, F. Schacherer, E. Wingender, Expanding the TRANSFAC database towards an expert system of regulatory molecular mechanisms, Nucleic Acids Res. 27 (1999) 318^322. [22] K.H. Emami, T.W. Burke, S.T. Smale, Sp1 activation of a TATAless promoter requires a species-speci¢c interaction involving transcription factor IID, Nucleic Acids Res. 26 (1998) 839^846. [23] B. Wasylyk, S.L. Hahn, A. Giovane, The Ets family of transcription factors, Eur. J. Biochem. 211 (1993) 7^18. [24] N. Li, S. Seetharam, B. Seetharam, Characterization of the human transcobalamin II promoter. A proximal GC/GT box is a dominant negative element, J. Biol. Chem. 273 (1998) 16104^16111.
[25] J. Dittmer, A. Gegonne, S.D. Gitlin, J. Ghysdael, J.N. Brady, Regulation of parathyroid hormone-related protein (PTHrP) gene expression. Sp1 binds through an inverted CACCC motif and regulates promoter activity in cooperation with Ets1, J. Biol. Chem. 269 (1994) 21428^21434. [26] I. Nuchprayoon, J. Shang, C.P. Simkevich, M. Luo, A.G. Rosmarin, A.D. Friedman, An enhancer located between the neutrophil elastase and proteinase 3 promoters is activated by Sp1 and an Ets factor, J. Biol. Chem. 274 (1999) 1085^1091. [27] A.S. Takaoka, T. Yamada, M. Gotoh, Y. Kanai, K. Imai, S. Hirohashi, Cloning and characterization of the human beta 4-integrin gene promoter and enhancers, J. Biol. Chem. 273 (1998) 33848^ 33855. [28] G. Behre, A.J. Whitmarsh, M.P. Coghlan, T. Hoang, C.L. Carpenter, D.E. Zhang, R.J. Davis, D.G. Tenen, c-Jun is a JNK-independent coactivator of the PU.1 transcription factor, J. Biol. Chem. 274 (1999) 4939^4946. [29] H.W. Bae, A.G. Geiser, D.H. Kim, M.T. Chung, J.K. Burmester, M.B. Sporn, A.B. Roberts, S.J. Kim, Characterization of the promoter region of the human transforming growth factor-beta type II receptor gene, J. Biol. Chem. 270 (1995) 29460^29468. [30] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248^254. [31] L. Hennighausen, H. Lubon, Interaction of protein with DNA in vitro, Methods Enzymol. 152 (1987) 721^735.
BBAEXP 91389 6-6-00
www.elsevier.com/locate/bba
Promoter paper
Analysis of the promoter region of the human FcRn gene Joanna E. Mikulska a b
a;
*, Neil E. Simister
b
Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, R. Weigla 12, 53-114 Wroclaw, Poland Rosenstiel Center for Basic Biomedical Sciences, W.M. Keck Institute for Cellular Visualization, and Biology Department, Brandeis University, Waltham, MA 02254-9110, USA Received 11 January 2000; accepted 2 March 2000
Abstract The 5P-flanking region of the human FcRn K-chain gene was analyzed for its ability to directly express the chloramphenicol acetyltransferase (CAT) reporter gene in NIH3T3 and Lu106 cells. Transient transfection of the CAT constructs revealed that there was promoter activity in the region 3660 to +300 of the 5P-flanking sequence. Electrophoretic mobility-shift assays showed that there are functional binding sites for Sp1 or Sp1-like factors, AP1 or a related factor, and additional unidentified proteins in the promoter region. ß 2000 Elsevier Science B.V. All rights reserved. Keywords : FcRn; Promoter; 5P-Flanking region ; Transcription factor; Gene regulation
The neonatal Fc receptor, FcRn, was identi¢ed as the receptor that transports IgG from milk across the intestinal epithelium of the suckling rat [1^3]. The human homologue of FcRn was ¢rst found in the placenta, where it is thought to transport maternal IgG to the fetus [4]. FcRn has since been found in several cell types, including capillary endothelium in some locations [5], where it appears to regulate plasma IgG concentrations [6^8]. FcRn structurally resembles the MHC class I molecules [3,9], but the FcRn K-chain is encoded outside the major histocompatibility complex [10^12]. We previously reported the sequence of the gene encoding human FcRn K-chain (HFGRT, GenBank AF200219 and AF200220) [13]. We mapped the transcription start sites, which were not associated with typical TATA or initiator sequences [13]. In the present work, we investigated the promoter activity of the 5P-£anking sequence of the human FcRn K-chain gene, and the binding of nuclear proteins to this region. These studies provide a starting point for examining the transcriptional regulation of the human FcRn gene. To test for promoter activity, fragments of the 5P-£anking sequence of the hFcRn gene were subcloned into the promoterless chloramphenicol acetyltransferase (CAT) expression vector pCAT-enhancer (Promega), as shown in Fig. 1. The plasmid CAT-1426 contained the 5P-region
* Corresponding author. Fax: +48-71-373-2587; E-mail : [email protected]
of the FcRn gene (bases 3660 to +766) in the HindIII/ AccI sites of pCAT-enhancer. CAT-712 contained the HindIII/AvrII fragment (3660 to +52) of the 5P-region in the HindIII/XbaI sites of pCAT-enhancer. The HindIII/AvrII fragment was also digested with SdaI to release two fragments: SdaI/AvrII (3230 to +52) was cloned into the PstI/XbaI sites of pCAT-enhancer creating CAT-281, and HindIII/SdaI (3660 to 3230) was inserted in the HindIII/PstI sites to make CAT-431. CAT-T2 was made by subcloning a TaqI fragment (373 to +300) into the AccI site of pCAT-enhancer. Additionally, this TaqI fragment was digested with AvrII: the TaqI/AvrII (373 to +52) and AvrII/TaqI (+52 to +300) fragments were subcloned into AccI/XbaI sites of pCAT-enhancer, creating CAT-124 and CAT-248, respectively. Each construct was sequenced to establish the orientation of the insert. The human cell lines HeLa, and Lu106 cells (embryo lung ¢broblast) were cultured in minimal essential medium (MEM), K-modi¢cation, containing L-glutamine (GibcoBRL), 100 U/ml of penicillin, 100 Wg/ml streptomycin, and 10% fetal bovine serum (Gibco-BRL). The mouse ¢broblast-like cell line NIH3T3 was grown in Dulbecco's modi¢ed Eagle's medium supplemented with antibiotics and fetal bovine serum as above. Cells were grown to 50^60% con£uence at 37³C in an atmosphere of 5% CO2 . Cells were washed twice with Opti-MEM I (GibcoBRL) and transfected with plasmids (10 Wg) using Lipofectin Reagent (Gibco-BRL). The pCAT-control vector containing the SV40 promoter and promoterless pCAT-
0167-4781 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 0 ) 0 0 0 6 8 - 3
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J.E. Mikulska, N.E. Simister / Biochimica et Biophysica Acta 1492 (2000) 180^184
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Fig. 1. Schematic diagram of the 5P-£anking region of the human FcRn K-chain gene (A) and CAT constructs (B). The restriction sites AvrII (A), BamHI (B), BsaHI (Bs), BstXI (Bx), EcoRI (E), HindIII (H), SmaI (S), SacI (Sa), SdaI (Sd), TaqI (T) are shown. The transcription initiation sites are indicated by arrows. Numbers 1^7 indicate the plasmid constructs. Constructs were generated by a restriction endonucleases digestion of the 5P-£anking region of the human FcRn K-chain gene and inserted to the promoterless CAT expression vector.
enhancer vector with no insert were used as positive and negative controls, respectively. After 5 h, Opti-MEM I was replaced with culture medium supplemented with 10% fetal bovine serum, and cells were grown for 43 h before being harvested. Cell extracts were prepared by three cycles of freeze^thaw in 0.25 M Tris^HCl, pH 8.0. Extracts were incubated at 60^65³C for 10 min to inactivate endogenous acetylases, then assayed for CAT activity [14]. The CAT induction from each plasmid in a given cell line was assayed in duplicate from three independent transfections, and was expressed relative to the CAT activity of the pCAT-control vector (100%). We found that a construct containing most of the cloned 5P-£anking region, CAT-1426 (bases 3660 to 6
Fig. 2. Functional analysis of the 5P-£anking region of the human FcRn K-chain gene. (A) Representative autoradiogram from analysis of promoter activity of the plasmid constructs transiently transfected into Lu106 cells: lane 1, control reaction containing only 1-deoxy [dichloroacetyl-1-14 C]chloramphenicol; lane 2, pCAT-control vector (positive control) ; lane 3, pCAT-enhancer vector (negative control) ; lanes 4^10, plasmid constructs, respectively, CAT-1426, CAT-712, CAT-281, CAT431, CAT-T2, CAT-124, and CAT-248. (B) Relative CAT activity of the promoter constructs transfected into NIH3T3 cells (open columns) and into Lu106 cells (¢lled columns). CAT activity (percentage of the acetylated form of chloramphenicol) of each construct in a given cell line was expressed relative to the CAT activity of the pCAT-control vector (100%). The pCAT-enhancer is the promoterless plasmid vector (negative control) used to prepare CAT constructs. Transfections were carried out three times with all plasmid constructs including positive and negative controls. The results shown here are the means of duplicate assays for three independent transfections.
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+766) supported transcription of the CAT reporter in NIH3T3 and Lu106 cells (Fig. 2). The construct was inactive in HeLa cells (data not shown). CAT-712 (3660 to +52) and CAT-T2 (373 to +300) had similar promoter activities to CAT-1426 (Fig. 2). When the insert in CAT712 was divided into two parts, in CAT-431 (3660 to 3230) and CAT-281 (3230 to +52), both were inactive (Fig. 2). Likewise, neither part of the 5P-£anking sequence from CAT-T2, in CAT-124 (373 to +52) and CAT-248 (+52 to +300) was capable of driving the transcription of the CAT gene (Fig. 2). These data indicate that there is promoter activity in the region 3660 to +300, and suggest that there are multiple positive regulatory elements throughout this region. Regulatory sequences downstream of the transcription start sites have been described for other genes, including osteocalcin [15], angiotensinogen [16], insulin-like growth factor-I [17], and elk-1 [18]. Thus the engagement both upstream and downstream elements in the regulation of expression is not unique to FcRn.
Potential binding sites for transcription factors within the 5P-£anking sequence were identi¢ed using TESS [19] in the string search mode and MatInspector Pro version 3.3 [20] (Genomatix, Munich, Germany) in the matrix search mode, in both cases using version 3.2 of the Transfac database [21]. Possible binding sites in the region 3660 to 3230 included a consensus Sp1 element at 3640 and an Sp1-like sequence at 3625. Sp1 is important in enhancing the expression of some TATA-less genes [22]. There was also a potential AP1 binding site at 3270. In the region +53 to +300, there was a potential Sp1 site at +80, and Ets family consensus sequences at +125 and +190. Ets family members may have a role in the formation of the transcription initiation complex on promoters lacking TATA and Inr [23,24]. Ets family members often require cooperation with Sp1 [25,26] or AP1 transcription factors [27,28] for selective and e¤cient activation of their target genes. To test the possible interactions of the putative sites in the sequence 3660 to 3230 and +52 to +300 with nuclear proteins, electrophoretic mobility shift assays were per-
Fig. 3. Electrophoretic mobility shift assays with: probe A, 197 bp fragment (3660 to 3464) of the 5P-£anking region of the human FcRn gene (A); probe B, 172 bp (3463 to 3292) (B); probe C, 62 bp (3291 to 3230) (C); probe D, 248 bp (+53 to +300) (D). Probes A^D, were end-labeled and incubated with nuclear extracts (12 Wg) from Lu106 cells, then analyzed by electrophoresis in non-denaturing 4% polyacrylamide gels. The binding reactions were carried out in the presence of 6 Wg of poly(dI-dC) and 4.5 Wg of BSA. Lane 1, probe alone (negative control) ; lane 2, labeled probe with nuclear extract. Competitors used were as follows: unlabeled probe (lane 3); AP1 consensus oligonucleotide (lane 4); Sp1 consensus (lane 5, except in B); CREB consensus (lane 6, and lane 5 of B). A 50-fold molar excess of the competitors was used except in lane 3, where a 35-fold excess was used.
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formed. Nuclear extracts of Lu106 cells were prepared as described by Bae et al. [29]. The protein concentrations of the extracts were determined by the Bradford assay [30], and aliquots were snap-frozen in liquid nitrogen and stored at 380³C until use. Gel mobility shift assays were done essentially as described [31], using 12 Wg of nuclear extract. Three DNA probes were used. Probe A is a 197bp DNA fragment (nucleotides 3660 to 3464) obtained by digesting the plasmid CAT-431 (above) with XmaI/HindIII. CAT-431 was also digested with BstXI and then XmaI/PstI to release a 172-bp fragment (3463 to 3292) and a 62-bp fragment (3291 to 3230), respectively probes B and C. Probe D is a 248-bp fragment (+53 to +300) made by cutting CAT-T2 (above) with AvrII and TaqI. After dephosphorylation, probes were end-labeled using [Q-32 P]ATP (NEN) and T4 polynucleotide kinase (Amersham). Double-stranded oligonucleotides containing the recognition sites for the transcription factors AP1, Sp1, and CREB (Promega) and unlabeled DNA probes were used as competitors. Probe A (3660 to 3464) formed ¢ve complexes with nuclear proteins from Lu106 cells (Fig. 3A, lane 2). Unlabeled probe competed with the labeled probe for binding, indicating that the interactions are speci¢c (Fig. 3A, lane 3). A 50-fold molar excess of DNA containing the consensus sequence for Sp1 competed away four complexes (I, II, III and V, Fig. 3A, lane 5), suggesting that these complexes contain Sp1 or Sp1-like factors. AP1 and CREB consensus oligonucleotides did not compete for binding of nuclear protein with probe A (Fig. 3A, lanes 4 and 6). Probe B (3463 to 3292) formed four speci¢c complexes (Fig. 3B). Neither AP1, nor CREB (Fig. 3B, lanes 4 and 5), nor Sp1 (not shown) consensus oligonucleotides competed for binding. Probe C (3291 to 3230) formed three complexes, which were competed by excess unlabeled probe (Fig. 3C, lanes 2 and 3). AP1 and CREB consensus oligonucleotides (Fig. 3C, lanes 4, 6), but not Sp1 competed for binding (Fig. 3C, lane 5). The target sequences for AP1 and CREB are very similar. Both AP1 and CREB oligonucleotides could therefore compete with probe C for binding to the same nuclear factor (complex I in Fig. 3C). Probe D (+53 to +300) formed two speci¢c complexes (Fig. 3D). Complex I was competed by the Sp1 consensus oligonucleotide (Fig. 3D, lane 5), but not AP1 or CREB oligonucleotides (Fig. 3D, lanes 4 and 6). These results were consistent with the binding of Sp1 or Sp1-like factors to the predicted sites at 3640, 3625, and +80, and with the binding of AP1 to the predicted site at 3270. Our results also showed the binding of additional unidenti¢ed proteins to sites in the 5P-£anking region. The presence of binding sites for several transcription factors suggests that expression of the human FcRn K-chain is controlled by a complex array of proteins. Further studies will be needed for a fuller understanding of the regulation of this physiologically important gene. The
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partial characterization of the promoter region presented in this report will serve as a basis for these studies. We thank Dr. M. Ugorski and Dr. D. Dus for cell lines, M. Banas for technical assistance in cell culture and Dr. T. Niedziela and Dr. W. Jachymek for help in preparing the ¢gures. This work was supported by the State Committee for Scienti¢c Research (KBN, Poland, Grant 4 P05A 033 08) and NIH Grants HD27691 and HD01146.
References [1] R. Rodewald, J.-P. Kraehenbuhl, Receptor-mediated transport of IgG, J. Cell Biol. 99 (1984) 159s^164s. [2] N.E. Simister, A.R. Rees, Isolation and characterization of an Fc receptor from neonatal rat small intestine, Eur. J. Immunol. 15 (1985) 733^738. [3] N.E. Simister, K.E. Mostov, An Fc receptor structurally related to MHC class I antigens, Nature 337 (1989) 184^187. [4] C.M. Story, J.E. Mikulska, N.E. Simister, A major histocompatibility complex class I-like Fc receptor cloned from human placenta: possible role in transfer of immunoglobulin G from mother to fetus, J. Exp. Med. 180 (1994) 2377^2381. [5] J. Borvak, J. Richardson, C. Medesan, F. Antohe, C. Radu, M. Simionescu, V. Ghetie, E.S. Ward, Functional expression of the MHC class I-related receptor, FcRn, in endothelial cells of mice, Int. Immunol. 10 (1998) 1289^1298. [6] V. Ghetie, J.G. Hubbard, J.K. Kim, M.F. Tsen, Y. Lee, E.S. Ward, Abnormally short serum half-lives of IgG in beta 2-microglobulinde¢cient mice, Eur. J. Immunol. 26 (1996) 690^696. [7] R.P. Junghans, C.L. Anderson, The protection receptor for IgG catabolism is the beta 2-microglobulin-containing neonatal intestinal transport receptor, Proc. Natl. Acad. Sci. USA 93 (1996) 5512^5516. [8] E.J. Israel, D.F. Wilsker, K.C. Hayes, D. Schoenfeld, N.E. Simister, Increased clearance of IgG in mice that lack L2-microglobulin possible protective role of FcRn, Immunology 89 (1996) 573^578. [9] W.P. Burmeister, L.N. Gastinel, N.E. Simister, M.L. Blum, P.J. î resolution of the MHC-related Bjorkman, Crystal structure at 2.2 A neonatal Fc receptor, Nature 372 (1994) 336^343. [10] J.J. Ahouse, C.L. Hagerman, P. Mittal, D.J. Gilbert, N.G. Copeland, N.A. Jenkins, N.E. Simister, A mouse MHC class-I-like Fc receptor encoded outside the MHC, J. Immunol. 151 (1993) 6076^6088. [11] E. Kandil, M. Noguchi, T. Ishibashi, M. Kasahara, Structural and phylogenetic analysis of the MHC class I-like Fc receptor gene, J. Immunol. 154 (1995) 5907^5918. [12] E. Kandil, M. Egashira, O. Miyosi, N. Niikawa, T. Ishibashi, M. Kasahara, The human gene encoding the heavy chain of the major histocompatibility complex class I-like Fc receptor (FCGRT) maps to 19q13.3, Cytogenet. Cell Genet. 73 (1996) 97^98. [13] J.E. Mikulska, L. Pablo, J. Canel, N.E. Simister, Cloning and analysis of the gene encoding the human neonatal Fc receptor, Immunogenetics, submitted for publication. [14] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning : A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. [15] B. Frenkel, M. Montecino, J.L. Stein, J.B. Lian, G.S. Stein, A composite intragenic silencer domain exhibits negative and positive transcriptional control of the bone-speci¢c osteocalcin gene: promoter and cell type requirements, Proc. Natl. Acad. Sci. USA 91 (1994) 10923^10927. [16] Y. Nibu, S. Takahashi, K. Tanimoto, K. Murakami, A. Fukamizu, Identi¢cation of cell type-dependent enhancer core element located in the 3P-downstream region of the human angiotensinogen gene, J. Biol. Chem. 269 (1994) 28598^28605.
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[17] C.I. Pao, J.L. Zhu, D.G. Robertson, K.W. Lin, P.K. Farmer, S. Begovic, G.J. Wu, L.S. Phillips, Transcriptional regulation of the rat insulin-like growth factor-I gene involves metabolism-dependent binding of nuclear proteins to a downstream region, J. Biol. Chem. 270 (1995) 24917^24923. [18] U. Lehmann, P. Brocke, J. Dittmer, A. Nordheim, Characterization of the human elk-1 promoter. Potential role of a downstream intronic sequence for elk-1 gene expression in monocytes, J. Biol. Chem. 274 (1999) 1736^1744. [19] J. Schug, G.C. Overton, TESS: Transcription Element Search System on the WWW, Technical Report CBIL-TR-1997-1001-v0.0, Computational Biology and Informatics Laboratory, School of Medicine, University of Pennsylvania, 1997. [20] K. Quandt, K. Frech, H. Karas, E. Wingender, T. Werner, MatInd and MatInspector ^ new fast and versatile tools for detection of consensus matches in nucleotide sequence data, Nucleic Acids Res. 23 (1995) 4878^4884. [21] T. Heinemeyer, X. Chen, H. Karas, A.E. Kel, O.V. Kel, I. Liebich, T. Meinhardt, I. Reuter, F. Schacherer, E. Wingender, Expanding the TRANSFAC database towards an expert system of regulatory molecular mechanisms, Nucleic Acids Res. 27 (1999) 318^322. [22] K.H. Emami, T.W. Burke, S.T. Smale, Sp1 activation of a TATAless promoter requires a species-speci¢c interaction involving transcription factor IID, Nucleic Acids Res. 26 (1998) 839^846. [23] B. Wasylyk, S.L. Hahn, A. Giovane, The Ets family of transcription factors, Eur. J. Biochem. 211 (1993) 7^18. [24] N. Li, S. Seetharam, B. Seetharam, Characterization of the human transcobalamin II promoter. A proximal GC/GT box is a dominant negative element, J. Biol. Chem. 273 (1998) 16104^16111.
[25] J. Dittmer, A. Gegonne, S.D. Gitlin, J. Ghysdael, J.N. Brady, Regulation of parathyroid hormone-related protein (PTHrP) gene expression. Sp1 binds through an inverted CACCC motif and regulates promoter activity in cooperation with Ets1, J. Biol. Chem. 269 (1994) 21428^21434. [26] I. Nuchprayoon, J. Shang, C.P. Simkevich, M. Luo, A.G. Rosmarin, A.D. Friedman, An enhancer located between the neutrophil elastase and proteinase 3 promoters is activated by Sp1 and an Ets factor, J. Biol. Chem. 274 (1999) 1085^1091. [27] A.S. Takaoka, T. Yamada, M. Gotoh, Y. Kanai, K. Imai, S. Hirohashi, Cloning and characterization of the human beta 4-integrin gene promoter and enhancers, J. Biol. Chem. 273 (1998) 33848^ 33855. [28] G. Behre, A.J. Whitmarsh, M.P. Coghlan, T. Hoang, C.L. Carpenter, D.E. Zhang, R.J. Davis, D.G. Tenen, c-Jun is a JNK-independent coactivator of the PU.1 transcription factor, J. Biol. Chem. 274 (1999) 4939^4946. [29] H.W. Bae, A.G. Geiser, D.H. Kim, M.T. Chung, J.K. Burmester, M.B. Sporn, A.B. Roberts, S.J. Kim, Characterization of the promoter region of the human transforming growth factor-beta type II receptor gene, J. Biol. Chem. 270 (1995) 29460^29468. [30] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248^254. [31] L. Hennighausen, H. Lubon, Interaction of protein with DNA in vitro, Methods Enzymol. 152 (1987) 721^735.
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