Genomic binding-site cloning reveals an estrogen ... - Europe PMC

Proc. Natl. Acad. Sci. USA Vol. 90, pp. 11117-11121, December 1993 Biochemistry

Genomic binding-site cloning reveals an estrogen-responsive gene that encodes a RING finger protein (transcription factor/target gene/zinc finger/estrogen-responsive element)

SATOSHI INOUE*, AKIRA OluMo*, TAKAYUKI HOSOIt, SHIGERU KONDO*, HIDEo TOYOSHIMA*, TAKASHI KONDO*, AKIRA IKEGAMIt, YASUYOSHI OUCHIt, HAJIME ORIMOt, AND MASAMI MURAMATSU*§ *Department of Biochemistry, Saitama Medical School, 38 Moro-Hongo, Moroyama-machi, Iruma-gun, Saitama, 350.04, Japan; and tDepartment of Geriatrics and *3rd Department of Internal Medicine,

Faculty of Medicine, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan

Communicated by Rachmiel Levine, August 12, 1993

genomic ER-binding sites. A gene that encodes a protein, referred to as the estrogen-responsive finger protein (efp), containing zinc finger motifs and that has been shown to be regulated by estrogen has now been isolated.

Estrogen receptor (ER)-binding fragments ABSTRACT were isolated from human genomic DNA by using a recombinant ER protein. Using one of these fragments as a probe, we have identified an estrgen-responsive gene that encodes a putative zinc finger protein. It has a RING finger motif present in a family of apparent DNA-binding proteins and is designated estrogen-responsive finger protein (efp). efp cDNA contains a consensus estrogen-responsive element at the 3' untranslated region that can act as a downstream estrogen-dependent enhancer. Moreover, efp is regulated by estrogen as demonstrated at both the mRNA and the protein level in ER-positive cells derived from mammary gland. These data suggest that efp may represent an estrogen-responsive transcription factor that mediates phenotypic expression of the diverse estrogen action. Thus, the genomic binding-site cloning may be applicable for isolation of the target genes of other transcription factors.

MATERIALS AND METHODS Isolation of ER-Binding Fragments and efp cDNA Clones. The EO fragment was isolated from human genomic DNA as described (3). Briefly, high molecular weight DNA from HeLa cells (10 &g) was digested with Pst I and BamHI, and the nitrocellulose filter binding selection was performed with the recombinant DNA-binding domain of the ER (10 pmol). The DNA trapped by the filter (-10 ng) was eluted, cloned into the plasmid vector pUC18 (Pst I-BamHI), and amplified. Then, the plasmid DNA (10 pg) was again incubated with the DNA-binding domain of ER (10 pmol), and the selection cycle was repeated five times. AgtlO and AZAPII cDNA libraries were prepared from poly(A)+ RNA of human placenta, transfected into Escherichia coli C600hfl or XL1-Blue, and screened by hybridization with the 32P-labeled EO fragment (see Results). Clone AC1, which had the largest insert including a long 3' untranslated region (accession no. D21205), and an overlapping AC3 clone, which had the longest open reading frame, were further characterized. Both strands of the cDNA insert of AC3 were completely sequenced by the dideoxynucleotide method (6) with Sequenase (United States Biochemical). Northern Blot Analysis and Immunological Procedure. For Northern blot analysis, extraction of RNA, fractionation on formaldehyde/agarose gels, and hybridization conditions were as described (7). The hybridization probe was the 32P-labeled EO, ER cDNA (8) or ,B-actin cDNA fragment. The IgG fraction of a rabbit anti-DDVRNRQQDVRMTANRKVEQ antisera was prepared as anti-efp antibody (by courtesy of Medical and Biological Laboratories, Ina, Japan) and used at 1:1000 dilution for immunoblotting. The cDNA insert of AC3 was cloned into the EcoRI site of the pSSRa expression vector (9) with the SRa promoter in the sense orientation to construct pSSRacefp. Either pSSRa (10 ug) or pSSRacefp (10 ug) was transfected into COS-7 cells by the calciumphosphate precipitation method (10). Nuclear extracts were prepared as described (11), and immunoblotting using the anti-efp antibody was performed as described (12). In some experiments, anti-efp antibody was preincubated with antigen (0.1 mg/ml) overnight at 4°C.

Estrogen is a hormone that is secreted from the ovary and causes the development of female organs. It regulates growth, differentiation, and the function of target cells. It is assumed that the estrogen receptor (ER), a member of the steroid/thyroid hormone receptor superfamily (1, 2), mediates this action by binding ligand dependently to the estrogenresponsive element (ERE) that exists in the enhancer region of target genes, regulating their transcription directly. However, in contrast to the diverse estrogen action on a variety of organs, tissues, and cells, relatively few genes are known to respond to ER. Those include vitellogenin, prolactin, pS2, ovalbumin, and the progesterone receptor (cited in ref. 3). Important genes that regulate the growth and differentiation of female organs such as mammary gland and uterus, for example, in response to estrogen have not yet been identified. Although greater than one-third of human breast cancers are known to be responsive to estrogen and hence to antihormone treatment (4), the mechanism of the growth promotion of these cancer cells by ER is still unclear. Moreover, the ER has been identified in various nuclei of brains, implicating some roles of estrogen in the central nervous system (5). Estrogen, ER, and the estrogen-responsive genes must play important roles in a number of vital organs besides female specifications. These circumstances prompted us to isolate more estrogen-responsive genes to understand the molecular physiology of estrogen action. Recently, we developed a method to isolate ER-binding fragments from human genomic DNA (3). All these fragments contained consensus EREs, some of which showed estrogen-dependent enhancer activity. We then isolated several more ER-binding fragments and used them as probes for identification of target genes adjacent to

Abbreviations: efp, estrogen-responsive finger protein; ER, estrogen receptor; ERE, estrogen-responsive element; CAT, chloramphenicol acetyltransferase; FBS, fetal bovine serum; vitERE, Xenopus vitellogenin gene ERE. §To whom reprint requests should be addressed. $The sequence reported in this paper has been deposited in the GenBank data base (accession no. D21205).

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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FIG. 1. (A) Restriction map of the EO fragment and the cDNAs (AC1, AC3) cloned thereby as probe. The ERE sequence (EREO) is indicated by the solid box and compared with vitERE and consensus ERE (conERE) sequences. The open reading frame is indicated by the open box, and the potential polyadenylylation site is denoted by a star. (B) Northern blot analysis of human placenta poly(A)+ RNA (5 ug) with the EO probe. Migration positions of ribosomal RNA markers are shown on the right. (C) Estrogen-dependent enhancer activity of the EO fragment. The reporter plasmids, tk-cat-EO, vitERE-tk-cat, and tk-cat (2 ug each), were cotransfected with (+) or without (-) 0.1 pg of the ER expression vector pSV2RcER(ER) into COS-7 cells. They were incubated in the presence (+) or absence (-) of 10 nM 17,B-estradiol (E2), and CAT activities were assayed.

treated FBS (3). The EO fragment was cloned into the Sma I site of pBLCAT2 (tk-cat) (14) in the sense orientation (5' -+ 3') to construct tk-cat-EO. The oligonucleotide containing the wild-type ERE ofthe Xenopus viteliogenin gene A2 enhancer (vitERE) was synthesized and inserted at the upstream position of pBLCAT2 to construct vitERE-tk-cat (3). CAT

Ceil Culture and Chloramphenicol Acetyltransferase (CAT) Assay. HBL-100 cells (13) were grown to a subconfluent state in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS). One day prior to estrogen treatment, the medium was changed to phenol red-free Dulbecco's modified Eagle's medium containing 10% dextran-coated charcoal-

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FIG. 2. Nucleotide sequence and deduced amino acid sequence (one-letter symbols) of efp determined from the AC3 cDNA clone. Four regions, RING finger (R), Bl box (B1), B2 box (B2), and coiled-coil domains (C), are shown on the right. Cysteine and histidine residues that may be involved in metal finger formation are circled.

Biochemistry: Inoue et al. assay was performed as described (3). Briefly, 1 x 106 COS-7 cells were plated in 60-mm Petri dishes and maintained in Eagle's minimal essential medium containing 10% FBS for 24 h. One hour prior to transfection, the medium was replaced with phenol red-free Eagle's minimal essential medium containing 10% dextran-coated charcoal-treated FBS. Cells were transfected by the calcium phosphate precipitation method (10) with 0.1 ,g of pSV2RcER (8), 2 ,g of reporter plasmids, and 2 ,g of the pCH110 /3-galactosidase expression vector (Pharmacia), used as an internal control to normalize for variations in transfection efficiency. In some experiments, pSV2RcER was omitted. The total amount of DNA transfected was made up to 20 Mg with carrier DNA pGEM3Zf(-) (Promega). After 12 h of incubation, the cells were divided into two dishes and cultured further in the absence or presence of 10 nM 17,3estradiol for 24 h. Cell extracts were assayed for protein concentration and CAT activity (15). The experiment was carried out four times, and a representative pattern is shown. RESULTS Isolation of Estrogen-Responsive Gene Fragments and cDNA. Using one of the ER-binding fragments named EO (Fig. 1A) as a probe, we detected positive signals by Northern blot analysis in human placenta mRNA (Fig. 1B). This suggested the existence of a transcribed region adjacent to this genomic DNA fragment. We then screened human placenta cDNA libraries with the EO probe and found >10 positive cDNAs out of 500,000 plaques. Restriction mapping and partial sequencing indicated that all the clones were derived from the same RNA. As shown in Fig. 1A, AC1 had the largest insert containing a poly(A) tail, and AC3 had the longest open reading frame (see below), a part of which overlapped the 5' region of AC1. AC1 contained the complete EO fragment, and AC3 contained the Pst I-EcoRI fragment of EO as an exon at the 3' untranslated region. The EO fragment contains the consensus ERE sequence (EREO), which is compared with the vitERE sequence (Fig. 1A). To confirm the estrogen-dependent enhancer activity of this region, the EO fragment was inserted into a downstream position of a reporter vector having a herpes simplex virus thymidine kinase promoter to construct the tk-cat-EO. The reporter plasmids were cotransfected with or without an ER expression vector into COS-7 cells. The CAT activity was stimulated significantly only in the presence of both the ER expression vector and 17p-estradiol (Fig. 1C). The estrogendependent enhancer activity of tk-cat-EO was thus demonstrated. Northern blot analysis of human placenta mRNA showed positive bands of 6 kb and 10 kb (Fig. 1B), and the lower band corresponded to the size of the cDNA picked up by AC1 and AC3. The 10-kb band may correspond to a high molecular mRNA precursor, a splicing variant, or an mRNA with alternative end. Alternatively, the 10-kb band may be derived from another gene with strong homology to efp in this region.

Structure of efp. Fig. 2 shows the nucleotide sequence and predicted amino acid sequence of the AC3 cDNA. Interestingly, the predicted protein contains a zinc finger motif called the RING finger (16, 17) and is named efp. The nucleotide sequence surrounding the putative initiation codon closely resembles the consensus sequence of Kozak (18). The predicted efp protein consists of 630 amino acids, with a calculated relative molecular mass (Mr) of 70,986. Computerassisted analysis and data base search show that efp contains structurally characteristic regions: a coiled-coil region and cysteine-rich regions including a RING finger, a Bl box, and a B2 box (17). There is no signal peptide sequence or a transmembrane region, suggesting that efp is an intracellular protein.


11119

efp belongs to a family of nuclear proteins containing a RING finger motif (Fig. 3A). These proteins not only have a common variant zinc finger structure but also appear to be related to cell regulation and differentiation proteins (see Discussion). These proteins are assumed to bind with Zn2+ and then to DNA using the zinc finger domains (17) (Fig. 3B). Some members of this family possess a second CH domain, the B-box domain, downstream of the RING finger (17, 19) (Fig. 3C). Interestingly, efp, PML (20-22), and T18 (23) contain two B box motifs and appear to form a subgroup as shown in Fig. 3D. Furthermore, all members of the B-boxcontaining family possess a predicted coiled-coil domain downstream of the B box (Fig. 3D).

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FIG. 3. (A) Alignment and comparison of proteins containing the RING finger domain. Conserved cysteine and histidine residues are denoted in bold type. In the T18 oncogene, the number 38 in parentheses indicates the position of 38 residues omitted to maintain the alignment. (B) Possible metal ion coordination of the RING finger motif. (C) Alignment and comparison of proteins containing the B box domain. (D) Schematic representation of the B-box-containing proteins. The solid boxes represent the RING finger, the open boxes represent the B boxes, and the wavy-lined boxes represent the coiled-coil domain. The brackets represent a gap. Members of each subgroup (A and B) are shown.

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FIG. 4. Immunoblotting using anti-efp antibody. Nuclear extracts (10 jg) were prepared from COS-7 cells transfected with the mock expression vector (lane 1), from HBL-100 cells (lane 2), and from COS-7 cells transfected with the AC3 expression vector (lanes 3 and 4). Anti-efp antibody (lanes 1-3) or anti-efp antibody preincubated with the antigen (lane 4) was used for immunoblotting. Migration positions of the molecular size markers (in kDa) are shown on the right.

Identification of the efp Protein in the Cell. To detect the specific efp product, a polyclonal antibody against a partial peptide sequence of the efp protein was prepared. By immunoblotting, this anti-efp antibody detected a specific band of 70 kDa in the nuclear extracts of HBL-100 cells derived from the human mammary gland (Fig. 4, lane 2). The molecular size of this band calculated from the relative mobility corresponded to the predicted Mr of efp. Moreover, the size of the band of the natural product agreed with that in COS-7 cells transfected with the AC3 expression vector (Fig. 4, lane 3). The band was not detected in COS-7 cells transfected with the control expression vector (Fig. 4, lane 1). The specific band in COS-7 cells transfected with the AC3 expression vector was blocked by preincubation of the anti-efp antibody with the synthetic peptide that was used for immunization (Fig. 4, lane 4). Immunostaining of COS-7 cells transfected with the efp expression vector demonstrated the nuclear localization of the efp products (data not shown). Estrogen Responsiveness of efp Expression. To demonstrate that efp is actually regulated by estrogen in vivo, we treated the HBL-100 cells with estrogen and followed the efp mRNA

level by Northern blot analysis (Fig. SA). The level of efp mRNA was elevated from 2 h after estrogen treatment, reached a peak (3.5 times) at 10 h, and then returned to the initial level by 20 h. The mRNA level of ER and ,B-actin did not change. Immunoblotting analysis showed that the efp protein was also increased, reaching the highest level at 10 h and then decreasing by 20 h (Fig. 5B). These results confirm the conclusion obtained by mRNA analysis that efp is regulated in vivo by estrogen.

DISCUSSION In this study, we used genomic binding-site cloning to isolate the estrogen-responsive gene efp, whose ERE is located in an exon corresponding to the 3' untranslated region of mRNA. CAT assay has shown that it can act as a downstream estrogen-dependent enhancer in the presence of ER in the cell. A number of enhancers are known to exist in introns or even in 3' untranslated regions of mRNA-e.g., the case of K-fgf (24). It is noteworthy that some target genes of Drosophila transcription factor Ultrabithorax (Ubx) are located adjacent to the Ubx-binding sites in genomic DNA (25), and one of the binding sites is present at the 3' region of a target gene (26). Here, we have shown that the genomic binding-site cloning is potentially useful to obtain the direct target genes of mammalian transcription factors. This study has shown that one of the target genes of ER is a zinc finger protein having a new class of motif, the RING finger. Members of the RING finger family are putative DNA-binding proteins, some of which are implicated in transcriptional regulation, DNA repair, and site-specific recombination. PML is a putative transcription factor that was found fused to the retinoic acid receptor a in promyelocytic leukemia translocations (20-22). rfp is a proposed regulator of spermatogenesis (27), rpt-1 is a potential transcription factor that regulates expression of the interleukin 2 receptor gene and human immunodeficiency virus 1 genes (28). Posterior sex comb (Psc) and suppressor two of zesta [Su(z)2] are

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FIG. 5. (A) efp is regulated by estrogen. Poly(A)+ RNA (5 pg) was prepared from the HBL-100 cells at the indicated hours after 171-estradiol treatment and analyzed by Northern blot hybridization using efp, ER, and 3-actin cDNA probes. Migration positions of ribosomal RNA markers are shown on the right. (B) efp protein is regulated by estrogen. Nuclear extracts (25 umg) were prepared from the HBL-100 cells at the indicated

hours after 17,B-estradiol treatment and analyzed by immunoblotting using anti-efp antibody. Migration positions of molecular size markers (in kDa) are shown on the right. (C) A model for the estrogen action through the estrogen-responsive transcription factor. In this model, an ER-regulated transcription factor mediates and possibly amplifies the estrogen effect.

Biochemistry: Inoue et al. Drosophila polycomb group (Pc-G) genes of which mammalian homologs include the bmi-J protooncogene (29, 30). RAD18 is a yeast protein required for DNA repair (31), and RAG-1 is a recombination-activating gene product (32). efp possesses the B box and coiled-coil domain characteristic of a subfamily of the RING finger family. This subfamily includes efp, PML, T18, rfp, rpt-1, SS-a/Ro (33), and xnf7 (19). Three of the seven subfamily members, PML, rfp, and T18, have transformation capabilities when found in chromosomal translocations. In each of these translocations, RING finger, B box, and coiled-coil domains are retained when fused to other proteins, suggesting that these domains play an important role in cell transformation. The coiled-coil region in which the negatively charged residues are exposed on one side of the helix is known to function as a transactivation domain (34). Alternatively, these domains may play a role in protein-protein interaction, including dimer formation. The fact that efp has both the potential DNA-binding and the dimerization-transactivation domains strongly suggests that it belongs to one of the transcription factors. Estrogen exerts a wide variety of effects on different organs, but ER, the putative sole mediator of estrogen action, was found as a single molecular species. To achieve the diversity of estrogen action, we may postulate a second mediator of estrogen action, the ER-regulated transcription factor. By this model, the estrogen-responsive transcription factor can mediate and amplify the estrogen action, forming a cascade of gene regulation and providing diverse and specific pathways in each target organ. efp is a good candidate for this model (Fig. 5C). The short response time of efp to estrogen (within 2 h) is compatible with this model. The progesterone receptor gene, of which the promoter region is responsive to ER (35), is another example that fits this model. The structure and function of the ER have been intensively studied, but the whole mechanism of estrogen action is still poorly understood. We propose the possibility that the second mediators such as estrogen-responsive transcriptional regulators have an important implication in the mechanism of estrogen action. We thank Mr. Inagaki (Pharmaceutical Laboratory, KIRIN, Maebashi, Japan) for kindly providing synthetic peptides; Dr. Tamai (Medical and Biological Laboratories, Ina, Japan) for preparing antibodies to synthetic peptides; Drs. H. Hamada, M. Hashimoto, T. Shimazaki, R. Sakai, S. Kato, T. Nishimura, and T. Matsuse for helpful discussion; and Ms. M. Goto and H. Yamaguchi for expert technical assistance. The HBL-100 cells were generously supplied by the RIKEN Cell Bank. This work was supported by grants from the Ministry of Education, Science and Culture, Japan. 1. Evans, R. M. (1988) Science 240, 889-895. 2. Green, S. & Chambon, P. (1988) Trends Genet. 4, 309-314. 3. Inoue, S., Kondo, S., Hashimoto, M., Kondo, T. & Muramatsu, M. (1991) Nucleic Acids Res. 19, 4091-4096. 4. Harris, J. R., Hellman, S., Henderson, I. C. & Kinne, D. W. (1987) Breast Diseases (Lippincott, Philadelphia). 5. Simerly, R. B., Chang, C., Muramatsu, M. & Swanson, L. W.

(1990) J. Comp. Neurol. 294, 76-95.


11121

6. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. 7. Hashimoto, M., Shoda, A., Inoue, S., Yamada, R., Kondo, T., Sakurai, T., Ueno, N. & Muramatsu, M. (1992) J. Biol. Chem. 267, 4999-5004. 8. Koike, S., Sakai, M. & Muramatsu, M. (1987) Nucleic Acids Res. 15, 2499-2513. 9. Takebe, Y., Seiki, M., Fujisawa, J.-I., Hoy, P., Yokota, K., Arai, K.-I., Yoshida, M. & Arai, N. (1988) Mol. Cell. Biol. 8, 466-472. 10. Graham, F. L. & van der Eb, A. J. (1973) Virology 52,456-467. 11. Schreber, E., Matthias, P., Muller, M. M. & Schaffner, W. (1989) Nucleic Acids Res. 17, 6419. 12. Hashimoto, M., Nakamura, T., Inoue, S., Kondo, T., Yamada, R., Eto, Y., Sugino, H. & Muramatsu, M. (1992) J. Biol. Chem. 267, 7203-7206. 13. Gaffney, E. V. (1982) Cell Tissue Res. 227, 563-568. 14. Luckow, B. & Schutz, G. (1987) Nucleic Acids Res. 15, 5490. 15. Gorman, C. M., Moffat, L. F. & Howard, B. H. (1982) Mol. Cell. Biol. 2, 1044-1051. 16. Freemont, P. S., Hanson, I. M. & Trowsdale, J. (1991) Cell 64, 483-484. 17. Reddy, B. A., Etkin, L. D. & Freemont, P. S. (1992) Trends Biol. Sci. 17, 344-345. 18. Kozak, M. (1987) Nucleic Acids Res. 15, 8125-8148. 19. Reddy, B. A. & Etkin, L. D. (1991) Nucleic Acids Res. 19, 6330. 20. de The, H., Lavau, C., Marchio, A., Chomienne, C., Degos, L. & Dejean, A. (1991) Cell 66, 675-684. 21. Kakizuka, A., Miller, W. H., Umesono, K., Warrell, R. P., Frankel, S. R., Murty, V. V. V. S., Dmitrovsky, E. & Evans, R. M. (1991) Cell 66, 663-674. 22. Goddard, A. D., Borrow, J., Freemont, P. S. & Solomon, E. (1991) Science 254, 1371-1374. 23. Miki, T., Fleming, T. P., Crescenzi, M., Molloy, C. J., Blam, S. B., Reynolds, S. H. & Aaronson, S. A. (1991) Proc. Natl. Acad. Sci. USA 88, 5167-5171. 24. Curatola, A. M. & Basilico, C. (1990) Mol. Cell. Biol. 10, 2475-2484. 25. Gould, A. P., Brookman, J. J., Strutt, D. I. & White, R. A. H.

(1990) Nature (London) 348, 308-312. 26. Grabe, G., Aragnol, D., Laurenti, P., Garzino, V., Charmot, D., Berenger, H. & Pradel, J. (1992) EMBO J. 11, 3375-3378. 27. Takahashi, M., Inaguma, Y., Hiai, H. & Hirose, F. (1988) Mol. Cell. Biol. 8, 1853-1856. 28. Patarca, R., Schwartz, J., Singh, R. P., Kong, Q.-T., Murphy, E., Anderson, Y., Sheng, F.-Y. W., Singh, P., Johnson, K. A., Guamagia, S. M., Durfee, T., Blattner, F. & Cantor, H. (1988) Proc. Natl. Acad. Sci. USA 85, 2733-2737. 29. van Lohuizen, M., Frasch, M., Wientjens, E. & Berns, A.

(1991) Nature (London) 353, 353-355.

30. Brunk, B. P., Martin, E. C. & Adler, P. N. (1991) Nature (London) 353, 351-353. 31. Jones, J. S., Weber, S. & Prakash, L. (1988) Nucleic Acids Res. 16, 7119-7131. 32. Schatz, D. A., Oettinger, M. A. & Baltimore, D. (1989) Cell 59, 1035-1048. 33. Chan, E. K. L., Hammel, J. C., Buyon, J. P. & Tan, E. M.

(1991) J. Clin. Invest. 87, 68-76.

34. Giniger, E. & Ptashne, P. (1987) Nature (London) 330, 670-672. 35. Kastner, P., Krust, A., Turcotte, B., Stropp, U., Tora, L., Gronemeyer, H. & Chambon, P. (1990) EMBO J. 9, 1603-1614.