Transcription of the Transforming Growth Factor-β2 Gene Is Dependent on an E-box Located between an Essential cAMP Response Element/Activating Transcription Factor Motif and the TATA Box of the Gene

Abstract

Transforming growth factor-␤2 (TGF-␤2) is an important regulator of cell proliferation and differentiation; however, its transcriptional regulation is not well understood. Here we report characterization of an essential E-box motif, positioned at ؊50/؊45 between a previously described functional cAMP response element/ activating transcription factor site and the TATA box of the human TGF-␤2 promoter. By site-directed mutagenesis, we demonstrate that this E-box motif is necessary for the promoter activity, not only in differentiated cells derived from embryonal carcinoma cells, but also in choriocarcinoma cells and in MCF-7 breast carcinoma cells. We also demonstrate that the transcription factors USF1 and USF2 bind to this E-box motif in vitro when nuclear extracts from each of these cell lines are examined by gel retardation assays. Moreover, using a dominant-negative USF2 protein, we show that USF proteins are critical for TGF-␤2 promoter activity in vivo. The importance of the E-box motif described in this study is supported by the presence of an E-box motif in the same position in the chicken TGF-␤2 gene promoter.Transforming growth factor-␤2 (TGF-␤2), 1 like other growth factors in the TGF-␤ family, is involved in the regulation of many different cellular functions, including cell proliferation and differentiation as well as production and maintenance of extracellular matrices (reviewed in Refs. 1 and 2). Through its multifaceted effects, TGF-␤2 plays important regulatory roles in a host of biological events, from embryogenesis through repair processes, to regulation of the immune system. Hence, the regulation of TGF-␤2 gene in various systems warrants detailed investigation. The studies presented here focus on the transcriptional regulation of the TGF-␤2 gene in embryonal carcinoma (EC) cells and their differentiated counterparts, which represent a model system of early embryonic development (reviewed in Ref. 3). Given the importance of TGF-␤2 production in implantation and in tumor malignancy (4 -6), we extended our studies to two choriocarcinoma cell lines and a breast carcinoma cell line. Previous work demonstrated the presence of a critical positive regulatory region in the TGF-␤2 gene promoter, localized between Ϫ77 and ϩ63, where ϩ1 is the transcription start site (7)(8)(9). This region contains a functional CRE/ATF motif, which is indispensable for the positive effect of this promoter region in different cell types and capable of binding activating transcription factor 1 (ATF-1) in vitro.Recently, computerized sequence analysis demonstrated that the human TGF-␤2 promoter also contains a CACGTG motif between Ϫ50 and Ϫ45, which conforms to the consensus sequence of an E-box motif, CANNTG (10). Interestingly, this E-box motif appears to be evolutionarily conserved, since the same CACGTG sequence is present in the chicken TGF-␤2 promoter, and it is located in the same position relative to the similarly conserved CRE/ATF site and the TATA box of the chicken promoter (11). Thus, the putative E-box motif l...

Transcriptional Regulation of the Transforming Growth Factor-β2 Promoter by cAMP-responsive Element-binding Protein (CREB) and Activating Transcription Factor-1 (ATF-1) Is Modulated by Protein Kinases and the Coactivators p300 and CREB-binding Protein

“…TGF-␤2 promoter/CAT chimeric gene constructs and their mutants (see Fig. 1) were described previously (16,(23)(24)(25). Unless otherwise noted, the null vector, pUC-19 (Invitrogen, Carlsbad, CA) was used to keep the total DNA concentration the same in each experiment.…”

“…Additionally, in electrophoretic gel mobility shift assays, ATF-1 and cAMP-responsive element binding protein (CREB) bind to the CRE/ATF motif, and the upstream stimulatory factors 1 and 2 (USF1 and -2) bind to the E-box element (26 -28). Previous studies have demonstrated that these transcription factors are present in nuclear extracts prepared from both F9 EC cells and F9-differentiated cells and can bind to their respective cis-regulatory elements in vitro (25,27,28). Expression of a dominant-negative USF expression plasmid reduces the expression of a TGF-␤2 promoter/reporter construct by approximately 80% (25).…”

“…Previous studies have demonstrated that these transcription factors are present in nuclear extracts prepared from both F9 EC cells and F9-differentiated cells and can bind to their respective cis-regulatory elements in vitro (25,27,28). Expression of a dominant-negative USF expression plasmid reduces the expression of a TGF-␤2 promoter/reporter construct by approximately 80% (25). Thus, it appears that the USF proteins are important for the proper expression of the TGF-␤2 gene, but prior to this study, the exact function of the E-box and CRE/ATF binding proteins in the case of the TGF-␤2 promoter in F9-differentiated cells had not been elucidated.…”

“…Important for the modulation of the TGF-␤2 gene during the differentiation of F9 EC cells is the presence of a critical positive regulatory region in the TGF-␤2 gene promoter, localized between Ϫ77 and ϩ63, where ϩ1 is the transcription start site (23,24). Within this positive regulatory region are two cis-regulatory elements: a CRE/ATF site positioned at Ϫ74 to Ϫ67 (23,24) and an E-box motif located between Ϫ50 and Ϫ45 (25). The nucleotide sequence of and spacing between both of these sites are evolutionary conserved in the human, chicken, and mouse promoters (16,26).…”

“…The nucleotide sequence of and spacing between both of these sites are evolutionary conserved in the human, chicken, and mouse promoters (16,26). 2 Mutation of either site reduces the expression of TGF-␤2 promoter/reporter gene constructs approximately 60 -80% (23)(24)(25). Additionally, in electrophoretic gel mobility shift assays, ATF-1 and cAMP-responsive element binding protein (CREB) bind to the CRE/ATF motif, and the upstream stimulatory factors 1 and 2 (USF1 and -2) bind to the E-box element (26 -28).…”