The human transforming growth factor alpha promoter directs transcription initiation from a single site in the absence of a TATA sequence.

Abstract

Transforming growth factor alpha (TGF-ot) is a transformation-responsive mitogenic polypeptide that is expressed in the brain, epithelial cells, and activated macrophages. We isolated and characterized the TGF-a promoter and localized the 5' end of the TGF-ea transcript to a unique position. Surprisingly, no apparent TATA box was present in the promoter sequence, suggesting that transcription from mammalian genes can initiate at unique and specific positions from promoters lacking this sequence motif.The transforming growth factors, which were first purified from retrovirally transformed cells, induce a reversible phenotypic transformation of some normal mammalian cells in culture (for reviews, see references 7 and 10). These factors have been classified into two types based on their biological and physical properties: transforming growth factor alpha (TGF-a), which in its fully processed form is a 50-amino-acid peptide sharing about 30% sequence homology with epidermal growth factor (EGF), and TGF-,1l (a prototype for a family of proteins [30]), which is a structurally unrelated homodimer of 112 amino acids. The synthesis of TGF-a has been detected in a variety of transformed cell lines and tumors (8, 10), prompting suggestions that the protein plays a role in the growth of some tumors through autocrine or paracrine mechanisms (29). However, recent studies with cDNAs encoding both human (9) and rat (21) TGF-a have led to the detection of this gene not only in tumor tissue but also in normal adult (5,18,23,33) and embryonic (13,20,31,32) cells. As a first step in dissecting the genetic requirements for expression of this gene, we localized the 5' end of the endogenous human mRNA to a single site of transcription initiation. We isolated genomic DNA 5' of this gene and identified by transient assays a 300-base-pair (bp) fragment within the flanking region that exhibits bidirectional promoter activity. Sequence analysis of this upstream region revealed that despite the identification of potential binding sites for several transcription factors, no canonical CCAAT or TATA sequences were apparent, suggesting that transcription from mammalian genes can initiate at unique and specific positions from promoters lacking this sequence motif.Analysis of the 5' end of TGF-a mRNA. To define the 3' boundary of the TGF-a promoter, we localized the 5' end of human TGF-a mRNA by primer extension analysis. For this purpose we used a 25-mer oligonucleotide complementary to sequences immediately downstream of the TGF-a initiator ATG, spanning positions +4 to +28 which were defined relative to the ATG codon. Validation of the specificity of * Corresponding author. the oligomer for TGF-a was first provided by primer extension analysis of total cytoplasmic RNA isolated from cells that were stably transfected with a vector containing TGF-a cDNA under transcriptional control of the simian virus 40 (SV40) early promoter (28). Three extension products with lengths of 160, 155, and 105 nucleotides were identified (Fig.

The EGF/TGFα response element within the TGFα promoter consists of a multi-complex regulatory element

“…Deletion to 201 bp of TGFa promoter which removes the adjacent EGF/ TGFa response element, signi®cantly decreases the level of basal CAT activity. Other groups have also reported that deletions which remove this element result in CAT constructs with reduced basal activity (Jakobovits et al, 1988;Raja et al, 1991). Therefore, this EGF/TGFa response element may also be important in maintaining a level of TGFa basal transcription which facilitates the action of more distal (`upstream') elements.…”

“…Therefore, this EGF/TGFa response element may also be important in maintaining a level of TGFa basal transcription which facilitates the action of more distal (`upstream') elements. Deletion of the ®rst 370 bp of DNA to generate a construct extending from 71140 to 7370 yields very low CAT activity (only 5% of the total activity) despite the presence of several potential Sp-1 and AP-2 sites within this fragment (Jakobovits et al, 1988). The remaining low-level basal transcription of the p201-CAT construct is probably due to Sp-1 binding at the two potential Sp-1 consensus sequences within this region.…”

“…In order to study TGFa transcriptional regulation, we cloned a 2813 bp fragment of the human TGFa promoter (Saeki et al, 1991). This fragment extended the TGFa promoter characterization by 1673 bp in the 5' direction over that reported by a previous group (Jakobovits et al, 1988). Where sequence overlap occurs, the sequence is identical.…”

“…There are several Sp-1 sites located within the proximal 600 bp of DNA. There are several potential AP-2 sites located in the more distal regions of the promoter between 7375 and 7850 (Jakobovits et al, 1988). Since EGFr activation stimulates TGFa expression, the characterization of the EGF/TGFa response element within the human TGFa promoter is important in understanding the control of this gene.…”

Cloning and Characterization of the Promoter Region of the Rat Epidermal Growth Factor Receptor Gene and Its Transcriptional Regulation by Nerve Growth Factor in PC12 Cells

“…The second type of TATAless promoter is the GϩC-ich class found in a number of genes. In contrast to TATA-containing promoters, which accurately initiate at a single site, only a few of these GϩC-rich promoters initiate transcription from a single site (29,30), whereas many demonstrate multiple transcription start sites (31)(32)(33). GϩC-rich sequences play a critical role in controlling the expression of various genes, including housekeeping genes and many cellular oncogenes.…”