Abstract
The gene encoding the putative zinc finger protein tristetraprolin (TTP), Zfp-36, is rapidly induced by a variety of mitogens and growth factors. We show here that 77 base pairs 5' of the transcription start site are sufficient for full serum inducibility of the mouse Zfp-36 promoter. This region of the promoter includes consensus sequences for the binding of the transcription factors EGR-1, AP2, and Sp1. In addition, we have identified a previously undescribed element, TTP promoter element 1 (TPE1); this 10-base pair sequence includes a palindrome and is identical in the human, bovine, and mouse genes. Each of the three binding elements, EGR-1, AP2, and TPE1, contribute to the serum induction of Zfp-36 and can confer serum-inducible expression on a heterologous minimal promoter. Gel mobility shift assays demonstrated the formation of complexes consisting of this region of the promoter and cellular nuclear proteins and demonstrated that the extent of complex formation could be altered by treatment of the cells with serum or insulin. These results suggest that the response of Zfp-36 to serum and other mitogens is mediated by a series of cis-acting elements acting in concert to confer full inducible transcription of this gene.
Highlights
An early response of many cells to serum and polypeptide growth factors is the activated transcription of specific genes in the absence of de novo protein synthesis
1 The abbreviations used are: TTP, tristetraprolin; PMA, phorbol 12-myristate 13-acetate; GH, growth hormone; chicken fibroblasts (CEF), chicken embryonic fibroblasts; FBS, fetal bovine serum; TPE1, TTP promoter element 1; 2) a zinc finger protein known as Nup475 [3] and TIS11 [4]
B, CEF cells were transfected with plasmids containing the full mouse mRNA coding region, the single TTP intron, and various lengths of genomic sequence 5Ј of the initiator codon: 1.7 kb (EcoRV), 0.9 kb (Sau3AI), and 137 bp (SstII) as indicated. 24 h after transfection, the cells were serum-deprived for 24 h and treated with control conditions (C) or stimulated with 10% FBS (S) for 60 min
Summary
A Balb/c mouse genomic library (Clontech, Palo Alto, CA) was screened using the mouse TTP cDNA [1] as a probe. The entire mouse genomic sequence has been deposited in GenBankTM (accession number L42317). A second mouse genomic library (129 sv, Stratagene) was screened to obtain a 3.8-kb Sau3aISalI fragment, which contained 0.9 kb of the 5Ј-flanking region, the entire mRNA coding sequence, and a single intron. DNA sequence analysis revealed only a 2-bp difference in the 5Ј-flanking region when compared with the clone from the Balb/c library. A human genomic clone was obtained by screening a human placental genomic library (Clontech) with the human TTP cDNA [2] (GenBankTM accession number for the human genomic sequence: M19844). Southern mapping, subcloning, and DNA sequencing were performed by standard techniques [8]
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