Somatostatin Promoter Research Articles

EivF, a factor required for transcription of the adenovirus EIV promoter, binds to an element involved in EIa-dependent activation and cAMP induction.

The isolation of a cellular factor that was specifically required for transcription from the promoter of the adenovirus early gene iv, an EIa-activated promoter, is described. This factor (EivF) was purified from HeLa cells using a functional transcription assay and identified as a 72,000- to 65,000-dalton protein. DNase I footprinting experiments demonstrated that purified EivF bound to the sequence 5'-GT(G/T)ACGT-3' present two times upstream of the Eiv TATA box. Nuclear extracts prepared from HeLa cells contained more than one factor capable of binding to the EivF recognition site. Previous studies have indicated that a sequence similar to the EivF-binding site was recognized by a 43,000-dalton protein and participated in the cAMP response of the somatostatin promoter. The purified and transcriptionally active EivF also bound to DNA sequence elements present in the somatostatin and alpha-gonadotropin promoters shown previously to be responsive to cAMP.

Three sequence-specific DNA-protein complexes are formed with the same promoter element essential for expression of the rat somatostatin gene.

We identified three sequence-specific DNA-protein complexes which are formed after in vitro binding of nuclear extracts, derived from neuronal (CA-77, rat brain) or non-neuronal (HeLa) cells, to positions -70 to -29 of the rat somatostatin promoter. The protein(s) responsible for the formation of the three sequence-specific complexes was fractionated from rat brain whole cell extracts by DEAE-Sepharose chromatography. The critical contact residues of the factor(s) in each complex, as determined by methylation interference analyses, are located within positions -59 to -35, which is protected from DNase I digestion; these include the G residues of a TGACGTCA consensus also found in the cAMP-responsive human enkephalin (positions -105 to -76) and E1A-inducible adenovirus type 5 E3 (positions -72 to -42) promoters. Competition assays with these heterologous promoters reveal that the factor(s) of each complex displays approximately 50-fold greater affinity for the somatostatin promoter-binding site. Synthetic oligonucleotides spanning positions -70 to -29 of the somatostatin promoter and containing single-base substitutions of the G residues in the TGACGTCA consensus were utilized in competition assays. The G residues located in the center of the module are the most critical determinants in the formation of the three sequence-specific complexes. Deletions disrupting the TGACGTCA consensus abolish not only formation of the three complexes in vitro but also expression of the somatostatin promoter in vivo, suggesting that formation of one or more of these complexes is essential for transcription of the rat somatostatin gene.

Identification of the promoter sequences involved in the cell specific expression of the rat somatostatin gene.

DNA sequences containing the 5' flanking region of the rat somatostatin gene were linked to the coding sequence of the bacterial chloramphenicol acetyl transferase gene. This recombinant plasmid is active in expressing CAT activity in the neuronally derived, somatostatin producing CA-77 cell line. Deletion analyses of the somatostatin promoter show that the sequences proximal to position -60, relative to the cap site are required for expression of this promoter. A 4 base pair deletion of residues -46 through -43 within the somatostatin promoter results in a down mutation in vivo suggesting the existence of an element critical for the expression of the promoter in CA-77 cells. In addition, the somatostatin recombinant and its 5' deletion constructs preferentially express CAT activity in CA-77 cells, whereas only basal level of expression is observed in HeLa, BSC40, and RIN-5F cell lines, pointing to the cell specific nature of this promoter.