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beta-cell type-specific expression of the upstream glucokinase promoter was studied by transfection of fusion genes and analysis of DNA-protein interactions. A construct containing 1,000 bp of 5'-flanking DNA was efficiently expressed in HIT M2.2.2 cells, a beta-cell-derived line that makes both insulin and glucokinase, but not in NIH 3T3 cells, a heterologous cell line. In a series of 5' deletion mutations between bases -1000 and -100 (relative to a base previously designated +1), efficient expression in HIT cells was maintained until -280 bp, after which transcription decreased in a stepwise manner. The sequences between -180 and -1 bp contributing to transcriptional activity in HIT cells were identified by studying 28 block transversion mutants that spanned this region in 10-bp steps. Two mutations reduced transcription 10-fold or more, while six reduced transcription between 3- and 10-fold. Three mutationally sensitive regions of this promoter were found to bind to a factor that was expressed preferentially in pancreatic islet beta cells. The binding sites, designated upstream promoter elements (UPEs), shared a consensus sequence of CAT(T/C)A(C/G). Methylation of adenine and guanine residues within this sequence prevented binding of the beta-cell factor, as did mutations at positions 2, 3, and 5. Analysis of nuclear extracts from different cell lines identified UPE-binding activity in HIT M2.2.2 and beta-TC-3 cells but not in AtT-20, NIH 3T3, or HeLa cells; the possibility of a greatly reduced amount in alpha-TC-6 cells could not be excluded. UV laser cross-linking experiments supported the beta-cell type expression of this factor and showed it to be approximately 50 kDa in size. Gel mobility shift competition experiments showed that this beta-cell factor is the same that binds to similar elements, termed CT boxes, in the insulin promoter. Thus, a role for these elements (UPEs or CT boxes), and the beta-cell factor that binds to them, in determining the expression of genes in the beta cells of pancreatic islets is suggested.
A novel protein tyrosine kinase (PTK) substrate, p120, has been previously implicated in ligand-induced signaling through the epidermal growth factor, platelet-derived growth factor and colony-stimulating factor 1 receptors, and in cell transformation by p60v-src. We have isolated a near full-length cDNA encoding murine p120. The encoded protein lacks significant homology with any reported protein, but it contains four copies of an imperfect 42 amino acid repeat that occurs 12.5 times in the protein encoded by Drosophila armadillo (arm), and its direct homologs, human plakoglobin (plak) and Xenopus laevis beta-catenin (beta-cat). The presence of this motif implies that p120 may share at least one aspect of its function with the arm protein and its homologs.
A unique cAMP regulatory sequence, -129/-96 base pairs (bp), associated with the gene encoding human cytochrome P450C21 (CYP21B) binds a nuclear protein designated ASP, as described previously (Kagawa, N., and Waterman, M. R. (1991) J. Biol. Chem. 266, 11199-11204). This putative transcription factor required for cAMP-dependent transcription of the human CYP21B gene has been purified from the nuclear extracts of mouse Y1 cells by using sequence-specific DNA-affinity chromatography. The purified ASP is 78 kDa as estimated by SDS-polyacrylamide gel electrophoresis and binds to its specific recognition site, -126/-113-bp CACTCTGTGGGCGG, which has been demonstrated to be the minimum cAMP regulatory sequence of the human CYP21B gene. To characterize ASP more precisely, an antibody was raised against the 78-kDa protein. This antibody led to a supershift of the DNA.ASP complex on gel shift analysis and inhibition of in vitro transcription promoted by the ASP binding sequence, thereby indicating that ASP is a 78-kDa transcription factor. Upon DNase I footprinting experiments, ASP showed a characteristic footprint which very closely resembles but is distinct from that of Sp1 which also occupies a binding site within -129/-96 bp. Furthermore, the addition of purified ASP enhanced the mRNA synthesis promoted by the minimum cAMP regulatory sequence in a cell-free transcription system using HeLa cell extracts, whereas added Sp1 does not. These results indicate that ASP is a primary transcription factor for the cAMP-dependent regulation of the human CYP21B gene.
The EFII cis element is a 38-bp sequence at the 5' end of the Rous sarcoma virus long terminal repeat, extending from nucleotides -229 to -192 (with respect to the viral transcription start site), which is recognized by sequence-specific DNA-binding proteins in avian fibroblast nuclear extracts (L. Sealy and R. Chalkley, Mol. Cell. Biol. 7:787-798, 1987). We demonstrate that multiple copies of the EFII cis element strongly activate transcription of a reporter gene in vivo. We correlate the region of the EFII cis element which activates transcription in vivo with the in vitro binding site for three nuclear factors, EFIIa, EFIIb, and EFIIc. The sequence motif recognized by EFIIa, -b, and -c is also found in consensus binding sites for members of a rapidly growing family of transcription factors related to the CCAAT/enhancer-binding protein (C/EBP). EFIIa, -b, and -c are present in fibroblast and epithelial cell lines from various species but are much less abundant in differentiated rat liver and kidney cells. The EFIIa binding activity is particularly abundant in an avian B-cell lymphoma line. As judged from molecular weight analysis, cell type distribution, and sequence recognition properties, the EFII factors under study appear to differ from most of the previously described C/EBP-related factors and thus may expand the diversity of the C/EBP family.
The bovine adrenodoxin gene gives rise to two species of mRNA differing only at their 5'-ends. The synthesis of these two types of mRNA in bovine adrenal cortical cells is regulated transcriptionally in part by ACTH via the action of cAMP. Examination of the 5'-end of the adrenodoxin gene revealed that each mRNA contains sequences derived from a different exon encoding the mitochondrial leader sequence. To define the sequences necessary for the synthesis of these two types of mRNA and to determine if the synthesis of each is driven by a separate promoter, 5'-regions of the adrenodoxin gene were inserted upstream from two different reporter genes and tested for promoter/enhancer regulatory activity by transient transfection into mouse adrenocortical Y1 tumor cells. The results clearly demonstrated that the bovine adrenodoxin gene contains two functional promoters; one, ADXP1, located in the 5'-flanking region gives rise to the minor form of mRNA, and a second, stronger promoter, ADXP2, which maps within intron 1 gives rise to the major form of mRNA. Unique cAMP-responsive sequences were found upstream from each promoter which share no sequence homology to the consensus CRE (cAMP-responsive element). Upon transient expression, the cAMP-responsive sequence associated with the ADXP2 promoter, termed CRS2, confers the cAMP responsiveness to stimulate the transcription of the linked beta-globin reporter gene regardless of whether the adrenodoxin ADXP2 promoter or the beta-globin promoter was utilized.(ABSTRACT TRUNCATED AT 250 WORDS)