Transcriptional regulation of alpha-fetoprotein expression by dexamethasone in human hepatoma cells

The level of alpha-fetoprotein (AFP) mRNA in HuH-7 human hepatoma cells is elevated by the addition of dexamethasone to the culture medium. To locate the DNA region involved in hormonal regulation of the AFP gene, we constructed recombinant plasmids in which various lengths of the 5'-flanking sequence of the human AFP gene were fused to the CAT gene. Various cell lines were transfected with the recombinant plasmids, incubated with or without 3 x 10(-6) M dexamethasone, and then assayed for chloramphenicol acetyltransferase expression. In hepatoma cells that produce AFP, the dexamethasone treatment resulted in the stimulated chloramphenicol acetyltransferase expression when the transfected plasmids contained 169 base pairs (bp) or longer AFP 5'-flanking sequence. No dexamethasone effect was observed when the 5'-flanking sequence was less than 98 bp long. The dexamethasone stimulation was effectively suppressed by the glucocorticoid antagonist RU486, indicating that this effect is mediated by glucocorticoid receptors. The 71-bp region between positions -169 and -98 contains a nucleotide stretch which is similar to the consensus sequence of the glucocorticoid responsive element (GRE). Partial alterations of this sequence resulted in decreased dexamethasone response. The GRE-containing region stimulated heterologous (SV40) promoter activity in response to dexamethasone treatment in an orientation- and position-independent manner. The GRE and the upstream AFP enhancer function independently from each other.     

Effect of flanking regions on the expression of the human interleukin-2 chromosomal gene in a murine myeloma cell line]

A human interleukin-2 (IL-2) gene was isolated from genomic DNA library. The isolated gene with 5'- and 3'-flanking sequences of various lengths was inserted into plasmids derived from the retroviral vector pPSneo. The recombinant plasmids were transfected into myeloma X63Ag8-653 cells. The transfected cells, harbouring the IL-2 gene with the shortened (to position -165) or totally deleted 5'-flanking sequence, constitutively expressed biologically active IL-2. Deletion of 3'-flanking region on did not affect the IL-2 expression.     

Interleukin-6 induction by tumor necrosis factor and interleukin-1 in human fibroblasts involves activation of a nuclear factor binding to a kappa B-like sequence.

Using variable-length deletion constructs of the 5'-flanking region of the human interleukin-6 (IL-6) gene linked to the chloramphenicol acetyltransferase gene, we showed that the region from positions -109 to -50 mediated the bulk of the response to tumor necrosis factor (TNF) or interleukin-1 (IL-1), while it was less responsive to forskolin. DNA mobility shift assays and DNase I footprinting analysis identified a nuclear protein from TNF- or IL-1-treated fibroblasts that bound to a region comprising a kappa B-like element located between positions -72 and -63 on the IL-6 gene. On the basis of these and other experiments, we conclude that TNF and IL-1 apparently activate IL-6 gene expression by closely related mechanisms involving activation of a NF-kappa B-like factor, whereas the pathway of IL-6 induction by forskolin is, in part, different.     

Analysis of the promoter region of the human FcRn gene

The 5'-flanking region of the human FcRn alpha-chain gene was analyzed for its ability to directly express the chloramphenicol acetyltransferase (CAT) reporter gene in NIH3T3 and Lu106 cells. Transient transfection of the CAT constructs revealed that there was promoter activity in the region -660 to +300 of the 5'-flanking sequence. Electrophoretic mobility-shift assays showed that there are functional binding sites for Sp1 or Sp1-like factors, AP1 or a related factor, and additional unidentified proteins in the promoter region.     

Functional serum response elements upstream of the growth factor-inducible gene zif268.

The zif268 gene, which encodes a protein with three typical zinc finger sequences, is induced in mouse 3T3 cells by serum, phorbol 12-myristate 13-acetate platelet-derived growth factor, and fibroblast growth factor. The induction is coordinate with that of c-fos. The 5'-flanking region of zif268 contains sequences that resemble known regulatory elements, including four CC(A or T)6GG sequences similar to the core serum response elements (SREs) found upstream of c-fos and actin genes. To determine whether the zif268 SRE-like elements mediate induction, CAT (chloramphenicol acetyltransferase) plasmids with different lengths of zif268 upstream sequences were tested for inducibility in 3T3 cells by serum, platelet-derived growth factor, or phorbol 12-myristate 13-acetate. In addition, double-stranded oligonucleotides corresponding to each of the four zif268 putative SREs were tested individually for responsiveness when placed upstream of a thymidine kinase gene promoter. Each of the four SREs conferred inducibility by the agents tested, and multiple SREs resulted in greater inducibility than did a single element. Each of the zif268 SREs also competed with the c-fos SRE for binding by serum response factor present in HeLa cell nuclear extract. We conclude that the zif268 SRE-like sequences are functional and probably account for the coordinate induction of zif268 and c-fos.