Enhancer and promoter elements directing activation and glucocorticoid repression of the alpha 1-fetoprotein gene in hepatocytes.

Mutations were introduced in 7 kilobases of 5'-flanking rat alpha 1-fetoprotein (AFP) genomic DNA, linked to the chloramphenicol acetyltransferase gene. AFP promoter activity and its repression by a glucocorticoid hormone were assessed by stable and transient expression assays. Stable transfection assays were more sensitive and accurate than transient expression assays in a Morris 7777 rat hepatoma recipient (Hepa7.6), selected for its strong AFP repression by dexamethasone. The segment of DNA encompassing a hepatocyte-constitutive chromatin DNase I-hypersensitive site at -3.7 kilobases and a liver developmental stage-specific site at -2.5 kilobases contains interacting enhancer elements sufficient for high AFP promoter activity in Hepa7.6 or HepG2 cells. Deletions and point mutations define an upstream promoter domain of AFP gene activation, operating with at least three distinct promoter-activating elements, PEI at -65 base pairs, PEII at -120 base pairs, and DE at -160 base pairs. PEI and PEII share homologies with albumin promoter sequences, PEII is a near-consensus nuclear factor I recognition sequence, and DE overlaps a glucocorticoid receptor recognition sequence. An element conferring glucocorticoid repression of AFP gene activity is located in the upstream AFP promoter domain. Receptor-binding assays indicate that this element is the glucocorticoid receptor recognition sequence which overlaps with promoter-activating element DE.     

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.     

Two elements of the rat prolactin 5' flanking region are required for its regulation by estrogen and glucocorticoids

The 5'-flanking region of the rat prolactin gene contains two DNase I-hypersensitive (HS) sites. We used gene transfer experiments to determine the nucleotide (nt) sequences within and around these two HS sites that may contain the information necessary for regulation of prolactin gene expression by estrogens and glucocorticoids. A chimeric gene construct (pPRL.CAT) was prepared by covalently linking the sequence of the rat prolactin gene to the bacterial chloramphenicol acetyltransferase-coding gene, cat. Rat GH3 cells were transfected with pPRL.CAT and six mutants that possess deletions within and around the two HS sites. Incubation of such transfectants with estrogen or dexamethasone indicated the existence of two functionally important elements within the 5'-flanking region of the rat prolactin gene. The element required for estrogen up-regulation of the prolactin gene is located between nt residues -1530 through -1950. The glucocorticoid down-regulatory element is located between nt -200 and +75.     

Glucocorticoid receptor binding and activation of a heterologous promoter by dexamethasone by the first intron of the human growth hormone gene.

In this study DNA-binding and gene transfer experiments were performed to examine a potential glucocorticoid regulatory element (GRE) in the human growth hormone gene. As assayed by nitrocellulose filter binding, only two regions of the human growth hormone gene, the 5'-flanking sequences and a fragment containing part of the first intron, were retained preferentially by purified glucocorticoid-receptor complexes. The relative binding by the transcribed sequences was three times greater than the relative binding by the 5'-flanking sequences, but less than the relative binding by a fragment containing the human metallothionein-IIA gene GRE. The intron, but not the 5'-flanking sequences, generated a "footprint" when the receptor complex was used to protect the segments against exonuclease III digestion; the protected sequence spanned nucleotides +86 to +115 in the first intron and contained a structure homologous in 14 of 16 nucleotides to a 16-nucleotide consensus GRE. The hexanucleotide 5'-TGTCCT-3', thought to be important for GRE activity, not only was found in this sequence and in the 5'-flanking region, but also was present twice in the 3' end of the gene that did not show specific receptor binding. The latter results suggest that the hexanucleotide alone is not sufficient to generate specific receptor binding tight enough to be assayed in this way. To test the biological activity of the intron binding site, a fragment containing these sequences was fused 5' to the human metallothionein-IIA gene promoter depleted of its GRE and linked to the structural sequences of the herpes simplex virus thymidine kinase (TK) gene. When this hybrid gene was transfected into Rat 2 TK- cells, its expression was induced threefold by the glucocorticoid dexamethasone, as assessed by transfection efficiency and RNA blotting analyses. Expression of the same gene without the human growth hormone gene segment was not affected by the steroid, whereas the wild-type human metallothionein-IIA gene promoter containing its GRE responded to the hormone by a sixfold increase in thymidine kinase mRNA. These results indicate that the human growth hormone gene contains a structure within its first intron that can function as a GRE.     

Introduction of the glucocorticoid binding sequences into the expression vector p delta SVE-CAT and its effect on the CAT gene expression in mammalian cells.

We have demonstrated that synthetic oligonucleotide representing glucocorticoid responsive element (GRE I) of MMTV inserted into the enhancerless early promoter of SV40 in p delta SVE-CAT expression vector, enhances transient expression of chloramphenicol acetyltransferase gene in HeLa and hepatoma cells cultivated in the presence of dexamethasone. The following changes in the structure of the core sequences (GTTACAAACTGTTCT) of the synthesized GRE eliminated its enhancing ability: i, changes in the left end of the core sequences from GTTACAAAATGTTCT to TCTTCAAACTGTTCT or to TACTCAAACTGTTCT; ii, the increase of gap between TGTTCT and the inverted repeat of this sequence. The above changes did not eliminate specific binding of glucocorticoid receptor to the synthetic oligonucleotides studied.