Identification of two distinct nuclear factors with DNA binding activity within the glucocorticoid regulatory region of the rat alpha-1-acid glycoprotein promoter

Efficient glucocorticoid induction of alpha-1-acid glycoprotein (AGP) mRNA in rat hepatoma cells HTC (JZ-1) requires the activity of one or more preexisting and labile proteins acting cooperatively with the glucocorticoid receptor. Inhibiting protein synthesis markedly diminishes the glucocorticoid induction of rat AGP mRNA without affecting the inducibility of other glucocorticoid inducible genes such as the mouse mammary tumor virus (MMTV) or tyrosine amino transferase (TAT). The sequences responsible for conferring glucocorticoid inducibility to the rat AGP gene have localized on the AGP promoter between nucleotides -121 and -42. A typical glucocorticoid regulatory element (GRE) is found between residues -121 and -105 and downstream of this are the sequences (-90 to -42) responsible for the cycloheximide inhibition of the hormonal induction (10). Using mobility shift assays we have characterized the binding of two proteins or complexes of proteins to this promoter region (-90 to -64). Our data show that the binding of these factors (called ANF-1 and ANF-2) to the DNA is highly specific, and is not directly affected by cycloheximide. Furthermore a second binding site for ANF-2 has been localized in the AGP regulatory region to a sequence that overlaps the GRE.     

A cAMP-responsive element regulates expression of the mouse steroid 11 beta-hydroxylase gene

In Y1 mouse adrenocortical tumor cells, expression of steroid 11 beta-hydroxylase (11 beta-OHase) is stimulated by cAMP following a delay of 4-6 h. Our results demonstrate that a cAMP-responsive element (CRE) within the 11 beta-OHase promoter region is a major determinant of this induction. The 5'-flanking sequences from the mouse 11 beta-OHase gene were placed in front of a human growth hormone reporter gene and transfected into Y1 cells. Treatment of transfected cells with 8-bromo-cAMP increased expression directed by the 11 beta-OHase 5'-flanking region by 3.8-fold. In 5'-deletion analyses, 123 base pairs of 5'-flanking sequences were sufficient for cAMP induction, whereas cAMP treatment did not affect expression of a plasmid with only 40 base pairs of 5'-flanking sequence. Within these 123 base pairs, a region from -56 to -49 matched 7 of 8 bases comprising the consensus sequence for the CRE. 11 beta-OHase 5'-flanking sequences from -65 to -42, including the CRE-like sequence, conferred cAMP inducibility to promoters from the thymidine kinase and chorionic gonadotropin alpha-subunit genes. DNase I footprinting and Southwestern blotting analyses demonstrated that the protein which interacted with the CRE in the 11 beta-OHase promoter region was similar to the CRE-binding protein associated with other cAMP-regulated genes. Together, these results suggest that an interaction between the 11 beta-OHase CRE and CRE-binding protein mediates cAMP induction of the 11 beta-OHase gene.     

Activation of a heterologous promoter in response to dexamethasone and cadmium by metallothionein gene 5'-flanking DNA

Human metallothionein-IIA (hMT-IIA) gene expression is regulated by heavy metals and glucocorticoids. When the cloned hMT-IIA gene or its 5'-flanking DNA structure fused to herpes simplex virus thymidine kinase (HSV-TK) structural gene sequences were transferred into TK- Rat 2 fibroblasts, both genes were inducible by Cd++ and/or dexamethasone. Placement of the hMT-IIA gene 5'-flanking region, either intact of deleted in its TATA box and cap site, upstream of the HSV-TK gene promoter rendered the latter both glucocorticoid- and heavy metal-inducible. Thus the structure that mediates both Cd++ and glucocorticoid responsiveness is present in the hMT-IIA gene 5'-flanking DNA, does not require its TATA box or cap site, and can activate a heterologous promoter.     

A conserved AU sequence from the 3' untranslated region of GM-CSF mRNA mediates selective mRNA degradation

The mRNAs of transiently expressed genes frequently contain an AU-rich sequence in the 3' untranslated region. We introduced a 51 nucleotide AT sequence from a human lymphokine gene, GM-CSF, into the 3' untranslated region of the rabbit beta-globin gene. Our experiments demonstrate that this caused the otherwise stable beta-globin mRNA to become highly unstable in vivo. The instability conferred by the AU sequence in the mRNA was partially alleviated by treatment of the cells with cycloheximide. We propose that the AU sequences are the recognition signal for an mRNA processing pathway which specifically degrades the mRNAs for certain lymphokines, cytokines, and proto-oncogenes.