One of the earliest cellular responses to growth factors is the rapid induction of primary response genes. One group of such genes was originally isolated as tetradecanoyl phorbol acetate (TPA) inducible sequences (TIS genes) from mouse 3T3 cells. Proteins encoded by the TIS genes include two transcription factors: TIS8 (also known as egr1/NGFIA/zif268) and TIS1 (also known as NGFIB/nur77/N10). We have examined the inducibility of these two genes in a skeletal muscle cell line in response to agents that have been reported to block muscle differentiation. We report here that basic fibroblast growth factor (bFGF) induced the expression of both TIS1 and TIS8 in mouse C2C12cells. Both genes were also inducible by TPA while forskolin which activates the cAMP-dependent pathway induced TIS1 but not TIS8. Down-regulation of protein kinase C (PKC) activity by TPA pretreatment repressed the bFGF induction of TIS1 but had little effect on the bFGF-stimulated expression of TIS8. Moreover, while both TPA and bFGF stimulated the hyperphosphorylation of c-RAF and the activity of MAP kinase, TPA pretreatment failed to block RAF phosphorylation or the stimulation of MAP kinase activity by bFGF. Induction of the two TIS genes in skeletal myoblasts therefore appeared to be dependent to different extents on the activation of protein kinase A (PKA), PKC and MAP kinase.

In Xenopus laevis, transcription of the gamma-fibrinogen subunit gene is activated by glucocorticoids. Hormone induction is regulated by three glucocorticoid response element (GRE) half-sites and an additional DNA sequence which binds a novel hepatocyte nuclear protein, Xenopus glucocorticoid receptor accessory factor (XGRAF). The XGRAF binding site (GAGTTAA) is located directly upstream of the most distal half-GRE. The proximity of the binding sites for XGRAF and the glucocorticoid receptor (GR) led to the hypothesis that these two sites form a glucocorticoid response unit (GRU). By transfecting DNA into primary hepatocytes, we showed that this GRU confers hormone responsiveness in the absence of other half-GREs. The XGRAF binding site enhances function of the half-GRE without itself responding to glucocorticoids. The GRU retains efficacy in other locations relative to the gamma-fibrinogen gene promoter, further increases transcription when present in multiple copies, and activates a heterologous promoter. Despite the contiguity of the XGRAF binding site and half-GRE, the two sites can be occupied simultaneously in vitro. The binding characteristics correlate with function since mutations that disrupt concurrent XGRAF and GR binding also impair transcription. This novel GRU represents a new regulatory mechanism that may be applicable to other glucocorticoid responsive genes that lack a full GRE.

In addition to the glucocorticoid receptor, DNA-binding proteins called accessory factors play a role in hormone activation of many glucocorticoid-responsive genes. Hormonal regulation of the ␥-fibrinogen subunit gene from the frog Xenopus laevis requires a novel DNA sequence that binds a liver nuclear protein called Xenopus glucocorticoid receptor accessory factor (XGRAF). Here we demonstrate that the recognition site for XGRAF encompasses GAGTTAA at positions ؊175 to ؊169 relative to the start site of transcription. This sequence is not closely related to the binding sites for known transcription factors. The two guanosines make close contact with XGRAF, as shown by the methylation interference assay. Single-point mutagenesis of every nucleotide in the 9-base pair region from positions ؊177 to ؊169 showed an excellent correlation between ability to bind XGRAF in vitro and ability to amplify hormoneinduced transcription from DNA transfected into Xenopus primary hepatocytes. Conversely, XGRAF had little or no effect on basal transcription of the ␥-fibrinogen gene. Maximal hormonal induction also requires three half-glucocorticoid response elements (half-GREs) homologous to the downstream half of the consensus GRE. Interestingly, the XGRAF-binding site is immediately adjacent to the most important half-GRE. This close proximity suggests a new mechanism for activation of a gene lacking a conventional full GRE.Steroid hormones, which include glucocorticoids and mineralocorticoids from the adrenal cortex and estrogens, progestins, and androgens from the gonads, regulate a vast array of physiological processes that are essential for development, differentiation, growth, metabolism, homeostasis, behavior, and reproduction in vertebrate organisms. In the classical model of steroid hormone action (1), the steroid ligands bind to specific intracellular protein receptors in target cells. The hormonereceptor complexes interact with particular short nucleotide sequences in the chromosomal DNA and modulate transcription of nearby genes. This model, however, cannot account fully for the complex tissue-specific and gene-specific actions of hormones. Transcriptional induction by steroids is influenced by many factors, such as local chromatin structure (2), stages of the cell cycle (3), cellular morphology or differentiation state (4, 5), specific hormone ligand (6), and physiological state (7). Differential hormone responsiveness depends in part on the availability of other transcriptional regulatory proteins including coactivators and corepressors, which do not themselves bind to DNA (8), and accessory factors, which are DNA-binding proteins (9). For glucocorticoid-regulated genes, several accessory factors have been identified (2, 10 -17), but the mechanisms by which these proteins potentiate hormonal activation of transcription are not known.To understand the role of accessory DNA-binding proteins in determining responsiveness to a steroid hormone signal, we are investigating glucocorticoid induction of fibrinogen gene expre...

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