The dependence of the muscle‐specific enhancer of the acetylcholine receptor alpha‐subunit gene on other domains of the promoter has been analysed by performing point mutagenesis and modular reconstitution of the enhancer‐‐promoter sequences. The enhancer is inactive in the absence of the proximal region containing an Sp1 binding site and an overlapping G‐C homopolymer binding factor site (referred to as GBF). The proximal region can be replaced by an Sp1 binding site from SV40 or an MEF‐2 binding site from the muscle creatine kinase gene. Specific mutation of the Sp1 site markedly affects transactivation by CMD1 or myogenin. Mutation of the GBF binding site leads to higher promoter activity in primary cultures of chick myotubes or in quail fibroblasts. In addition, binding of a purified Sp1 protein prevents the binding of GBF in vitro. It is proposed that in the case of the alpha‐subunit promoter, the myogenic factors activate transcription in cooperation with Sp1, and that GBF contributes to muscle‐specific expression of the promoter by interfering with Sp1 binding in nonmuscle muscle cells or myoblasts.

We have analyzed the potential role of myogenin in the regulation by electrical activity of the expression of the acetylcholine receptor (AChR) alpha-subunit gene in cultured chick embryonic myotubes. The state of phosphorylation of myogenin was followed by 32P-labeling and immunoprecipitation with an anti-myogenin antibody. In electrically active myotubes myogenin is phosphorylated, while it is dephosphorylated in electrically silent myotubes following tetrodotoxin (TTX) treatment. Accordingly, nuclear protein kinase C (PKC) activity decreases in TTX-treated myotubes. Myogenin dephosphorylation is also observed upon incubation of myotubes with GF109203X, a pharmacological agent which specifically inhibits PKC activity. Both treatments cause similar increases in the expression of the AChR protein. The effects are not additive. Thus TTX and GF109203X most probably affect a common process. Recombinant chick myogenin binds to myogenic sites (E boxes) present in the AChR alpha-subunit promoter but loses this binding capacity after phosphorylation. As a working hypothesis we propose that repression of AChR biosynthesis by electrical activity results, at least partly, from phosphorylation of myogenin via the PKC pathway.

The proximal promoter region of the human transferrin gene contains an hepatocyte-specific cis-element (PRI, nucleotides -76 to -51) whose DNA sequence is homologous to a sequence (nucleotides -89 to -68) present in the transcriptionally essential 5' region of the human antithrombin III gene and to another hepatocyte-specific sequence (A domain) of the human alpha 1-antitrypsin gene promoter. The results reported here lead to the conclusion that the liver trans-acting factor Tf-LF1, binding to the transferrin PRI cis-element interacts with the homologous antithrombin III region, but is different from the transcription factor LF-A1 interacting with the A domain of the alpha 1-antitrypsin promoter. The distal region DRI (nucleotides -480 to -454) of the human transferrin gene promoter presents in its core the same 10 nucleotide-long sequence as the PRI cis-element. We have previously shown that the liver protein Tf-LF2, binding to the DRI element is different from the Tf-LF1 trans-acting factor. In this paper we also show that Tf-LF2 is different from the transcription factor LF-A1 interacting with the alpha 1-antitrypsin promoter. The results allow us to conclude that at least three distinct liver nuclear proteins bind to different subsets of 5' DNA regions containing similar sequences. These sequences are present in genes expressed essentially in liver.

We have studied the liver-specific transcriptional activity of the human transferrin gene promoter. Results of competition experiments, site-directed mutagenesis, and 5' deletion analysis have demonstrated that a TATA box and a binding site for the liver-specific protein Tf-LF1 are the only elements needed to direct hepatic-specific transcription in vitro. Thus, Tf-LF1 behaves as other previously described proteins, HNF-1, DBP and LF-A1, in that it is sufficient to confer liver-specific transcriptional activity to a promoter in vitro. This results contrast with observations made in transient expression experiments, in which Tf-LF1 binding alone cannot direct hepatic-specific expression, and the binding of at least one more protein, similar to C/EBP, is needed. Thus, as described for other hepatic genes, the number of elements necessary to confer tissue specificity is different in vivo and in vitro.