Identification of two nuclear factor-binding domains on the chicken cardiac actin promoter: implications for regulation of the gene.

The cis-acting regions that appear to be involved in negative regulation of the chicken alpha-cardiac actin promoter both in vivo and in vitro have been identified. A nuclear factor(s) binding to the proximal region mapped over the TATA element between nucleotides -50 and -25. In the distal region, binding spanned nucleotides -136 to -112, a region that included a second CArG box (CArG2) 5' to the more familiar CCAAT-box (CArG1) consensus sequence. Nuclear factors binding to these different domains were found in both muscle and nonmuscle preparations but were detectable at considerably lower levels in tissues expressing the alpha-cardiac actin gene. In contrast, concentrations of the beta-actin CCAAT-box binding activity were similar in all extracts tested. The role of these factor-binding domains on the activity of the cardiac actin promoter in vivo and in vitro and the prevalence of the binding factors in nonmuscle extracts are consistent with the idea that these binding domains and their associated factors are involved in the tissue-restricted expression of cardiac actin through both positive and negative regulatory mechanisms. In the absence of negative regulatory factors, these same binding domains act synergistically, via other factors, to activate the cardiac actin promoter during myogenesis.

A 40-base-pair sequence in the 3' end of the beta-actin gene regulates beta-actin mRNA transcription during myogenesis.

In an earlier report, evidence was presented that the down-regulation of beta-actin mRNA during myogenesis was controlled by a region 3' to the promoter of the gene. In this paper we report the location of this regulatory sequence, determined by deletion analysis and the use of chimeric genes, transfected stably into the mouse myogenic cell line C2C12. The domain responsible for the reduction in beta-actin mRNA levels is at most 40 base pairs long and is located just 5' to the canonical polyadenylylation signal in the gene. Placement of this sequence in the corresponding 3' position both in the alpha-cardiac-actin gene and in the neomycin-resistance gene in pSV2-neo confers the beta-actin mRNA regulatory pattern when these constructs are stably introduced into C2C12 cells. Nuclear run-on experiments indicate that transcriptional control can account for the decrease observed in beta-actin mRNA levels during myogenesis for both the endogenous as well as the transfected beta-actin gene constructs. This 3' transcriptional control sequence is conserved in all of the vertebrate beta-actin genes sequenced and is not similar to any of the 3' processing-adenylylation or termination sequences described previously. This mode of gene regulation may reflect a more general mechanism involved in the process of gene suppression during development.

The beta actin promoter. High levels of transcription depend upon a CCAAT binding factor.

Although beta actin mRNA is down-regulated during myogenesis, the beta actin promoter confers constitutive expression when joined to heterologous genes transfected into a variety of different cell backgrounds, including differentiated muscle. Normal promoter activity is dependent upon the binding of a ubiquitous factor to the CCAAT-box element. Loss or reduction in factor binding correlates with a major reduction in promoter activity both in vivo and in vitro. The binding domain covers approximately 23 base pairs as determined by DNase footprinting. Methylation of A and G residues in and adjacent to the CCAAT box results in the loss of factor binding. Mutations across the binding domain indicate that the sequence GCCAATCAG within the domain is sufficient as a recognition sequence for factor binding. This binding is not competed by the alpha cardiac actin CCAAT sequence. Bandshift experiments demonstrate a predominant single band of similar mobility in nuclear extracts from various cells and tissues, with the exception of HeLa cells. The prevalence of the factor and its recognition sequence in a variety of promoters suggests that this factor has a common role in the transcriptional activation of several eukaryotic promoters.