Discrimination among potential activators of the beta-globin CACCC element by correlation of binding and transcriptional properties.

Abstract

Adult K-globin-like promoters contain a cis-acting element, CCACACCC, that is conserved across species and is required for wild-type levels of transcription. We have studied the contribution of this element and proteins that interact with it to activate 13-globin transcription. We found that an erythroid-like cell line, MEL, contains several proteins that specifically bind the CACCC element. By comparing the DNA-binding properties of promoters with mutations in the CACCC element with the transcriptional activities of these mutant promoters, we found that two CACCC-binding proteins did not bind to mutant promoters that direct decreased levels of transcription. One of these proteins is the transcriptional activator Spl, and the other we have designated CACD (CACCC-binding species D). We subjected CACD to a binding site selection procedure and obtained high-affinity CACD binding sites that are identical to that of the 13-globin CACCC element. This result, combined with our finding that CACD binds the CACCC element with a higher affinity than does Spl, argues that the CACCC element is a target of CACD rather than Spl. The strategy of correlating the results of a binding site selection experiment with those of in vivo expression and in vitro binding studies may allow evaluation of the relative potential of different proteins to activate transcription through a single cis-acting site.The adult P-globin-like promoters contain several ele-

The GATA-4 transcription factor transactivates the cardiac muscle-specific troponin C promoter-enhancer in nonmuscle cells.

“…For example, GATA-1 functions in conjunction with NF-E2 in regulating the transcription of the globin locus control region (1,3,55,79). In addition, the cx-and ,-globin promoters contain binding sites for both GATA and CACCC box-binding factors (21,57). In this light, it is of interest that the cTnC promoter-enhancer contains at least three additional nuclear protein-binding sites, each of which has been shown to be important for the activity of this enhancer in cardiac myocytes (54).…”

Positive- and Negative-acting Krüppel-like Transcription Factors Bind a Transforming Growth Factor β Control Element Required for Expression of the Smooth Muscle Cell Differentiation Marker SM22α in Vivo

“…GKLF is a member of an expanding subfamily of Krü ppellike factors that includes erythroid Krü ppel-like factor (44), basic Krü ppel-like factor (45), LKLF (49), and BTEB factors, all of which share extensive homology in their carboxyl-terminal DNA-binding domains and the ability to bind to a CACCC motif (29,43,45,46). Indeed, several of our observations indicate that removal of GKLF repressor function alone is not sufficient for TGF-␤-dependent increases in expression of SM22␣ or ␣-SM actin.…”

Context-dependent EKLF responsiveness defines the developmental specificity of the human ɛ-globin gene in erythroid cells of YAC transgenic mice

“…Several proteins interact with CAC sequences in vitro (Hartzog and Myers 1993). These include the ubiquitous transcription factor Sp1 (Gumucio et al 1991;Yu et al 1991;Hartzog and Myers 1993) and the erythroid Krü ppel-like factor (EKLF; Miller and Bieker 1993). EKLF shows extensive homology to Sp1 and binds to the proximal CAC box in the adult ␤-globin gene promoter with high affinity and can activate ␤-globin transcription in vitro (Miller and Bieker 1993;Feng et al 1994).…”

“…The critical nature of this element to ␤-globin transcription was discovered many years ago, when specific human ␤-thalassemias were shown to be associated with mutations in the proximal CAC box of the adult gene (Orkin et al 1982(Orkin et al , 1984. Several proteins interact with CAC sequences in vitro (Hartzog and Myers 1993). These include the ubiquitous transcription factor Sp1 (Gumucio et al 1991;Yu et al 1991;Hartzog and Myers 1993) and the erythroid Krü ppel-like factor (EKLF; Miller and Bieker 1993).…”