Characterization of the major regulatory element upstream of the human alpha-globin gene cluster.

The major positive regulatory activity of the human alpha-globin gene complex has been localized to an element associated with a strong erythroid-specific DNase I hypersensitive site (HS -40) located 40 kb upstream of the zeta 2-globin mRNA cap site. Footprint and gel shift analyses of the element have demonstrated the presence of four binding sites for the nuclear factor GATA-1 and two sites corresponding to the AP-1 consensus binding sequence. This region resembles one of the major elements of the beta-globin locus control region in its constitution and characteristics; this together with evidence from expression studies suggests that HS -40 is a primary element controlling alpha-globin gene expression.     

Nuclear factor EF-1A binds to the adenovirus E1A core enhancer element and to other transcriptional control regions.

We have identified a cellular enhancer-binding protein, present in nuclear extracts prepared from human and rodent cells, that binds to the adenovirus E1A enhancer element I sequence. The factor has been termed EF-1A, for enhancer-binding factor to the E1A core motif. EF-1A was found to bind to two adjacent, related sequence motifs in the E1A enhancer region (termed sites A and B). The binding of EF-1A to these adjacent sites, or to synthetic dimerized sites of either motif, was cooperative. The cooperative binding of EF-1A to these sites was not subject to strict spacing constraints. EF-1A also bound to related sequences upstream of the E1A enhancer region and in the polyomavirus and adenovirus E4 enhancer regions. The EF-1A-binding region in the E1A enhancer stimulated expression of a linked gene in human 293 cells when multimerized. Based on the contact sites for EF-1A binding determined by chemical interference assays, this protein appears to be distinct from any previously characterized nuclear binding protein.     

Specificity of nuclear protein binding to a CYP1A1 negative regulatory element

Primary cultures of rat epidermal keratinocytes lose the ability to respond to chemicals with the induction of CYP1A1 gene expression after approximately 15 passages. This repression is mediated by a CT-rich direct repeat negative regulatory DNA (NeRD) element present in the upstream regulatory region of the CYP1A1 gene. Competitive gel retardation analysis using keratinocyte nuclear extracts and mutant NeRD oligonucleotides revealed the presence of two specific protein-NeRD complexes and revealed the specific nucleotides important for the formation of each complex. These studies demonstrate that these two factors bind to overlapping sites within the NeRD element. Nucleotide specificity of complex A formation is similar to that of previously identified nuclear silencing factors, while that of complex B appears to represent a unique CT-rich binding factor. These results suggest that repression of CYP1A1 gene expression in high passage keratinocytes may involve the interplay between at least two specific NeRD binding factors.     

Identification of the binding sites for potential regulatory proteins in the upstream enhancer element of the Drosophila fushi tarazu gene.

With a view to identifying proteins that regulate the expression of the Drosophila ftz gene we have sequenced its enhancer-like upstream element (USE) and determined the binding sites for embryonic nuclear proteins within this region by in vitro DNAaseI footprinting. We find that greater than 50% of this element is bound by nuclear protein. By footprinting and gel-retardation studies in embryonic extracts from different developmental stages, we have characterised a number of USE/protein complexes whose nature alters in concert with changes in the ftz expression pattern, suggesting that these USE-binding proteins may be involved in the regulation of gene activity. In some cases this suggestion is substantiated by the observation that the protected DNA sequences show homology to the binding sites for ftz regulating DNA-binding proteins such as the pair-rule gene product even-skipped.