The mouse elongation factor-2 gene: isolation and characterization of the promoter

Elongation factor 2 (EF-2) is a protein involved in peptide chain elongation in eukaryotes. We isolated the mouse EF-2 gene and characterized its promoter. We showed that the majority of enhancer elements were located within 500 bp of the flanking sequence and identified a factor binding site sequence (CGTCACGTGACGC) located between nucleotides -58 and -47 containing two CGTCA motifs separated by two nucleotides. The motif represents a half-site for binding of the cAMP response element (CRE) binding protein (CREB). Mutation analysis indicated that the presence of one CGTCA site alone conferred cAMP inducibility, but the presence of one or two CGTCA sites and spacing nucleotides elicited cAMP-independent, constitutive expression. UV cross-linking and DNA affinity chromatography revealed that three 40-, 43-, and 65-kD proteins bound to the CRE-like element. Of these, the 65-kD protein was unique to the CRE-like element. The 40-kD protein was ATF1 and the 43-kD protein with the molecular size of CREB was not CREB, on the basis of reactivity to their respective antibodies. Because ATF1 responds poorly to cAMP induction, it is likely the contributor to the constitutive expression rather than inductive expression of the CRE-like element, and, thus, the EF-2 gene.     

C/EBP, c-Myb, and PU.1 cooperate to regulate the neutrophil elastase promoter

The murine neutrophil elastase (NE) gene is expressed specifically in immature myeloid cells. A 91-bp NE promoter region contains three cis elements which are conserved evolutionarily and are essential for activation of the promoter in differentiating 32D cl3 myeloid cells. These elements bound c-Myb (at -49), C/EBPalpha (at -57), and PU.1 (at -82). In NIH 3T3 cells, the NE promoter was activated by c-Myb, C/EBPalpha, and PU.1, via their respective binding sites. Cooperative activation was seen by any combination of c-Myb, C/EBPalpha, and PU.1, including all three together, again via their DNA-binding sites. In CV-1 cells, but not in NIH 3T3 cells, cooperation between Myb and C/EBPalpha depended on the integrity of the PU.1-binding site. In addition to C/EBPalpha, C/EBPdelta strongly activated the NE promoter, alone or with c-Myb, but C/EBPbeta was less active. Either of C/EBPalpha's two transactivation domains cooperatively activated the promoter with c-Myb, in both NIH 3T3 and 32D c13 cells. Synergistic binding to DNA in a gel shift assay between C/EBPalpha, c-Myb, and PU.1 could not be demonstrated. Also, separation of the C/EBP- and c-Myb-binding sites by 5 or 10 bp did not prevent cooperativity. These results suggest that a coactivator protein mediates cooperative activation of the NE promoter by a C/EBP and c-Myb. These factors, together with PU.1, direct restricted expression of the NE promoter to immature myeloid cells.