Activity of the rat liver-specific aldolase B promoter is restrained by HNF3.

Although it contains binding sites for HNF1, NFY and C/EBP/DBP, the proximal promoter of the aldolase B gene is surprisingly weak when tested by transient transfection in differentiated hepatoma cells. This low activity could be due to overlapping between HNF1 and HNF3 binding sites in element PAB, from -127 to -103 bp with respect to the cap site. Replacement of the PAB region by a consensus HNF1 binding site unable to bind HNF3, results in a 30 fold activation of the promoter, in accordance with the hypothesis that activity of the wild-type promoter is normally restrained by HNF3 binding to PAB competitively with HNF1. Consistently, transactivation of the wild-type promoter by excess HNF1 is very high, most likely due to the displacement of HNF3, while the construct with the exclusive HNF1 binding site is weakly transactivated by HNF1. The inhibitory effect of HNF3 on HNF1-dependent transactivation is clearly due to competition between these two factors for binding to mutually exclusive, overlapping sites; indeed, when HNF1 and HNF3 sites are contiguous and not overlapping, the resulting promoter is as active as the one containing an exclusive HNF1 binding site. A construct in which PAB has been replaced by an exclusive HNF3 binding site is weakly expressed and is insensitive to HNF3 hyperexpression. DBP-dependent transactivation, finally, is independent of the nature of the element present in the PAB region.     

Expression of the rat aldolase B gene: a liver-specific proximal promoter and an intronic activator

The nature and location of the cis-acting DNA sequences regulating expression of the rat aldolase B gene has been investigated. Two liver-specific DNAse I hypersensitive sites were detected, one located just upstream from the cap site, the second in the middle of the first, 4.8-kbp-long, intron. A fragment of 190 bp 5' to the cap site behaved as a tissue-specific but weak core promoter: it directed a detectable reporter gene expression in the Hep G2 cells and hepatocytes, but not in fibroblasts. The tissue-specific expression was stimulated at least 16 fold when constructs contained the entire first intron. The intronic activating sequences could be ascribed to an inner 2 kbp fragment in which the downstream liver-specific DNAse I hypersensitive site was located.     

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.     

Construction of rat aldolase C expression plasmid and the hybrid formation between rat aldolase C and human aldolase A or B co-expressed in Escherichia coli

Rat aldolase C cDNA was inserted in an Escherichia coli expression vector to construct the rat aldolase C expression plasmid, pRAC42. This plasmid produces active rat aldolase C in the transfected E. coli host cells. The characteristics of the purified enzyme, e.g. mol. wt, electrophoretic mobilities and kinetic parameters, are indistinguishable from those of authentic rat brain aldolase C. Three different tetrameric hybrid forms, C3A, C2A2 and CA3, in addition to C4 and A4, were found to be produced in the host cell when E.coli was co-transfected with expression plasmids for rat aldolase C and for human aldolase A. Similarly, the hybrid forms, C3B, C2B2 and CB3, in addition to C4 and B4, were also produced in the cells when co-transfected with the plasmids for rat aldolase C and for human aldolase B.