Deletion mapping of the human presenilin-1 (PS1) promoter delineated the most active fragment from ؊118 to ؉178 in relation to the transcription start site mapped in this study, in both human neuroblastoma SK-N-SH and hepatoma HepG2 cells. 5 deletions revealed that a crucial element controlling over 90% of the promoter activity in these cell lines is located between ؊22 and ؊6. A mutation altering only two nucleotides of the ETS consensus sequence present at ؊12 (GGAA to TTAA) has a similar effect. Electrophoretic mobility shift assays showed that a set of specific complexes between nuclear factors and the PS1 promoter are eliminated by this point mutation, as well as by competition with an ETS consensus oligonucleotide. Competition experiments in DNase I footprinting correlated with electrophoretic mobility shift assays and showed that only one of several footprints over the PS1 promoter is eliminated by competition with an ETS consensus oligonucleotide. It extends from ؊14 to ؊6 and surrounds the ETS motif present at ؊12. Thus, a crucial ETS element is present at ؊12 and binds a protein(s) recognizing specifically the ETS consensus motif. At least one such complex is eliminated by preincubating the nuclear extract with an antibody with broad cross-reactivity with Ets-1 and Ets-2 proteins, thus confirming that an ETS transcription factor(s) recognizes the ؊12 motif. Several Sp1 binding motifs at positions ؊70, ؊55, and ؉20 surround this ETS element. Competition DNase I footprinting showed that Sp1-like nuclear factors recognize specifically these sites in both cell lines. Furthermore, a combination of 5 and 3 deletions indicated the presence of positive promoter elements between ؊96 and ؊35 as well as between ؉6 and ؉42. Thus, transfection and footprinting assays correlate to suggest that Sp1 transcription factor(s) bind at several sites upstream and downstream from the initiation site and activate the transcription of the PS1 promoter. Sequences downstream from the transcription initiation site also contain major control elements. 3 deletions from ؉178 to ؉107 decreased promoter activity by 80%. However, further deletion to ؉42 increased promoter activity by 3-4-fold. Collectively, these data indicate that sequences upstream and downstream from the transcription start site each control over 80% of the promoter activity. Hence, this suggests that protein-protein interactions between factors recognizing downstream and upstream sequences are involved.

The expression of the human presenilin-1 cellular gene is suppressed by the p53 protooncogene. The rapid kinetic of the down-regulation has suggested that it may result from a primary mechanism. We show here that p53 also suppresses the transcription of a presenilin-1 promoter-chloramphenicol acetyltransferase reporter synthetic gene in transient infection assays in neuroblastoma (SK-N-SH) and hepatoma (HepG2) cell lines. Only a minimum promoter including sequences from ؊35 to ؉ 6 from the transcription initiation is sufficient to confer down-regulation. We have previously defined a crucial DNA element controlling 90% of the expression of the gene within the same short area, and the identification of the transcription factors involved should also provide insights into the regulation of PS1 by p53. This region contains an Ets transcription factor binding motif, and a 2-base pair alteration within the core sequence (GGAA to TTAA) of the Ets consensus also reduced transcription by more than 90%. We now show that Ets1 and Ets2 indeed transactivate a PS1 promoter-chloramphenicol acetyltransferase reporter including the (؊35 to ؉6) fragment. Furthermore, in vitro translated Ets2 binds specifically to the ؊10 Ets motif in electrophoretic mobility shift assays. Therefore, Ets1/2 factors bind specifically to the ؊10 Ets element and activate PS1 transcription. We also show that the coactivator p300 enhances the activation by Ets1 and Ets2 as well as the repression by p53. p300 is known to interact with p53 as well as with Ets1 and Ets2. We show that p53 does not bind directly to the PS1 promoter. Hence the repression of PS1 transcription by p53 is likely to be mediated through protein-protein interactions.Presenilin genes (PS1 and PS2) encode highly homologous integral membrane proteins (1, 2). A majority of early onset or familial Alzheimer's disease (FAD) 1 cases results from mutations in PS1, PS2, or amyloid precursor protein (APP) with a majority of cases in PS1 (3, 4). The pathogenesis of FAD includes as an early invariant the development of amyloid plaques containing specifically A␤42/43 polypeptide (3, 5). A␤42 is produced by sequential proteolytic cleavage of ␤ APP (6). PS1 appears to play a crucial role in the normal metabolism of APP as well as in the pathological increase of A␤42 (7). The exact function of PS1 in the processing of APP is still unclear. PS1 appears to be tightly associated within a multiprotein complex with ␥ secretase, the second of two proteases that cleave APP and that has not yet been identified (8). Recent evidence has suggested that PS1 itself may contain ␥ secretase catalytic activity (9 -11). Furthermore, evidence that an endoprotease activity crucial for normal biological function is contained in PS1 or requires PS1 has also been derived from examining the function of PS1 in Notch receptor cleavage and activation. The PS1 homologue in Caenorhabditis elegans, SEL-12, is required for Notch receptor signaling and cell fate determination (12). Similar to APP, Notch undergoes intramembrane p...

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We have identified DNA sequences required for the expression of the presenilin 1 (PS1) gene. A promoter region has been mapped in SK‐N‐SH cells and includes sequences between −118 and +178 flanking the major initiation site (+1). The PS1 gene is also efficiently transcribed in the SH‐SY5Y subclone of SK‐N‐SH cells. However the promoter appears to be utilized in alternative ways in both cell types. Sequences both upstream as well as downstream from the initiation site mapped in SK‐N‐SH cells were shown by 5′‐ and 3′‐deletion analysis to play a crucial role in both cell lines. However, in SH‐SY5Y cells either upstream or downstream sequences are sufficient to direct transcription, whereas in SK‐N‐SH cells 5′‐deletions past the +1 site eliminate over0 95% of transcription. Several Ets motifs (GGAA) as well as Sp1 motifs [(G/T)GGCGGRRY] are juxtaposed both upstream and downstream from +1. To understand how the promoter may be utilized alternatively in different cell types we have examined the effect of point mutations in these elements. Altering an Ets motif at −10 eliminates 80% of transcription in SK‐N‐SH cells whereas the same mutation has only a minor effect in SH‐SY5Y cells. Conversely, mutation of the Ets element at +90, which eliminates 70% of transcription in SH‐SY5Y cells, has a lesser effect in SK‐N‐SH cells. In both cell types a promoter including mutations at both −10 and +90 sites loses over 90% transcription activity indicating the crucial importance of these two Ets motifs. The effect of Sp1 mutations appears to be similar in both cell types. Hence the differential expression in each cell type may be at least partially determined by Ets factors and the −10/+90 sites. We have identified several Ets factors that recognize specifically the −10 Ets motif by the yeast one‐hybrid selection including avian erythroblastosis virus E26 oncogene homologue 2, Ets‐like gene 1, Ets translocation variant 1 and Ets related molecule (ERM). We show here that ERM specifically recognizes Ets motifs on the PS1 promoter located at −10 as well as downstream at +90, +129 and +165 and activates PS1 transcription with promoter fragments containing or not the −10 Ets site.