We have previously defined a crucial DNA element controlling 90% of the expression of the presenilin 1 gene at (-35 to +6). This region contains an Ets transcription factor binding motif, and a two-basepair alteration within the core sequence (GGAA to TTAA) of the Ets consensus also reduced transcription by over 90%. We have shown that Ets1/2 transcription factors bind specifically to the -10 Ets element and activate PS1 transcription. The identification of other transcription factors recognizing specifically this promoter area should provide insights into the regulation of PS1. We have used the -10 Ets element as a bait in yeast one hybrid screening of a human brain cDNA library using a His3 reporter construct. We describe an abbreviated one-hybrid protocol to screen cDNA libraries. This assay selected four factors from the Ets family: Ets2, ER81, ERM and Elk1. We have also shown that specific DNA binding activity to the -10 Ets element of PS1 could easily be detected in yeast clones by EMSAs including protein extracts from yeast cells, thus confirming specific DNA binding activity without further sequencing and subcloning into suitable expression vectors. Ultimately the identity of putative clones was confirmed by DNA sequencing. We also confirmed the specific DNA binding properties of the factors identified by showing that the proteins produced by in vitro translation of the entire cDNAs from Elk1 and ER81 indeed binds specifically to the -10 region of the PS1 promoter.
|Number of pages||6|
|Journal||Brain Research Protocols|
|State||Published - Aug 2003|