An abbreviated procedure for the cloning and identification of Ets transcription factors regulating the expression of the human presenilin 1 gene.
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.
Pastorcic
Martine
M
Department of Pharmacology and Neuroscience, University of North Texas Health Science Center at Fort Worth, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107, USA.
Das
Hriday K
HK
D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D053764:Presenilin-1; D011401:Promoter Regions, Genetic / Q000235:genetics / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000235:genetics; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D050783:Proto-Oncogene Proteins c-ets; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D050807:ets-Domain Protein Elk-1
D004268:DNA-Binding Proteins; C494112:ELK1 protein, human; C494041:ETS1 protein, human; C095239:ETV1 protein, human; D008565:Membrane Proteins; C508853:PSEN1 protein, human; D053764:Presenilin-1; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D014157:Transcription Factors; D050807:ets-Domain Protein Elk-1; D004247:DNA