Cloning And Functional Analysis Of The Promoter Of Mouse PP2A-Aα Gene

Protein phosphatases-2A(PP-2A)is a major serine/threonine phosphatase and accounts for more than 50%serine/threonine phosphatase activity in eukaryotes.The holoenzyme of PP-2A consists of three different subunits:the scaffold A subunit,the catalytic C subunit and the regulatory B subunit.The scaffold subunit appears in two isoforms,which are encoded by two genes:PP2A-Aa and PP2A-Aβ. The scaffold subunits provide a platform for both C and B subunits to bind,thus playing a crucial role in providing specific PP-2A activity. Mutation of both genes causes human diseases such as various types of cancers.Regulation of these genes by various factors,both extracellular and intracellular,remains largely unknown.In the present study,we have cloned the promoter of the mouse PP2A-Aa gene and made some deletion mutants from the promoter 5' terminal and did in vitro mutagenesis on cis-elements for the binding of transcription factor Ets-1, CREB,AP-2a and Spl.We transfect ARPE-19 and FHL124 cell lines with these plasmids and then check their relative luciferase activity with dual-luciferase reporter assay system.Our results demonstrate that:(1).Transcription factor Ets-1,CREB,AP-2a and Spl are all present in ARPE-19 and FHL124 cell lines;(2).The core promoter of the mouse PP2A-Aa gene consists of 677 bp and contains numerous cis-elements for the binding of Ets-1,CREB, AP-2a and SP-1.The promoter region of mouse PP2A-Aa gene was found to lack TATA box but have a CAAT box and -625--+52 region provide the main activity to drive its expression;(3).When the binding site for transcription factor Ets-1 was mutated, compared with mouse wild type core promoter its relative luciferase activity was dramatically decreased to 40%;(4).When the binding site for transcription factor CREB was mutated,compared with mouse wild type core promoter its relative luciferase activity was dramatically decreased to 50%;(5).When all the two binding sites for transcription factor AP-2a was mutated,compared with mouse wild type core promoter its relative luciferase activity was dramatically decreased to 80%;(6).When the binding site for transcription factor Sp1 was mutated, compared with mouse wild type core promoter its relative luciferase activity was increased to 120%;(7).Transcription factor Ets-1,CREB,AP-2a and Sp1 dose-dependent luciferase assay reveal that transcription factor Ets-1, CREB and AP-2a all positively regulate the promoter of the PP2A-Aa gene while transcription factor SP-1 displays negative regulation;(8).Gel mobility shifting assays revealed that these four transcription factors can all bind to mouse PP2A-Aapromoter;(9).ChIP assay further confirms that both Ets-1 and CREB play important role in regulating mouse PP2A-Aa gene promoter.Taking it together,our results reveal that multiple transcription factors,such as Ets-1,CREB,AP-2a and Sp1,regulate the mouse PP2A-Aa gene.