Cloning And Functional Analysis Of The Promoter Of PP2A-Aβ Gene

Protein phosphatases-2A (PP-2A) is a major serine/threonine phosphatase that is abundantly expressed in eukaryotes. PP-2A is a heterotrimer that contains a 65kD scaffold A subunit, a 36kD catalytic C subunit and a regulatory B subunit of variable isoforms with 54～130kD. The scaffold subunit A has PP2A-Aαand PP2A-Aβtwo isoforms, act as platforms for both C and B subunits to bind, and thus are key structural components for PP-2A activity. Mutations in both genes encoding PP2A-Aαand PP2A-Aβlead to carcinogenesis and likely other human diseases.In our previous work we cloned the PP2A-Aαpromoter and found that four major cis-elements including the binding sites for Ets-1, CREB, AP-2αand Sp1/Sp3 were presented in the proximal promoter of the mouse PP2A-Aαgene. It showed that the gene coding for PP2A-Aαis positively regulated by multiple transcription factors including Ets-1, CREB and AP-2αbut negatively regulated by Sp1/Sp3.In our present study, we have conducted functional dissection of the promoter of the mouse PP2A-Aβgene. Our results demonstrate that:(1) The luciferase reporter gene activity assays demonstrated that B4 which contains 480bp (-430 to +50) sequence is the proximal promoter of PP2A-Aβgene. (2) Three major cis-elements including the binding sites for Ets-1, Sp1/Sp3, and RXRα/βare present in the proximal promoter of the mouse PP2A-Aβgene. (3) Gel mobility shifting assays revealed that Ets-1, Sp1/Sp3, and RXRα/βall bind to PP2A-Aβgene promoter in vitro. (4) ChIP assays demonstrated that Ets-1, Sp1/Sp3, and RXRα/βcan all bind to PP2A-Aβgene promoter in vivo. (5) In vitro mutagenesis and reporter gene activity assays demonstrated that while Ets-1, Spl/Sp3, and RXRα/βall positively regulate the promoter of the PP2A-Ap gene. (6) Finally, Over-expression of the cDNAs encoding Ets-1, Sp1/Sp3 or RXRα/βinαTN4-1 cells, further confirm their control over the PP2A-Aβgene by Real-time PCR and Western blot to exa mine the mRNA and protein expression of the endogenous PP2A-Aβ.Together, our results reveal that multiple transcription factors Ets-1, Sp1/Sp3 and RXRa/βregulate the PP2A-Aβgene. Moreover, our results provide important information to explain why PP2A-Aa and PP2A-A(3 display distinct expression levels.