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Methylation Status of CpG Islands Flanking a cAMP Response Element Motif on the Protein Phosphatase 2Ac Promoter Determines CREB Binding and Activity¹

Division of Rheumatology in Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115

Protein phosphatase 2A (PP2A) is a major serine/threonine protein phosphatase in eukaryotic cells and is involved in many essential aspects of cell function. The catalytic subunit of the enzyme (PP2Ac), a part of the core enzyme, has two isoforms, (PP2Ac) and β (PP2Acβ), of which PP2Ac is the major form expressed in vivo. Deregulation of PP2A expression has been linked to several diseases, but the mechanisms that control the expression of this enzyme are still unclear. We conducted experiments to decipher molecular mechanisms involved in the regulation of the PP2Ac promoter in human primary T cells. After preparing serially truncated PP2Ac promoter luciferase constructs, we found that the region stretching around 240 bases upstream from the translation initiation site was of functional significance and included a cAMP response element motif flanked by three GC boxes. Shift assays revealed that CREB/phosphorylated CREB and stable protein 1 could bind to the region. Furthermore, we demonstrated that methylation of deoxycytosine in the CpG islands limited binding of phosphorylated CREB and the activity of the PP2Ac promoter. In contrast, the binding of stable protein 1 to a GC box within the core promoter region was not affected by DNA methylation. Primary T cells treated with 5-azacitidine, a DNA methyltransferase inhibitor, showed increased expression of PP2Ac mRNA. We propose that conditions associated with hypomethylation of CpG islands, such as drug-induced lupus, permit increased PP2Ac expression.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Public Health Service National Institutes of Health Grant RO1 AI068787.

² Address correspondence and reprint requests to Dr. George C. Tsokos or Dr. Yuang-Taung Juang, Department of Medicine, Division of Rheumatology, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, E/CLS 937, Boston, MA 02115. E-mail addresses: gtsokos{at}bidmc.harvard.edu or yjuang{at}bidmc.harvard.edu

³ Abbreviations used in this paper: PP2A, protein phosphatase 2A; PP2Ac, PP2A catalytic subunit; PP2Ac, PP2Ac isoform; PP2Acβ, PP2Ac β isoform; SLE, systemic lupus erythematosus; pCREB, phosphorylated CREB; DNMT, DNA methyltransferase; dC, deoxycytosine; CRE, cAMP response element; 5-azaC, 5-azacitidine; Sp1, stable protein 1; M. SssI, CpG methyltransferase; SAM, S-adenosyl methionine; CREM, cAMP response element modulator; ChIP, chromatin immunoprecipitation assays; dmC, deoxymethylcytosine; Mt, mutant.