title: DNA methylation of the 5'-untranslated region at +298 and +351 represses BACE1 expression in mouse BV-2 microglial cells.
abstract: BACE1, which cleaves the amyloid precursor protein, is the rate-limiting enzyme for β-amyloid peptide production, leading to the pathogenesis of Alzheimer's disease (AD). A high plasma level of homocysteine, acting as a potent methyltransferase inhibitor, is assumed to be a risk factor for AD onset. Using the demethylating drug 5-aza-2'-deoxycytidine (5-Aza), we tested whether and how BACE1 expression is regulated in mouse BV-2 microglial cells. 5-Aza increased both BACE1 mRNA and protein levels in a dose-dependent manner. Bisulfite-sequencing analysis revealed that two CpG sites at positions +298 and +351 in the 5'-untranslated region (5'-UTR) of the BACE1 gene were specifically demethylated in BV-2 cells treated with 5-Aza. In silico analysis showed that the +351 site is the STAT3/CTCF-binding site; the function of the +298 site has not been identified. To assess whether these two CpG sites play an important role in 5-Aza-induced transcriptional activation of BACE1, we constructed a BACE1 gene promoter including the 5'-UTR (-1136 to +500) fused to a CpG-free luciferase gene (pCpGL-BACE1) and its mutant pCpGL-BACE1-AA, which has substituted CG dinucleotides at the two CpG sites of pCpGL-BACE1 to AA. Promoter analysis showed a significant decrease (∼30%) in the activity of pCpGL-BACE1-AA compared with that of pCpGL-BACE1. Furthermore, in vitro methylation of these two reporter constructs showed a complete silencing of their promoter activities. Our data demonstrate that BACE1 gene expression is regulated by DNA methylation of at least two CpG sites at positions +298 and +351 in the 5'-UTR in BV-2 microglial cells.
authors.affiliation: Department of Molecular Medicine, Ewha Womans University Medical School, Seoul, South Korea.
authors.forename: Catherine Jeonghae
authors.initials: CJ
authors.lastname: Byun
authors.forename: Jungwon
authors.initials: J
authors.lastname: Seo
authors.forename: Sangmee Ahn
authors.initials: SA
authors.lastname: Jo
authors.forename: Yoon Jung
authors.initials: YJ
authors.lastname: Park
authors.forename: Maja
authors.initials: M
authors.lastname: Klug
authors.forename: Michael
authors.initials: M
authors.lastname: Rehli
authors.forename: Moon-Ho
authors.initials: MH
authors.lastname: Park
authors.forename: Inho
authors.initials: I
authors.lastname: Jo
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics*; D053829:Amyloid Precursor Protein Secretases / Q000235:genetics*; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000235:genetics*; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D002460:Cell Line; D018899:CpG Islands; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors; D000077209:Decitabine; D015870:Gene Expression; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D017628:Microglia / Q000378:metabolism*
chemical_list: D020121:5' Untranslated Regions; D000077209:Decitabine; D015254:DNA Modification Methylases; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; C509282:Bace1 protein, mouse; D001374:Azacitidine