Chromatin-Modifying Agents Increase Transcription of CYP46A1,a Key Player in Brain Cholesterol Elimination

Abstract

The major mechanism of brain cholesterol elimination is the conversion of cholesterol into 24S-hydroxycholesterol by CYP46A1, a neuron-specific cytochrome P450. Since increasing evidence suggests that upregulation of CYP46A1 may be relevant for the treatment of Alzheimer's disease, we aim to identify the molecular mechanisms involved in CYP46A1 transcription. Our previous studies demonstrated the role of Sp transcription factors in basal expression and histone deacetylase (HDAC) inhibitor-dependent derepression of CYP46A1. Here, we show that the demethylating agent 5'-Aza-2'-deoxycytidine (DAC) is a CYP46A1 inducer and that pre-treatment with DAC causes a marked synergistic activation of CYP46A1 transcription by trichostatin A. Surprisingly, bisulfite sequencing analysis revealed that the CYP46A1 core promoter is completely unmethylated in both human brain and non-neuronal human tissues where CYP46A1 is not expressed. Therefore, we have investigated Sp expression levels by western blot and real-time PCR, and their binding patterns to the CYP46A1 promoter, by electrophoretic mobility shift assay and chromatin immunoprecipitation assays, after DAC treatment. Our results showed that DAC decreases not only Sp1 and Sp3 protein levels, but also the binding activity of Sp3 to the +1 region of the CYP46A1 locus. Concomitantly, HDAC1 and HDAC2 were also significantly dissociated from the promoter. In conclusion, DAC induces CYP46A1 gene expression, in a DNA methylation-independent mechanism, decreasing Sp3/HDAC binding to the proximal promoter. Furthermore, by affecting the expression of the Sp3 transcription factor in neuronal cells, DAC might affect not only brain cholesterol metabolism, but also the expression of many other neuronal genes.