Sp proteins play a critical role in histone deacetylase inhibitor-mediated derepression of CYP46A1 gene transcription.
We investigated whether the CYP46A1 gene, a neuronal-specific cytochrome P450, responsible for the majority of brain cholesterol turnover, is subject to transcriptional modulation through modifications in histone acetylation. We demonstrated that inhibition of histone deacetylase activity by trichostatin A (TSA), valproic acid and sodium butyrate caused a potent induction of both CYP46A1 promoter activity and endogenous expression. Silencing of Sp transcription factors through specific small interfering RNAs, or impairing Sp binding to the proximal promoter, by site-directed mutagenesis, led to a significant decrease in TSA-mediated induction of CYP46A1 expression/promoter activity. Electrophoretic mobility shift assay, DNA affinity precipitation assays and chromatin immunoprecipitation assays were used to determine the multiprotein complex recruited to the CYP46A1 promoter, upon TSA treatment. Our data showed that a decrease in Sp3 binding at particular responsive elements, can shift the Sp1/Sp3/Sp4 ratio, and favor the detachment of histone deacetylase (HDAC) 1 and HDAC2 and the recruitment of p300/CBP. Moreover, we observed a dynamic change in the chromatin structure upon TSA treatment, characterized by an increase in the local recruitment of euchromatic markers and RNA polymerase II. Our results show the critical participation of an epigenetic program in the control of CYP46A1 gene transcription, and suggest that brain cholesterol catabolism may be affected upon treatment with HDAC inhibitors.
Nunes
Maria João
MJ
Faculty of Pharmacy, iMed.UL - Research Institute for Medicines and Pharmaceutical Sciences, University of Lisbon, 1649-003 Lisbon, Portugal.
Milagre
Inês
I
Schnekenburger
Michael
M
Gama
Maria João
MJ
Diederich
Marc
M
Rodrigues
Elsa
E
D000818:Animals; D002460:Cell Line; D000072556:Cholesterol 24-Hydroxylase; D047369:Chromatin Immunoprecipitation; D004331:Drosophila melanogaster; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D006655:Histone Deacetylases / Q000378:metabolism*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000187:drug effects; D034741:RNA, Small Interfering / Q000494:pharmacology; D051703:Sp Transcription Factors / Q000502:physiology*; D018709:Statistics, Nonparametric; D013250:Steroid Hydroxylases / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection / Q000379:methods
D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D034741:RNA, Small Interfering; D051703:Sp Transcription Factors; C012589:trichostatin A; D013250:Steroid Hydroxylases; D000072556:Cholesterol 24-Hydroxylase; D006655:Histone Deacetylases