Identification of the regulatory mechanisms and factors involved in the brain-specific expression of CYP46A1

• Author(s): Silva, Inês Milagre da, 1980-
• Date: 2010
• Persistent ID: http://hdl.handle.net/10451/3006
• Origin: Repositório da Universidade de Lisboa
• Subject(s): Teses de doutoramento - 2011
• Document Type: Doctoral thesis
• Language: English
• Advisor(s): Rodrigues, Elsa, 1969-; Gama, Maria João Carlos da Silva, 1958-
• Contributor(s): Repositório da Universidade de Lisboa

Description

Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2011

Cholesterol has a crucial role in central nervous system physiology and cell signaling, and many studies correlate alterations in brain cholesterol homeostasis with neurodegenerative diseases. The CYP46A1 gene codes for the cholesterol 24-hydroxylase (CYP46A1), a cytochrome P450 specifically expressed in neurons, that is responsible for the majority of brain cholesterol turnover. However, despite its physiological importance, the molecular mechanisms underlying the human CYP46A1 expression had not been characterized. Therefore, our work aimed to identify regulatory elements and factors involved the CYP46A1 brain-specific expression, and further assess if CYP46A1 transcription is regulated by epigenetic modifications, such as DNA methylation. Moreover, we aimed to identify a human cell model that could be a valuable tool for the study of cholesterol homeostasis in human neurons. In our initial studies, we cloned and characterized the human CYP46A1 promoter. Functional deletion analysis, over-expression studies, site-directed mutagenesis and gel-shift assays identified that not only Sp transcription factors control CYP46A1 transcription, but are most probably responsible for cell type specificity. To test our hypothesis that an increase in the (Sp3+Sp4)/ Sp1 ratio would result in CYP46A1 transcriptional activation, we differentiated Ntera2/clone D1 (NT2) human teratocarcinoma cells into post-mitotic neurons (NT2N). We demonstrated for the first time a significant increase in CYP46A1 mRNA and protein levels in a human cell culture, and identified a concomitant decrease in the levels of Sp1 associated with the proximal promoter of this gene. Nevertheless, we did not observe any conserved pattern in Sp protein binding to other Sp-regulated gene promoters, suggesting that Sp-DNA binding and transcriptional activity is highly dependent on the neuronal chromatin context. Moreover, we showed that throughout NT2 differentiation, HMG-CoA Abstract xxiv synthase, HMG-CoA reductase, SREBP2 and LDLr, key players in brain cholesterol homeostasis, present expression profiles that mimic what is thought to occur in vivo. Our results also suggest that progenitor cells eliminate cholesterol in the form of 27-hydroxycholesterol while neurogenesis induces a shift to the 24-hydroxylase-dependent elimination pathway. Finally, we showed that the demethylating agent 5’-Aza-2’-deoxycytidine (DAC) is a CYP46A1 inducer. 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. We demonstrated that DAC induces CYP46A1 expression, in a DNA methylation- independent mechanism, by decreasing Sp3/HDAC binding to the proximal promoter. Collectively, our results provide new insights on the regulatory circuits that control CYP46A1 transcription, and contribute to the identification of potential therapeutic approaches that can modulate CYP46A1 expression.

Fundação para a Ciência e a Tecnologia:SFRH/BD/27660/2006; PPCDT/SAU-MMO/55919/2004 and PTDC/SAU-GMG/64176/2006 (to Professor Elsa Margarida Teixeira Rodrigues) from FCT and FEDER.

Fundação para a Ciência e Tecnologia (FCT),(SFRH/BD/27660/2006), Lisbon, Portugal; PPCDT/SAU-MMO/55919/2004; PTDC/SAU-GMG/64176/2006. FCT; FEDER.