Publicação
Identification of the regulatory mechanisms and factors involved in the brain-specific expression of CYP46A1
| Resumo: | 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. |
|---|---|
| Autores principais: | Silva, Inês Milagre da, 1980- |
| Assunto: | Teses de doutoramento - 2011 |
| Ano: | 2010 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| _version_ | 1877771600384753664 |
|---|---|
| author | Silva, Inês Milagre da, 1980- |
| author_facet | Silva, Inês Milagre da, 1980- |
| author_role | author |
| contributor_name_str_mv | Rodrigues, Elsa, 1969- Gama, Maria João Carlos da Silva, 1958- Repositório Científico de Acesso Aberto da ULisboa |
| country_str | PT |
| creators_json_txt | [{\"Person.name\":\"Silva, Inês Milagre da, 1980-\"}] |
| datacite.contributors.contributor.contributorName.fl_str_mv | Rodrigues, Elsa, 1969- Gama, Maria João Carlos da Silva, 1958- Repositório Científico de Acesso Aberto da ULisboa |
| datacite.creators.creator.creatorName.fl_str_mv | Silva, Inês Milagre da, 1980- |
| datacite.date.Accepted.fl_str_mv | 2010-01-01T00:00:00Z |
| datacite.date.available.fl_str_mv | 2011-04-05T16:49:57Z |
| datacite.date.embargoed.fl_str_mv | 2011-04-05T16:49:57Z |
| datacite.rights.fl_str_mv | http://purl.org/coar/access_right/c_abf2 |
| datacite.subjects.subject.fl_str_mv | Teses de doutoramento - 2011 |
| datacite.titles.title.fl_str_mv | Identification of the regulatory mechanisms and factors involved in the brain-specific expression of CYP46A1 |
| dc.contributor.none.fl_str_mv | Rodrigues, Elsa, 1969- Gama, Maria João Carlos da Silva, 1958- Repositório Científico de Acesso Aberto da ULisboa |
| dc.creator.none.fl_str_mv | Silva, Inês Milagre da, 1980- |
| dc.date.Accepted.fl_str_mv | 2010-01-01T00:00:00Z |
| dc.date.available.fl_str_mv | 2011-04-05T16:49:57Z |
| dc.date.embargoed.fl_str_mv | 2011-04-05T16:49:57Z |
| dc.format.none.fl_str_mv | application/pdf |
| dc.identifier.none.fl_str_mv | http://hdl.handle.net/10451/3006 |
| dc.language.none.fl_str_mv | eng |
| dc.rights.none.fl_str_mv | http://purl.org/coar/access_right/c_abf2 |
| dc.subject.none.fl_str_mv | Teses de doutoramento - 2011 |
| dc.title.fl_str_mv | Identification of the regulatory mechanisms and factors involved in the brain-specific expression of CYP46A1 |
| dc.type.none.fl_str_mv | http://purl.org/coar/resource_type/c_db06 |
| description | 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. |
| dirty | 0 |
| eu_rights_str_mv | openAccess |
| format | doctoralThesis |
| fulltext.url.fl_str_mv | https://repositorio.ulisboa.pt/bitstreams/a286a682-2e27-4853-9fe0-4e6744890e70/download |
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| identifier.url.fl_str_mv | http://hdl.handle.net/10451/3006 |
| inst_facet_str | urn:organizationAcronym:ul{{{_:::_}}}Universidade de Lisboa |
| instacron_str | ul |
| institution | Universidade de Lisboa |
| instname_str | Universidade de Lisboa |
| language | eng |
| network_acronym_str | ul |
| network_name_str | Repositório da Universidade de Lisboa |
| oai_identifier_str | oai:repositorio.ulisboa.pt:10451/3006 |
| organization_str_mv | urn:organizationAcronym:ul |
| person_str_mv | Silva, Inês Milagre da, 1980- |
| publishDate | 2010 |
| repo_facet_str | urn:repositoryAcronym:ul{{{_:::_}}}Repositório da Universidade de Lisboa |
| reponame_str | Repositório da Universidade de Lisboa |
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| spelling | engporCholesterol 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.application/pdfporIdentification of the regulatory mechanisms and factors involved in the brain-specific expression of CYP46A1Silva, Inês Milagre da, 1980-Rodrigues, Elsa, 1969-Gama, Maria João Carlos da Silva, 1958-HostingInstitutionOrganizationalRepositório Científico de Acesso Aberto da ULisboae-mailmailto:repositorio@ulisboa.ptrepositorio@ulisboa.pt2011-04-05T16:49:57Z20102010-01-01T00:00:00ZHandlehttp://hdl.handle.net/10451/3006http://purl.org/coar/access_right/c_abf2open accessTeses de doutoramento - 20113998060 bytesliteraturehttp://purl.org/coar/resource_type/c_db06doctoral thesishttp://purl.org/coar/access_right/c_abf2application/pdffulltexthttps://repositorio.ulisboa.pt/bitstreams/a286a682-2e27-4853-9fe0-4e6744890e70/downloadLisboa |
| spellingShingle | Identification of the regulatory mechanisms and factors involved in the brain-specific expression of CYP46A1 Silva, Inês Milagre da, 1980- Teses de doutoramento - 2011 |
| status | SINGLETON |
| subject.fl_str_mv | Teses de doutoramento - 2011 |
| title | Identification of the regulatory mechanisms and factors involved in the brain-specific expression of CYP46A1 |
| title_full | Identification of the regulatory mechanisms and factors involved in the brain-specific expression of CYP46A1 |
| title_fullStr | Identification of the regulatory mechanisms and factors involved in the brain-specific expression of CYP46A1 |
| title_full_unstemmed | Identification of the regulatory mechanisms and factors involved in the brain-specific expression of CYP46A1 |
| title_short | Identification of the regulatory mechanisms and factors involved in the brain-specific expression of CYP46A1 |
| title_sort | Identification of the regulatory mechanisms and factors involved in the brain-specific expression of CYP46A1 |
| topic | Teses de doutoramento - 2011 |
| topic_facet | Teses de doutoramento - 2011 |
| url | http://hdl.handle.net/10451/3006 |
| visible | 1 |
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