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Genome-wide methylation analysis demonstrates that 5-aza-2-deoxycytidine treatment does not cause random DNA demethylation in fragile X syndrome cells
BACKGROUND: Fragile X syndrome (FXS) is caused by CGG expansion over 200 repeats at the 5′ UTR of the FMR1 gene and subsequent DNA methylation of both the expanded sequence and the CpGs of the promoter region. This epigenetic change causes transcriptional silencing of the gene. We have previously de...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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BioMed Central
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4806452/ https://www.ncbi.nlm.nih.gov/pubmed/27014370 http://dx.doi.org/10.1186/s13072-016-0060-x |
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author | Tabolacci, Elisabetta Mancano, Giorgia Lanni, Stella Palumbo, Federica Goracci, Martina Ferrè, Fabrizio Helmer-Citterich, Manuela Neri, Giovanni |
author_facet | Tabolacci, Elisabetta Mancano, Giorgia Lanni, Stella Palumbo, Federica Goracci, Martina Ferrè, Fabrizio Helmer-Citterich, Manuela Neri, Giovanni |
author_sort | Tabolacci, Elisabetta |
collection | PubMed |
description | BACKGROUND: Fragile X syndrome (FXS) is caused by CGG expansion over 200 repeats at the 5′ UTR of the FMR1 gene and subsequent DNA methylation of both the expanded sequence and the CpGs of the promoter region. This epigenetic change causes transcriptional silencing of the gene. We have previously demonstrated that 5-aza-2-deoxycytidine (5-azadC) treatment of FXS lymphoblastoid cell lines reactivates the FMR1 gene, concomitant with CpG sites demethylation, increased acetylation of histones H3 and H4 and methylation of lysine 4 on histone 3. RESULTS: In order to check the specificity of the 5-azadC-induced DNA demethylation, now we performed bisulphite sequencing of the entire methylation boundary upstream the FMR1 promoter region, which is preserved in control wild-type cells. We did not observe any modification of the methylation boundary after treatment. Furthermore, methylation analysis by MS-MLPA of PWS/AS and BWS/SRS loci demonstrated that 5-azadC treatment has no demethylating effect on these regions. Genome-wide methylation analysis through Infinium 450K (Illumina) showed no significant enrichment of specific GO terms in differentially methylated regions after 5-azadC treatment. We also observed that reactivation of FMR1 transcription lasts up to a month after a 7-day treatment and that maximum levels of transcription are reached at 10–15 days after last administration of 5-azadC. CONCLUSIONS: Taken together, these data demonstrate that the demethylating effect of 5-azadC on genomic DNA is not random, but rather restricted to specific regions, if not exclusively to the FMR1 promoter. Moreover, we showed that 5-azadC has a long-lasting reactivating effect on the mutant FMR1 gene. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13072-016-0060-x) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-4806452 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-48064522016-03-24 Genome-wide methylation analysis demonstrates that 5-aza-2-deoxycytidine treatment does not cause random DNA demethylation in fragile X syndrome cells Tabolacci, Elisabetta Mancano, Giorgia Lanni, Stella Palumbo, Federica Goracci, Martina Ferrè, Fabrizio Helmer-Citterich, Manuela Neri, Giovanni Epigenetics Chromatin Research BACKGROUND: Fragile X syndrome (FXS) is caused by CGG expansion over 200 repeats at the 5′ UTR of the FMR1 gene and subsequent DNA methylation of both the expanded sequence and the CpGs of the promoter region. This epigenetic change causes transcriptional silencing of the gene. We have previously demonstrated that 5-aza-2-deoxycytidine (5-azadC) treatment of FXS lymphoblastoid cell lines reactivates the FMR1 gene, concomitant with CpG sites demethylation, increased acetylation of histones H3 and H4 and methylation of lysine 4 on histone 3. RESULTS: In order to check the specificity of the 5-azadC-induced DNA demethylation, now we performed bisulphite sequencing of the entire methylation boundary upstream the FMR1 promoter region, which is preserved in control wild-type cells. We did not observe any modification of the methylation boundary after treatment. Furthermore, methylation analysis by MS-MLPA of PWS/AS and BWS/SRS loci demonstrated that 5-azadC treatment has no demethylating effect on these regions. Genome-wide methylation analysis through Infinium 450K (Illumina) showed no significant enrichment of specific GO terms in differentially methylated regions after 5-azadC treatment. We also observed that reactivation of FMR1 transcription lasts up to a month after a 7-day treatment and that maximum levels of transcription are reached at 10–15 days after last administration of 5-azadC. CONCLUSIONS: Taken together, these data demonstrate that the demethylating effect of 5-azadC on genomic DNA is not random, but rather restricted to specific regions, if not exclusively to the FMR1 promoter. Moreover, we showed that 5-azadC has a long-lasting reactivating effect on the mutant FMR1 gene. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13072-016-0060-x) contains supplementary material, which is available to authorized users. BioMed Central 2016-03-24 /pmc/articles/PMC4806452/ /pubmed/27014370 http://dx.doi.org/10.1186/s13072-016-0060-x Text en © Tabolacci et al. 2016 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Research Tabolacci, Elisabetta Mancano, Giorgia Lanni, Stella Palumbo, Federica Goracci, Martina Ferrè, Fabrizio Helmer-Citterich, Manuela Neri, Giovanni Genome-wide methylation analysis demonstrates that 5-aza-2-deoxycytidine treatment does not cause random DNA demethylation in fragile X syndrome cells |
title | Genome-wide methylation analysis demonstrates that 5-aza-2-deoxycytidine treatment does not cause random DNA demethylation in fragile X syndrome cells |
title_full | Genome-wide methylation analysis demonstrates that 5-aza-2-deoxycytidine treatment does not cause random DNA demethylation in fragile X syndrome cells |
title_fullStr | Genome-wide methylation analysis demonstrates that 5-aza-2-deoxycytidine treatment does not cause random DNA demethylation in fragile X syndrome cells |
title_full_unstemmed | Genome-wide methylation analysis demonstrates that 5-aza-2-deoxycytidine treatment does not cause random DNA demethylation in fragile X syndrome cells |
title_short | Genome-wide methylation analysis demonstrates that 5-aza-2-deoxycytidine treatment does not cause random DNA demethylation in fragile X syndrome cells |
title_sort | genome-wide methylation analysis demonstrates that 5-aza-2-deoxycytidine treatment does not cause random dna demethylation in fragile x syndrome cells |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4806452/ https://www.ncbi.nlm.nih.gov/pubmed/27014370 http://dx.doi.org/10.1186/s13072-016-0060-x |
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