Quantitative analysis of DNA demethylation and transcriptional reactivation of the FMR1 gene in fragile X cells treated with 5-azadeoxycytidine

Pietrobono, Roberta; Pomponi, Maria Grazia; Tabolacci, Elisabetta; Oostra, Ben A.; Chiurazzi, Pietro; Neri, Giovanni

doi:10.1093/nar/gkf434

Cited by 107 publications

(105 citation statements)

References 35 publications

(61 reference statements)

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“…However, HDAC inhibitors alone were unable to induce any reactivation at all [Chiurazzi et al, 1999], suggesting that DNA methylation is dominant over histone hypoacetylation at the FMR1 locus, as also reported for other heavily methylated genes [Cameron et al, 1999]. These initial observations were confirmed by measuring FMR1 transcript with real-time RT-PCR and confirming DNA demethylation at the FMR1 promoter after 5-azadC treatment [Pietrobono et al, 2002]. We then investigated histone modifications in the promoter, exon 1 and exon 16 of FMR1 by chromatin immunoprecipitation (ChIP) before and after pharmacological treatment of FXS lymphoblasts with 5-azadC and acetyl-L-carnitine .…”

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confidence: 68%

“…2), effectively transforming a methylated full mutation into an unmethylated full mutation (UFM) Tabolacci et al, 2008a]. 5-azadC effects increased with dose and treatment time [Chiurazzi et al, 1999;Pietrobono et al, 2002], but 4 weeks after treatment was discontinued we observed regain of DNA methylation in the FMR1 promoter and loss of transcription [Pietrobono et al, 2002]. Further follow-up experiments are war-ranted in order to understand if some clones actually remained demethylated or if all cells reverted to the heterochromatic status.…”

Section: Transcriptional Therapy Of Fragile X and Other "Epigenetic" mentioning

confidence: 85%

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Epigenetics, fragile X syndrome and transcriptional therapy

Tabolacci

Chiurazzi

2013

American J of Med Genetics Pt A

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Epigenetics refers to the study of heritable changes in gene expression that occur without a change in DNA sequence. Epigenetic mechanisms therefore include all transcriptional controls that determine how genes are expressed during development and differentiation, but also in individual cells responding to environmental stimuli. The purpose of this review is to examine the basic principles of epigenetic mechanisms and their contribution to human disorders with a particular focus on fragile X syndrome (FXS), the most common monogenic form of developmental cognitive impairment. FXS represents a prototype of the so-called repeat expansion disorders due to "dynamic" mutations, namely the expansion (known as "full mutation") of a CGG repeat in the 5 0 UTR of the FMR1 gene. This genetic anomaly is accompanied by epigenetic modifications (mainly DNA methylation and histone deacetylation), resulting in the inactivation of the FMR1 gene. The presence of an intact FMR1 coding sequence allowed pharmacological reactivation of gene transcription, particularly through the use of the DNA demethylating agent 5 0 -aza-2 0 -deoxycytydine and/or inhibitors of histone deacetylases. These treatments suggested that DNA methylation is dominant over histone acetylation in silencing the FMR1 gene. The importance of DNA methylation in repressing FMR1 transcription is confirmed by the existence of rare unaffected males carrying unmethylated full mutations. Finally, we address the potential use of epigenetic approaches to targeted treatment of other genetic conditions. Ó 2013 Wiley Periodicals, Inc.Key words: fragile X syndrome; epigenetics; transcriptional therapy INTRODUCTION When in 1942 Conrad H. Waddington coined the term "epigenetics," the exact nature of genes and their role in heredity and in transcriptional regulation was not known; he used it as a conceptual model of how genes might interact with their surroundings to produce a phenotype [Waddington, 1942]. The term is a portmanteau of the words epigenesis (differentiation of cells from their initial totipotent state in embryonic development) and genetics. Only in 2008 at a Cold Spring harbor meeting, consensus was reached on the definition of epigenetic trait, as "stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence" [Berger et al., 2009]. The greek prefix epi-refers to features that are "on top of" genetics, that is, all those stable but reversible changes to DNA, RNA and proteins that regulate gene expression and allow cells with the same DNA to do different things at different times.Broadly speaking, epigenetic mechanisms include all transcriptional controls that regulate gene expression, whether during development and differentiation or in mature cells responding to the environment. Epigenetic changes can be inherited (e.g., imprinting) and be relatively stable (e.g., chromosome X inactivation), but are often rapidly imposed or removed on a given locus according to cell needs. Four major layers of epigenetic controls have ...

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confidence: 68%

Section: Transcriptional Therapy Of Fragile X and Other "Epigenetic" mentioning

confidence: 85%

Epigenetics, fragile X syndrome and transcriptional therapy

Tabolacci

Chiurazzi

2013

American J of Med Genetics Pt A

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“…However, even though epigenetic states are potentially reversible, treatment with agents generally affecting DNA methylation have their natural limitation due to their non-selectivity and sometimes also because of their inefficiency to demethylate particular CpG sequences. 41,42 Moreover, simultaneous change of a whole network of epigenetically silenced genes by inhibitory drugs may negatively interfere with the final outcome of a proper immunological response. 43 In fact, preincubation with sodium butyrate did not unmask those cells selected by our method (Fig.…”

Section: Discussionmentioning

confidence: 99%

Genetic redundancy in human cervical carcinoma cells: Identification of cells with “normal” properties

Bachmann

Zawatzky

Rösl

2007

Intl Journal of Cancer

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Although it is generally assumed that cancer arises from a singular cell, a tumor must be considered as a dynamic and emergent biological structure, whose organizing principle is determined by genetic and epigenetic modifications, occurring variably in response to microenvironmental selection conditions. As previously shown, HPV-positive cervical carcinoma cells have lost their ability to induce IFN-b upon TNF-a treatment. However, regarding cancer as a non-linear system, which may, even in the absence of an apparent selection pressure, fluctuate between different ''metastable'' phenotypes, we demonstrate that TNF-a mediated IFN-b induction is not irreversibly disturbed in all cells. Using the IFN-b sensitive Encephalomyocarditis virus (EMCV) as a tool to monitor antiviral activity in long-term established malignant HeLa cells, rare IFN-b expressing clones were rescued from a population of non-responsive and EMCV-sensitive cells. Antiviral activity was mediated by the re-expression of IRF-1 and p48 (IRF-9), both key regulatory molecules normally found to be suppressed in cervical carcinoma cells. Upon inoculating of selected clones into immunocompromised animals, a reduced or even an absence of tumorigenicity of initially highly malignant cells could be discerned. These data indicate that both the absence of interferon signaling and the ability to form tumors were reversed in a minority of cells. We provide a paradigm for the existence of innate genetic redundancy mechanisms, where a particular phenotype persists and can be isolated without application of drugs generally changing the epigenetic context. ' 2007 Wiley-Liss, Inc.Key words: human papillomaviruses; interferon-b regulation; EMCVcytolysis; tumor necrosis factor alpha; redundancy mechanisms; oscillation; chaotic cell systems; tumorigenicity Infections with certain types of human papillomaviruses (HPV) are the major cause for the development of cervical cancer. 1 Epidemiological studies show that immunodeficient women have a significantly higher risk of developing a tumor than age-matched controls. Hence, cancer of the cervix uteri is the final outcome of a long term selection and escape process, where HPV positive cells are no longer controlled by immunological surveillance. 2 Consequently, a physiological intact communication route between inflammatory and HPV-containing cells is an indispensable prerequisite for a proper antiviral response. 3 Targeting IFN-signaling actually provides a general selective advantage for tumor formation, because it favors the disruption of the intercellular cross-talk between HPV positive and immunological effector cells. 4 In a previous investigation, we demonstrated that the antiviral effect of TNF-a, which is mediated by IFN-b gene activation, 5 is disturbed in HPV positive cervical carcinoma cells. 6 The reason for this failure is the lack of interferon-regulatory factor (IRF)-1 and p48 (IRF-9) expression, two key regulators, tightly involved in the transcriptional control of the intermediate and the delayed interfero...

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“…The FMR1 amplicon is a 89-bp product spanning the junction between exon 13 and 14 of the gene (positions 1432 -1520 of GenBank sequence NM_002024). The primers and TaqMan probe employed were as described by Pietrobono et al 33 The relative amount of FMR1-mRNA was assessed by comparison with the human HPRT-mRNA detected with the Pre-Developed TaqMan Assay Reagent (ABI 4310890E: huHPRT endogenous control).…”

Section: Methodsmentioning

confidence: 99%

Epigenetic analysis reveals a euchromatic configuration in the FMR1 unmethylated full mutations

Tabolacci¹,

Moscato

Zalfa

et al. 2008

Eur J Hum Genet

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Fragile X syndrome (FXS) is caused by the expansion of a CGG repeat in the 5 0 UTR of the FMR1 gene and the subsequent methylation of all CpG sites in the promoter region. We recently identified, in unrelated FXS families, two rare males with an unmethylated full mutation, that is, with an expanded CGG repeat (4200 triplets) lacking the typical CpG methylation in the FMR1 promoter. These individuals are not mentally retarded and do not appear to be mosaic for premutation or methylated full mutation alleles. We established lymphoblastoid and fibroblast cell lines that showed essentially normal levels of the FMR1-mRNA but reduced translational efficiency of the corresponding mRNA. Epigenetic analysis of the FMR1 gene demonstrated the lack of DNA methylation and a methylation pattern of lysines 4 and 27 on histone H3 similar to that of normal controls, in accordance with normal transcription levels and consistent with a euchromatic configuration. On the other hand, histone H3/H4 acetylation and lysine 9 methylation on histone H3 were similar to those of typical FXS cell lines, suggesting that these epigenetic changes are not sufficient for FMR1 gene inactivation. These findings demonstrate remarkable consistency and suggest a common genetic mechanism causing this rare FMR1 epigenotype. The discovery of such a mechanism may be important in view of therapeutic attempts to convert methylated into unmethylated full mutations, restoring the expression of the FMR1 gene.

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Quantitative analysis of DNA demethylation and transcriptional reactivation of the FMR1 gene in fragile X cells treated with 5-azadeoxycytidine

Cited by 107 publications

References 35 publications

Epigenetics, fragile X syndrome and transcriptional therapy

Epigenetics, fragile X syndrome and transcriptional therapy

Genetic redundancy in human cervical carcinoma cells: Identification of cells with “normal” properties

Epigenetic analysis reveals a euchromatic configuration in the FMR1 unmethylated full mutations

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