Reciprocal changes in DNA methylation and hydroxymethylation and a broad repressive epigenetic switch characterize FMR1 transcriptional silencing in fragile X syndrome.

Sarah Brasa,Izabela Rozenberg,Florian Hahne,Sébastien Jacquemont,Fabrizio Gasparini,Baltazar Gomez-Mancilla,Salah-Dine Chibout,Jonathan Moggs,Rémi Terranova,Olivier Grenet,Thomas Peters,Yunsheng He,Christine Mccormack,Arne Mueller

doi:10.1186/s13148-016-0181-x

Sarah Brasa, Izabela Rozenberg + Show 12 more

Open Access

https://doi.org/10.1186/s13148-016-0181-x

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Abstract

BackgroundFragile X syndrome (FXS) is the most common form of inherited intellectual disability, resulting from the loss of function of the fragile X mental retardation 1 (FMR1) gene. The molecular pathways associated with FMR1 epigenetic silencing are still elusive, and their characterization may enhance the discovery of novel therapeutic targets as well as the development of novel clinical biomarkers for disease status.ResultsWe have deployed customized epigenomic profiling assays to comprehensively map the FMR1 locus chromatin landscape in peripheral mononuclear blood cells (PBMCs) from eight FXS patients and in fibroblast cell lines derived from three FXS patient. Deoxyribonucleic acid (DNA) methylation (5-methylcytosine (5mC)) and hydroxymethylation (5-hydroxymethylcytosine (5hmC)) profiling using methylated DNA immunoprecipitation (MeDIP) combined with a custom FMR1 microarray identifies novel regions of DNA (hydroxy)methylation changes within the FMR1 gene body as well as in proximal flanking regions. At the region surrounding the FMR1 transcriptional start sites, increased levels of 5mC were associated to reciprocal changes in 5hmC, representing a novel molecular feature of FXS disease. Locus-specific validation of FMR1 5mC and 5hmC changes highlighted inter-individual differences that may account for the expected DNA methylation mosaicism observed at the FMR1 locus in FXS patients. Chromatin immunoprecipitation (ChIP) profiling of FMR1 histone modifications, together with 5mC/5hmC and gene expression analyses, support a functional relationship between 5hmC levels and FMR1 transcriptional activation and reveal cell-type specific differences in FMR1 epigenetic regulation. Furthermore, whilst 5mC FMR1 levels positively correlated with FXS disease severity (clinical scores of aberrant behavior), our data reveal for the first time an inverse correlation between 5hmC FMR1 levels and FXS disease severity.ConclusionsWe identify novel, cell-type specific, regions of FMR1 epigenetic changes in FXS patient cells, providing new insights into the molecular mechanisms of FXS. We propose that the combined measurement of 5mC and 5hmC at selected regions of the FMR1 locus may significantly enhance FXS clinical diagnostics and patient stratification.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-016-0181-x) contains supplementary material, which is available to authorized users.

Highlights

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability, resulting from the loss of function of the fragile X mental retardation 1 (FMR1) gene
The syndrome is commonly associated with the expansion of cytosine-guanine-guanine (CGG) trinucleotide repeats in the 5′ untranslated region (5′UTR) of the human fragile X mental retardation 1 (FMR1) gene
In patients with more than 200 CGG repeats, the aberrant, CGG-repeat expanded FMR1 messenger ribonucleic acid mediates FMR1 gene silencing [3] resulting in the absence of fragile X mental retardation protein (FMRP) expression, a translational regulator involved in neurotransmitter mediated synaptic maturation and plasticity [2]

Summary

Introduction

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability, resulting from the loss of function of the fragile X mental retardation 1 (FMR1) gene. Aberrant deoxyribonucleic acid (DNA) hypermethylation of the FMR1 promoter CpG island and CGG repeats is strongly associated with FMR1 gene silencing and represents a molecular hallmark of full-mutation FXS patients [6,7,8,9,10,11]. A small number of full-mutation, unmethylated individuals have been reported [23] These patients displayed a FMR1 promoter epigenetic pattern comparable to that of normal controls, in accordance with normal transcription levels and consistent with a euchromatic configuration. The mechanisms preventing the initial methylation and protecting against a repressive chromatin configuration are unknown, and their identification may help understand the key pathways affected in the majority of full-mutation patients and might lead to new therapeutic opportunities for restoring normal FMR1 expression levels in FXS patients [12, 24]

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