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

Abstract

Background: 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 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.

Results: We 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.

Conclusions: We 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.

Keywords: 5-hydroxymethylation (5hmC); Chromatin profiling; Clinical biomarker; Epigenetic silencing, FMR1; Fragile X syndrome (FXS).

Fig. 1

FMR1 locus (h)MeDIP profiling identifies novel regions of 5mC and 5hmC changes in FXS patient PBMC samples. a. Experimental overview. Methylated DNA immunoprecipitation (MeDIP) assay was used to profile DNA methylation (5mC), DNA hydroxymethylation (5hmC). Chromatin immunoprecipitation (ChIP) was used to profile histone post-translational modifications (PTM). Antibody (Ab) b–c. The graphs illustrate the relative enrichment of the indicated mark (log2 fold enrichment) in FXS (red) and control (blue) PBMCs. The upper panel illustrates the chromosomal and genomic location locations as well as the indicated referenced Refseq genes: FMR1, and referenced antisense non coding RNAs (FMR1AS1 and L29074.3). This snapshot illustrates the epigenetic landscape over a region covering 79 kb on ChrX: 146971000-147050000 (c). RPL19 and GAPDH provide controls regions of no changes in 5mC and 5hmC (b). In the graphs, the thicker lines indicate higher deviation between biological replicates (controls n = 4; FXS n = 8) (aggregation mean, sliding window 529 bp). Regions I to V were selected as regions of strongest apparent epigenetic variation across epigenetic marks and cell types based on epigenomic landscape visualization illustrated in Figs. 1 and 3. The chromosome coordinates of these regions are provided in Table 1

Fig. 2

Inter-individual variation in 5mC and 5hmC levels across the FMR1 locus of FXS patient PBMC samples. a Selected FMR1 genomic regions of methylation change (Table 1) throughout the FMR1 locus were interrogated by (h)MeDIP-PCR in DNA extracted from four control (blue) and eight FXS (red) patient PBMCs samples, data represent the mean of relative enrichment to input in log2 with standard deviation SD (left panels) and individual data for the 2I3-4 locus (right panels). Significance levels of the mean difference in control and FXS PBMCs is indicated by triple asterisks p ≤ 0.001, double asterisks p ≤ 0.01, single asterisk p ≤ 0.05, or no star, p > 0.05 using a t test with unequal variance (Additional file 11: Table S4). The location of qPCR primer pairs (Additional file 9: Table S2) used in this study is illustrated. Data for inter-individual 5mC and 5hmC variation across all selected FMR1 loci is available in Additional file 2: Figure S1 and Additional file 3: Figure S2. Arrows in a, b, and c illustrate observed anti-correlation between 5mC and 5hmC enrichment in two FXS patients samples B and E. b (h)MedIP-array methylation profiles surrounding FMR1 transcriptional start site are represented in eight individual FXS patient samples (A to H in red) as compared to control samples (blue) for 5mC and 5hmC. c Pyrosequencing at the FMR1 promoter region, using a commercial assay (HsFMR1, QIAGEN) located at proximity of the FMR1 start site and a newly designed assay (B3) in a novel region of methylation changes located 738 bp downstream of the TSS confirm DNA methylation enrichment at the base resolution level in all eight patients. The genomic location of measured CpGs relative to the TSS is indicated (see Additional file 10: Table S3)

Fig. 3

Broad, cell type-specific, epigenetic changes at the FMR1 locus in FXS cells. The graphs illustrate the relative enrichment (log2 fold enrichment) of the indicated chromatin marks detected by ChIP (H3K27me3, H3K9me2 and -me3, H4K20me1 and -me3 H3K36me3, H3K4me2, H3K9ac, H4K16ac) and (h)MeDIP (5mC and 5hmC) in FXS (red) and control (blue) fibroblasts. The upper panel illustrates the chromosomal and genomic location (region covering 79 kb on ChrX: 146971000–147050000) as well as the indicated referenced Refseq genes: FMR1 and referenced antisense non coding RNAs (FMR1AS1 and L29074.3). In the graphs, the thicker lines indicate higher deviation between biological replicates (FXS n = 3) (aggregation mean, sliding window 529 bp). ChIP-PCR validation data is available Additional file 4: Figure S3

Fig. 4

Repressive epigenetic switch spanning entire FMR1 locus in FXS PBMCs. Relative ChIP-qPCR enrichment of the indicated chromatin histone post-translational modifications (H3K4me2, H3K9me2, H3K9me3, and H4K20me1) in three control (blue) and eight (red) patients samples (A to H, as in Fig. 2). Data represent the mean of relative enrichment to input in log2 with standard deviation SD (left panels). Significance levels of the mean difference in control and FXS PBMCs is indicated by triple asterisks p ≤ 0.001, double asterisks p ≤ 0.01, single asterisk p ≤ 0.05, or no star p > 0.05 using a t test with unequal variance (Additional file 11: Table S4). The bar graphs on the right hand-side represents individual patient data for the indicated marks in region 2I3-4 located in the first intron of the FMR1 gene. The upper panel illustrates the FMR1 locus organization with pyrosequencing (HsFMR1 and B3) as well as PCR assays locations (listed in Additional file 1: Table S1 and Additional file 9: Table S2)

Fig. 5

FMR1 5hmC levels correlate with transcriptionally active chromatin and is reciprocal to 5mC levels at the FMR1 TSS. a Hierarchical clustering using Ward’s method for the different indicated molecular endpoints at the region surrounding FMR1 TSS. Yellow color represents the highest values, blue color the lowest, and the black the mean. b–c Linear regression analysis of indicated molecular assays (5mC, 5hmC, or FMR1 mRNA expression) in eight FXS patients PBMC samples. The coefficient of determination denoted RSq and the p value are indicated. The dots gradient coloring represents the mRNA expression level for each sample, black for the highest expression level, and light grey for the lowest level. All results from linear regression analyses are available from Additional file 6: Table S5

Fig. 6

FMR1 5hmC levels anti-correlate with FXS patient disease severity. a FMR1 locus representation with indicated assay location (Additional file 9: Table S2 and Additional file 10: Table S3). b–d Linear regression analysis of indicated molecular assays (5mC vs 5hmC) and ABC-C clinical score endpoints in eight FXS patients PBMC samples. The coefficient of determination denoted RSq and the p value are indicated. The dots gradient coloring represents the mRNA expression level for each sample, black for the highest expression level, and light grey for the lowest level. Aberrant Behavior Checklist (ABC-C) (sub)-scores (the higher, the more severe). All results from linear regression analyses are available from Additional file 8: Table S6