FMR1 epigenetic silencing commonly occurs in undifferentiated fragile X-affected embryonic stem cells

Abstract

Fragile X syndrome (FXS) is the most common heritable form of cognitive impairment. It results from epigenetic silencing of the X-linked FMR1 gene by a CGG expansion in its 5'-untranslated region. Taking advantage of a large set of FXS-affected human embryonic stem cell (HESC) lines and isogenic subclones derived from them, we show that FMR1 hypermethylation commonly occurs in the undifferentiated state (six of nine lines, ranging from 24% to 65%). In addition, we demonstrate that hypermethylation is tightly linked with FMR1 transcriptional inactivation in undifferentiated cells, coincides with loss of H3K4me2 and gain of H3K9me3, and is unrelated to CTCF binding. Taken together, these results demonstrate that FMR1 epigenetic gene silencing takes place in FXS HESCs and clearly highlights the importance of examining multiple cell lines when investigating FXS and most likely other epigenetically regulated diseases.

Figure 1

CGG Expansion Size and Methylation State in FXS HESC Lines (A) CGG expansion size and methylation state were determined by a methylation-sensitive Southern blot assay. This test distinguishes between unmethylated normal (2.8 kb), premutation (2.9–3.4 kb), and full-mutation alleles (3.4–5.8 kb) and their methylated equivalents, as indicated by 5.2 kb, 5.3–5.8 kb, and fragments larger than 5.8 kb, respectively. Full mutations were identified in nine different cell lines (including HEFX): four females and five males. Note that SZ-FX7, SZ-FX12 and LS-FX9 are mosaic for the full mutation (carry both full and premutations) and that most cell lines are aberrantly methylated (SZ-FX1,3,6,8,14 and LS-FX9). Xi patterns are observed within XX FXS cells. (B) Schematic illustration of the regions analyzed next to the repeats (top) and the bisulfite pyrosequencing results for DNA methylation levels at CpG sites localized 5′ (16 CpGs) and 3′ (6 CpGs) to the CGGs (bottom).

Figure 2

Histone Modifications, FMR1 Expression and CTCF Binding in FXS HESCs (A) Real-time PCR ChIP analysis for H3K9me3 and H3K4me2 in WT and FXS-affected HESCs. APRT and HOXA9 were used as negative and positive controls for H3K9me3, respectively, while CRYSTALIN and APRT were used as negative and positive controls for H3K4me2, respectively (both positive controls were set to one and are not presented). The data in each panel represents an average of three to five independent ChIP experiments. Error bars represent standard error (paired t test, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001). (B) Mean real-time RT-PCR analysis of FMR1 transcription in XY (left) and XX (right) WT, FXS affected, and premutation (SZ-FX4) HESC lines. The expression level of each cell line represents an average of three to seven independent experiments. Cycle threshold (Ct) values were normalized to the corresponding Ct value of GAPDH. XY WT cell lines are B200, and the XX WT cell line is B123. Error bars represent standard error (t test for unequal variances, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001). (C) Western blot analysis of FXS HESCs confirming the expressions of FMRP (∼72 kD) in WT and XY FXS HESCs. GAPDH (40 kD) was used as a loading control. (D) ChIP analysis of CTCF relative enrichment along the FMR1 locus (−300 to +10 Kb relative to the transcription start site) in WT and FXS-affected HESCs. APRT was used as a negative control, and the average of DMPK (DM1) and FRATAXIN (FXN) genes was used as a positive control (the average of positive controls was set to one). The data in each panel represent an average of three independent ChIP experiments. Error bars represent standard error.

Figure 3

FMR1 Transcription, Methylation Levels and Histone Modifications in Subclones of FXS HESCs (A) Mean relative FMR1 transcription levels of subclones from SZ-FX6 (left) and SZ-FX14 (right). Two independent qRT-PCR experiments were performed on a single passage culture. Ct values were normalized to the corresponding Ct value of GAPDH. Error bars represent standard error. (B) Bisufite pyrosequencing for methylation levels near the CGGs (5′ left and 3′ right) in several of FXS HESC (SZ-FX6 and SZ-FX14) subclones. (C) Real-time PCR ChIP analysis of H3K9me3 and H3K4me2 immediately upstream to the CGGs, in hypomethylated (SZ-FX6 cl.12B) versus hypermethylated (SZ-FX6 cl.15B) isogenic subclones. APRT and CRYSTALIN were used as negative controls for H3K9me3 and H3K4me2, respectively. The data in each panel represent an average of three to five independent ChIP experiments (paired t test, ^∗∗p < 0.01, ^∗∗∗p < 0.001). Error bars represent standard error.