Fig. 3.

Temporal requirement for FMR1 mRNA binding to the FMR1 promoter. (A) The small molecule 1a blocks the drop in FMRP levels during days 31 to 60 of differentiation. To determine when FMR1 mRNA is required for FMR1 silencing, we applied 1a to FXS hESCs during days 31 to 60 of differentiation. 1a maintained FMRP expression in FXS neurons. (B and C) 1a prevents FMR1 silencing. In FXS neurons, application of 1a during days 31 to 60 of differentiation was sufficient to maintain FMR1 mRNA levels [(B), quantitative reverse transcription polymerase chain reaction (qRT-PCR), n = 4 per condition], high levels of H3K4me2 and low levels of H3K9me2 histone modifications [(C), chromatin immunoprecipitation, n = 4 per condition]. Control hESCs were unaffected by 1a. Application of 1a (10 μM) during days 1 to 30 of differentiation failed to prevent FMR1 silencing (see fig. S12). Data are means ± SEM; Student's t test (two-tailed distribution, **P < 0.01, ***P < 0.001). Different conditions on the same cell line were considered as two samples with equal variance.