Figure 4.

Long-lasting effect of 5-azadC and methylome analysis after treatment. In A are reported the transcription and translation results of a recent study [66]. The upper panel reports the scheme of 5-azadC treatment with the relative time points. Relative quantification of FMR1-mRNA by RT-PCR after treatments with 5-azadC of 2 different FXS lymphoblastoid cell lines showed increased FMR1-mRNA expression at T3 (8 days after last drug administration), decreasing at T6 (18 days after last drug administration) and T7 (22 days after last drug administration). Western blot with antibody against FMRP and GAPDH on FXS cell extracts demonstrated that after treatment the expression of FMRP was restored and disappeared after 22 days (T7) from the end of the treatment (Panel A). Panel B. Bisulphite sequencing of the methylation boundary including the CpG island of the FMR1 promoter region before (top-left) and after (bottom-left) treatment with 5-azadC of WT cells showed no substantial modification of the methylation profile, while an almost complete demethylation of the promoter region, not affecting the methylation boundary was observed in FXS cells (top right). The methylation boundary is indicated by the arrow. Dendrogram of the methylation profile of the analyzed samples demonstrated a clustering of untreated samples compare to treated ones. The heat map shows some changes in the methylation profile after treatment, that however do not reach statistical significance (p > 0.05). Data derived from ref. [67].