Figure 7

MeCP2 recruits HDAC activity, promoting local histone hypoacetylation and exon inclusion. (A) Schematic representation showing the partial structure of the FAM204A gene. The red squares (a, b, and c) below the gene indicate the locations of qPCR probes used for ChIP assays. (B) Comparison of expression level of FAM204A isoforms that excludes (red) or includes (blue) exon 2. Expression of the FAM204A isoforms was determined by RNA-Seq data analyzed by MISO (confirmed by qPCR, data not shown). The normalized expression levels are displayed as a percentage, where the total expression of the two isoforms sum to 100%. WT, HCT116-WT cells; DKO, HCT116-DKO cells; TSA, TSA-treated HCT116-WT cells; MeCP2-KD, MeCP2-knockdown HCT116-WT cells. (C) Knockdown of MeCP2 decreased MeCP2 binding to the exon 1 (probe a) and ASE (probe b) regions of the FAM204A gene. MeCP2 occupancy at different DNA regions was measured using the ChIP assays with chromatin harvested from control (shLuc) or MeCP2-knockdown (MeCP2-KD) HCT116 cells, followed by quantification using the qPCR assays. (D) Knockdown of MeCP2 resulted in a significant increase of histone acetylation over the ASE region (probe b). Acetylation level was measured using ChIP-qPCR assays with chromatin harvested from the cells as described in C. (E) DNA demethylation by DNMT-deficiency resulted in decreased binding of MeCP2 to the ASE region (probe b). MeCP2 binding was measured using ChIP-qPCR assays with chromatin harvested from HCT116-WT and HCT116-DKO cells. (F) DNA demethylation by DNMT-deficiency resulted in increased histone acetylation over the ASE region (probe b). MeCP2 binding was measured using ChIP-qPCR assays with chromatin harvested from HCT116-WT and HCT116-DKO cells. (G) Inhibition of HDAC activity did not significantly change the binding of MeCP2 to the ASE region. MeCP2 binding was measured using ChIP-qPCR assays with chromatin from untreated (CTR) and TSA-treated HCT116-WT cells. Error bars in C-G represent SE. P-values shown in C-G were calculated using one-sided Student's t-test. N.S., not significant. (H) Cartoon showing the three independent treatments that downregulate the inclusion of ASEs: (1) inhibition of DNA methylation, (2) knockdown of MeCP2 and (3) inhibition of HDAC activity using TSA treatment. (I) Model depicting the relationship of DNA methylation, MeCP2 binding, HDAC recruitment and Pol II elongation in exon recognition.