Mbd1 mutant adult NSPCs exhibited increased Fgf-2 expression. A–D, NSPCs cultured under proliferating conditions expressed the neural progenitor marker Nestin (cytoplasmic; red), and Sox2 (nuclear; green). 4′,6-diamidino-2-phenylindole (dapi) (blue) stained all nuclei. E, quantitative real time PCR analyses showing that the Fgf-2 mRNA level was significantly higher in Mbd1^-/- (KO) NSPCs compared with WT NSPCs (n = 3; ^*, p < 0.05). F, Western blotting analysis showing that Mbd1 protein was absent in the KO NSPCs (top) and that both nuclear (nu; middle) and cytoplasmic (cyto; bottom) Fgf-2 were significantly higher in KO NSPCs compared with WT NSPCs. β-Actin was used as a protein loading control.

Acute knockdown of Mbd1 led to increased Fgf-2 expression in adult NSPCs. A, transient cotransfection of a His-tagged Mbd1 expression plasmid with four different Mbd1-RNAi duplexes (D1, D2, D3, and D4) into HEK293 cells. The result indicated that the Mbd1-RNAi duplex D1 and to a lesser extent D4 significantly inhibited the expression of Mbd1 protein. Mbd1 expression was detected by an anti-His tag antibody. β-Actin was used as a control. B, semiquantitative reverse transcription-PCR showing that transfected Mbd1-RNAi D1 resulted in reduced endogenous Mbd1 mRNA levels in adult WT NSPCs. Gapdh was used as an internal control. C, quantitative real time PCR assays showing that transfected Mbd1-RNAi D1 resulted in an increased endogenous Fgf-2 mRNA level in adult NSPCs, and the higher transfection efficiency corresponded to further increases in Fgf-2 mRNA levels. T.E., transfection efficiency; ^*, p < 0.05; ^**, p < 0.01. D–G, phase-contrast (D and F) and fluorescence (E and G) images showing adult NSPCs infected with recombinant lentivirus expressing either a control RNAi (D and E) or Mbd1-RNAi (F and G). GFP was coexpressed from the lentiviral vectors. Note that recombinant lentiviruses infected adult WT NSPCs with high efficiency, and Mbd1-RNAi lentivirus-infected WT NSPCs adopted a distinctive morphology compared with control RNAi-infected cells. H and I, infection of adult WT NSPCs with two different Mbd1-RNAi lentiviruses (RNAi-A and RNAi-B) led to decreased Mbd1 expression and increased Fgf-2 expression at both mRNA levels (H; PCR) and protein levels (I; Western blotting).

Overexpression of Mbd1 led to decreased Fgf-2 expression. A, reverse transcription-PCR analyses showing that overexpression of Mbd1 carried by lentivirus led to decreased Fgf-2 mRNA expression in both KO and WT NSPCs. B, real time quantitative PCR results confirmed that Fgf-2 mRNA levels were significantly higher in KO NSPCs compared with WT NSPCs when both of them were infected with control lentivirus (GFP), consistent with results shown in Fig. 1. Mbd1 overexpression led to decreased Fgf-2 expression in both KO and WT NSPCs (^*, p < 0.05; ^**, p < 0.01). C and D, chromatin immunoprecipitation assays demonstrated that endogenous Mbd1 bound to Fgf-2 promoter in both mouse (C) and rat (D) adult NSPCs. Input DNA, positive control. Con Ab, an antibody against GFP was used as a negative control for immunoprecipitation. No Ab, rabbit IgG was used as negative control for immunoprecipitation. The PCR primers used to amplify precipitated Fgf-2 promoter, corresponding to “Region 2” of the Fgf-2 promoter, are indicated in Fig. 4A.

The Fgf-2 promoter was hypomethylated in Mbd1^-/- NSPCs, and such differential methylation was specific to the Fgf-2 promoter. A, schematic view of mouse Fgf-2 promoter, focusing on two regions that were analyzed in this study. We found no difference in DNA methylation in the CpG-rich Region 1 containing 45 CpGs. Region 2 contains nine CpGs, and CpG#1 is adjacent to the putative binding sites of several transcription factors. PCR primers used for bisulfate sequencing analysis are indicated (Region 1, Fgf 2.5′D + Fgf 2.3′D; Region 2, Fgf 2.5′+ Fgf 2.3′). B, the percentage of methylation at each CpG in Region 1 was compared between WT and KO NSPCs. The analyses were done using four independent samples, and the result from one experiment is shown as an example; the reduced methylation of CpG#1 was further confirmed using methylation-sensitive PCR (inset; M, methylated product; U, unmethylated product). C, Fgf-2 promoter methylation analyzed from four independent experiments showing significant reductions in the percentage of promoter methylation at all CpG sites (All CpGs; ^**, p < 0.01) and at CpG#1 (^*, p < 0.05) in KO NSPCs. D, the promoter of imprinted H19 genes was not differentially methylated in KO NSPCs. E, global DNA methylation was not altered in KO NSPCs, as demonstrated by Southern blotting using methylation-sensitive HpaII (H) and methylation-insensitive MspI (M) restriction enzyme-digested genomic DNA and a pericentric minor satellite probe.

Down-regulation of Mbd1 and overexpression of Fgf-2 in WT adult NSPCs inhibited neuronal differentiation. A–H, compared with lentivirus-nonsilencing (NC) control RNAi-infected NSPCs (A–C), lentivirus-Mbd1-RNAi-infected NSPCs (E–H) differentiated into fewer TuJ1-positive (red) neurons (A–D). Green, GFP coexpressed with RNAi. D, quantitative data comparing the percentages of TuJ1-positive neurons differentiated from lentivirus-infected (GFP+) NSPCs indicated that down-regulation of Mbd1 in NSPCs leads to decreased neuronal differentiation. The data were from two independent experiments with triplicates performed. I–L, nearly all NSPCs infected by lentivirus-GFP were GFP-positive. M–P, Fgf-2 lentivirus-infected WT NSPCs. Note that although both GFP lentivirus- and Fgf-2 lentivirus-infected NSPCs could differentiate into Tuj1-positive neurons (red), the percentage of Tuj1-positive cells was significantly decreased in Fgf-2-overexpressing NSPCs (E–G). H, quantitative data comparing the percentages of TuJ1-positive neurons differentiated from either GFP- or Fgf-2-overexpressing NSPCs indicated that overexpression of Fgf-2 inhibited the neuronal differentiation of NSPCs. ^*, p < 0.05 (scale bar, 100 μm). DAPI, 4′,6-diamidino-2-phenylindole.