Figure 7.

AKT Activation Suppresses Mbd3

(A–F) Summary of the procedure used to culture cells and isolate samples for microarray analysis (B–F). Gonads were surgically isolated from E11.5 AKT-mer embryos. After making a single cell suspension, PGCs were isolated based on SSEA-1 expression using a FACS AriaII cell sorter. Purified PGCs were seeded onto m220 feeder cells in ESC medium containing bFGF. One day after seeding, the indicated chemicals were added. On day 2, pluripotent candidate cells were sorted based on SSEA-1 expression for microarray analysis (Nagamatsu and Suda, 2013). 2i comprised inhibitors of MEK and glycogen synthase kinase-β (GSK3-β). A83 indicates the transforming growth factor-β (TGF-β) type 1R inhibitor. (B) Array heatmap analysis for pluripotent candidate cells at day 2 of each treatment. Mbd3 and Mbd3 targets of MEFs in which four reprogramming factors (Oct3/4, Sox2, Klf4, and c-Myc [OSKM]) were transduced are shown. These target genes are from Rais et al. (2013). (C) The number of MBD3 targets of OSKM-transduced MEFs in BLIMP-1 modules. Bars indicate the total number of genes in each BLIMP-1 module (Figure 4A). Red indicates MBD3 target genes of OSKM-transduced MEFs. (D) Number of MBD3 targets of OSKM-transduced MEFs among all BLIMP-1 targets (Magnúsdóttir et al., 2013). (E) Number of MBD3 targets of OSKM-transduced MEFs in ESC module (Kim et al., 2010). Bars indicate the total number of genes in each regulated module of ESCs. Red indicates MBD3 target genes of OSKM-transduced MEFs. (F) GSEA of BLIMP-1 targets for pluripotent candidate cells at day 2 of each treatment. BLIMP-1 modules were the gene sets identified in Figure 4.

(G) Western blotting of MBD3 after 4-hydroxytamoxifen (4OHT) treatment of MEFs isolated from WT and Akt-mer embryos. MEFs were treated with 4OHT (100 nM). At the indicated time points, MEFs were harvested and MBD3 expression was analyzed by western blot.

(H) The number of 3F (Oct3/4, Sox2 and Klf4)-induced ESC-like colonies formed from Akt-mer MEFs, with or without 4OHT treatment at day 22. 4OHT was added at day 2 and allowed to react until day 6 after the 3F induction. Data represent the mean ± SD of independent experiments. Statistical significance was determined using Student’s t test (n = 6). ^∗∗p < 0.01.

(I) Gene expression regulation of cellular dynamics in the process of the acquisition of pluripotency in PGCs. Events identified in both the current study and a previous study (Nagamatsu et al., 2012a) are shown. The efficient dedifferentiation of Mbd3 deficient PGCs was previously reported (Rais et al., 2013).

See also Figure S3.