FIGURE 3. REST maintains the expression of critical self-renewal regulators and represses expression of a set of miRNAs in mouse ES cells.

a, b, Heterozygous deletion of Rest results in decreased expression of self-renewal genes. a, RT–PCR analysis of total RNA from ES, RRC and YHC cells. Genes are indicated on the left side of each panel. Gapdh was used as a loading control. b, Western blot analysis of whole-cell extracts from ES, RRC and YHC cells. Antibodies specific for REST, c-Myc and Oct4 proteins were used for the analysis. -Tubulin was used as a loading control. c, Exogenously added Rest, but not GFP, maintained the expression of c-Myc and Oct4 in ES cells growing without LIF. Shown is a western blot analysis of whole-cell extracts from mouse ES cells transfected with plasmids encoding GFP or REST and then grown in the absence of LIF. Antibodies specific for REST, c-Myc and Oct4 proteins were used for the analysis. -Tubulin was used as a loading control. d–h, REST suppresses the expression of miRNAs, which are associated with the absence of self-renewal regulators. d, e, ChIP (d) and quantitative ChIP (e) assays showed that REST binds to the gene chromatin of a set of miRNAs in ES cells (1, input; 2, IgG, negative control; 3, anti-H3 antibody, positive control; 4, anti-REST antibody; + and - represent predicted RE-1 binding sites occupied and unoccupied by REST, respectively). *, P < 0.0001; **, P = 0.001. The values of three replicates are represented as mean  s.d. f, qRT–PCR analysis of ES, YHC, RRC, EB and EB plus REST showed that expression of the miRNAs (shown in d and e) was lower in ES than in YHC, RRC and EB cells. The higher expression of miRNAs in EB cells could be extinguished in a gain-of-function experiment in the presence of exogenously added REST. *, P < 0.0001. The values are represented as mean  s.d. (n = 3). g, The expression of the miRNAs shown in d was upregulated in a loss-of-function experiment when mouse ES cells were treated with siRest but not when they are treated with non-targeting siRNA (NT). *, P < 0.0001. The values are represented as mean  s.d. (n = 3). Analysis was performed 3 days after transfection. h, siRest-mediated knockdown of REST that produced increased expression of the miRNAs in g corresponded to the decreased expression of Rest and the known self-renewal genes Oct4, Nanog and Sox2. A qRT–PCR assay with mouse ES cells treated with siRest and NT siRNA is shown. Analysis was performed 5 days after transfection. *, P < 0.0001. The values are represented as mean  s.d. (n = 3).