a, RT-PCR analysis and b, Q.RT-PCR analysis of total RNA isolated from ES and YHC cells grown under self-renewing conditions were performed using primers specific for markers color-coded for ectoderm, mesoderm, endoderm, and trophectoderm. Gapdh was used as an internal control. * p = 0.001; ** p = 0.003; *** p = 0.006; **** p = 0.012. The values are represented as mean +/− SD (n=3). c, EB cells derived from YHC cells show lineage markers that are either absent or significantly reduced in EB cells derived from wild-type mES cells. RT-PCR analysis of total RNA isolated from wild-type EB cells and YHC EB cells was performed using specific primers for various differentiated lineage genes (shown above each lane). Gapdh was used as a loading control. d, Q.RT-PCR analysis of total RNA isolated from mES cells treated with either non-targeting siRNA (NT) or siREST was performed using primers specific for different lineage markers. Analysis was performed 5 d after transfection. * p < 0.0001; ** p = 0.001; *** p = 0.002; **** p = 0.004; **** p = 0.005; ****** p = 0.01. The values are represented as mean +/− SD (n=3). e, Inner cell mass of blastocysts showed coexpression of REST and self-renewal markers Oct4, Nanog, and Sox2. Blastocysts were stained for nuclei (DAPI, blue), REST (red), and self-renewal markers (green). Merged and differential interference contrast (DIC) images are also shown. Scale bars represent 25 μm.

a, Addition of exogenous precursor-miR-21 (pre-miR-21), but not non-targeting siRNA (NT) or pre-miR-26a, caused a decrease of self-renewal in mES cells. * p < 0.0001. The error bars correspond to three replicates (n=3). b, Addition of exogenous pre-miR-21 and exogenous pre-miR-26a but not NT to mES cells resulted in increased levels of their corresponding miR expression levels. Results of Q.RT-PCR assays performed with NT-, pre-miR-26a- and pre-miR-21-transfected cells 1 d after transfection are shown. * p < 0.0001. The values are represented as mean +/− SD (n=3). c, The effect of miR-21 on loss of self-renewal was specific. Addition of exogenous pre-miR-21 along with anti-miR-21 rescued the loss of self renewal by pre-miR-21 alone. * p < 0.0001 (there was no significant difference between NT and Pre-miR-21 plus anti-miR-21 values). The error bars correspond to three replicates (n=3). d, Addition of exogenous pre-miR-21, but not NT, to mES cells resulted in loss of self-renewal markers Oct4, Nanog, Sox2, and c-Myc. Q.R-PCR after transfection of mES cells with pre-miR-21 and NT was performed 5 d after transfection to detect the expression levels of the self-renewal markers. * p < 0.0001; ** p = 0.002; *** p = 0.007; **** p = 0.008. The values are represented as mean +/− SD (n=3). e, Proposed model of REST-mediated maintenance of self-renewal through miR-21. REST maintains self-renewal and pluripotency of mES cells by suppressing miR-21 expression.

a, EB cells (ES cells grown in the absence of LIF under nonadherent conditions for 4 d) showed reduced levels of self-renewal markers and REST compared with ES cells (grown in the presence of LIF under adherent conditions). Western blot analysis of whole-cell extracts prepared from ES cells and EB cells showed an association between expression of REST and self-renewal markers. Actin was used as a loading control. b–f, Mouse ESC lines with heterozygous deletion of REST (RRC and YHC) show loss of self-renewal. b, RT-PCR analysis and c, Q.RT-PCR analysis with different primer sets of total RNA isolated from ES, RRC, and YHC cells showed reduced REST transcripts in REST^+/− cells. Gapdh was used as a loading control. * p < 0.0001. The values are represented as mean +/− SD (n=3). d, Western blot analysis of whole-cell extracts from ES, RRC, and YHC cells showed reduced REST protein in REST^+/− cells. α-Tubulin was used as a loading control. e, Alkaline phosphatase staining of ES colonies showed loss of self-renewal in REST^+/− cells as compared with wild-type ES cells f, Percentages of self-renewing colonies of ES, RRC, and YHC cells calculated after alkaline phosphatase assays when cultured under self-renewing conditions showed significant reductions in the self-renewal capacity of both REST^+/− cell lines compared with ES. * p < 0.0001. The error bars correspond to three replicates (n=3). g–i, siRNA-mediated knockdown of REST caused loss of self-renewal in mES cells. g, Specific knockdown of targeted genes was achieved using siRNA. Q.RT-PCR of total RNA purified from mES cells treated with siREST or siOct4 showed knockdown of specific genes. Analysis was performed 5 d after transfection. * p < 0.0001. The values are represented as mean +/− SD (n=3). h, Western blotting showed reduced REST protein levels in siREST treated cells compared with control (NT siRNA). i, siREST- and siOct4-treated cells showed less self-renewal than NT-treated cells did. mES cell colonies were screened by alkaline phosphatase assays. * p < 0.0001; ** p < 0.001. The error bars correspond to three replicates (n=3). j, Exogenously added REST, but not GFP, maintained self-renewal in mES cells cultured under differentiation conditions. mES cells were transfected with plasmids encoding GFP or REST and grown in the absence of LIF. Percentages of self-renewing colonies from three independent experiments were averaged after alkaline phosphatase assay and are shown for each transfected gene. *** p < 0.01. The error bars correspond to three replicates (n=3).

a and 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. α-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. Western blot analysis of whole-cell extracts from mES 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. α-Tubulin was used as a loading control. d–h, REST suppresses the expression of miRs, which are associated with the absence of self-renewal regulators. d, Chromatin immunoprecipitation assays and e, quantitative chromatin immunoprecipitation assays showed that REST binds to the gene chromatin of a set of miRs 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 +/− SD. f, Q.RT-PCR analysis of ES, YHC, RRC, EB, and EB+REST showed that expression of the miRs (shown in D and E) was lower in ES than in YHC, RRC, and EB cells. The higher expression of miRs 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 +/− SD (n=3). g, The expression of the miRs shown in (D) was upregulated in a loss-of-function experiment when mES 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 +/− SD (n=3). Analysis was performed 3 d after transfection. h, siREST-mediated knockdown of REST that produced increased expression of the miRs in (G) corresponded to the decreased expression of REST and the known self-renewal genes Oct4, Nanog, and Sox2. Q.RT-PCR assay with mES cells treated with siREST and NT are shown. Analysis was performed 5 d after transfection. * p < 0.0001. The values are represented as mean +/− SD (n=3).