REST maintains self-renewal and pluripotency of embryonic stem cells

The neuronal repressor REST ( RE1- silencing transcription factor; also called NRSF) is expressed at high levels in mouse embryonic stem ( ES) cells(1), but its role in these cells is unclear. Here we show that REST maintains self- renewal and pluripotency in mouse ES cells through suppression of the microRNA miR- 21. We found that, as with known self- renewal markers, the level of REST expression is much higher in self- renewing mouse ES cells than in differentiating mouse ES ( embryoid body, EB) cells. Heterozygous deletion of Rest ( Rest(+/-)) and its short- interfering-RNA- mediated knockdown in mouse ES cells cause a loss of self- renewal - even when these cells are grown under self- renewal conditions - and lead to the expression of markers specific for multiple lineages. Conversely, exogenously added REST maintains self- renewal in mouse EB cells. Furthermore, Rest(+/-) mouse ES cells cultured under self- renewal conditions express substantially reduced levels of several self- renewal regulators, including Oct4 ( also called Pou5f1), Nanog, Sox2 and c- Myc, and exogenously added REST in mouse EB cells maintains the self- renewal phenotypes and expression of these self- renewal regulators. We also show that in mouse ES cells, REST is bound to the gene chromatin of a set of miRNAs that potentially target self- renewal genes. Whereas mouse ES cells and mouse EB cells containing exogenously added REST express lower levels of these miRNAs, EB cells, Rest(+/-) ES cells and ES cells treated with short interfering RNA targeting Rest express higher levels of these miRNAs. At least one of these REST- regulated miRNAs, miR- 21, specifically suppresses the self- renewal of mouse ES cells, corresponding to the decreased expression of Oct4, Nanog, Sox2 and c- Myc. Thus, REST is a newly discovered element of the interconnected regulatory network that maintains the self- renewal and pluripotency of mouse ES cells.