1. Department of Cancer Genetics,
2. Department of Biochemistry and Molecular Biology,
3. Department of Neuro-Oncology,
4. The Brain Tumor Center,
5. Center for Stem Cell and Developmental Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA
6. Program in Genes and Development, The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas 77030, USA
7. These authors contributed equally to this work.
8. Present address: Laboratory of Genetics, Salk Institute for Biological Studies, La Jolla, California 92037, USA.

Correspondence to: Sadhan Majumder^1,3,4,5,6 Correspondence and requests for materials should be addressed to S.M. (Email: smajumder@mdanderson.org).

The neuronal repressor REST (RE1-silencing transcription factor; also called NRSF) is expressed at high levels in mouse embryonic stem (ES) cells¹, 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.