Novel STAT3 target genes exert distinct roles in the inhibition of mesoderm and endoderm differentiation in cooperation with Nanog

The Leukemia Inhibitory factor (LIF) activates the transcription factor STAT3, which results in the maintenance of mouse embryonic stem cells in the pluripotent state by inhibiting both mesodermal and endodermal differentiation. How the LI/STAT3 pathway inhibits commitment to both mesoderm and endoderm lineages is presently unknown. Using a hormone-dependent STAT3 and with microarray analysis, we identified 58 targets of STAT3 including 20 unknown genes. Functional analysis showed that 22 among the 23 STAT3 target genes analysed contribute to the maintenance of the undifferentiated state, as evidenced by an increase in the frequency of differentiated colonies in a self-renewal assay, and a concomitant elevation of early differentiation markers upon knockdown. Fourteen of them, including Dact1, Klf4, Klf5, Rgs16, Smad7, Ccrn4l, Cnnm1, Ocln, Ier3, Pim-1, Cyr61, and Sgk, were also regulated by Nanog. Analysis of lineage-specific markers showed that the STAT3 target genes fell into three distinct categories depending on their capacity to inhibit either mesoderm or endoderm differentiation, or both. The identification of genes which harness self-renewal, and are downstream targets of both STAT3 and Nanog, shed light on the mechanisms underlying functional redundancy between STAT3 and Nanog in mouse ES cells.