We next sought to investigate whether lincRNAs play a role in regulating the ESC state. Regulation of the ESC state involves two components, maintaining the pluripotency program and repressing differentiation programs15. To determine whether lincRNAs play a role in the maintenance of the pluripotency program, we studied their effects on the expression of Nanog, a key transcription factor that is required to establish28 and uniquely marks the pluripotent state29,30. We infected ESCs carrying a luciferase reporter gene expressed from the endogenous Nanog promoter31 with shRNAs targeting lincRNAs or protein-coding genes. We monitored loss of reporter activity after 8 days relative to 25 negative control hairpins across biological replicates (see Methods). To ensure that the observed effects were not simply due to a reduction in cell viability, we excluded shRNAs that caused a reduction in cell numbers (see Methods, Supplemental Figure 4, Supplemental Table 7). Altogether, we identified 26 lincRNAs that had major effects on endogenous Nanog levels with many at comparable levels to the knockdown of the known protein-coding regulators of pluripotency such as Oct4 and Nanog (Figure 2a, Supplemental Table 7). This establishes that these lincRNAs have a role in maintaining the pluripotent state.
