ABSTRACT: Embryonic stem cell (ESC) self-renewal and pluripotency is controlled by the coordinated action of transcription factors and chromatin regulators. Compared to the pluripotency transcription factors, the function of the chromatin regulators, especially the ATP-dependent chromatin remodelers, remains poorly understood in ESCs. Here, we show that INO80, a SWI/SNF family chromatin remodeling complex, is essential for ESC self-renewal, pluripotency, somatic cell reprogramming, and embryonic development. Ino80, the ATPase of the complex, forms an auto-regulatory loop with the ESC master transcription factors Oct4, Nanog, and Sox2. More importantly, it co-occupies the enhancer regions of most key pluripotency genes with the master transcription factors, and positively regulates their expression by maintaining an open chromatin structure. Our data suggests that INO80 is an integral component of the pluripotency transcription network, and plays a critical role in both the maintenance and establishment of pluripotency Mouse J1 cells were transfected with non-targeting (NT), Ino80, Ino80b, Ino80c or Ino80e siRNAs, and RNA was isolated 96 hours after transfection. agent: non-targeting siRNA: Mock_1, Mock_2 agent: Ino80 siRNA: Ino80a_1, Ino80a_2 agent: Ino80b siRNA: Ino80b_1, Ino80b_2 agent: Ino80c siRNA: Ino80c_1, Ino80c_2 agent: Ino80e siRNA: Ino80e_1, Ino80e_2 cell line: mouse J1 ESC: Mock_1, Mock_2, Ino80a_1, Ino80a_2, Ino80b_1, Ino80b_2, Ino80c_1, Ino80c_2, Ino80e_1, Ino80e_2 time: 96 hours: Mock_1, Mock_2, Ino80a_1, Ino80a_2, Ino80b_1, Ino80b_2, Ino80c_1, Ino80c_2, Ino80e_1, Ino80e_2 biological replicate: Ino80a_1, Ino80a_2 biological replicate: Ino80b_1, Ino80b_2 biological replicate: Ino80c_1, Ino80c_2 biological replicate: Ino80e_1, Ino80e_2 biological replicate: Mock_1, Mock_2