Functional regulation of Nanog transcription factor by transcriptional corepressors, CtBP1 and HIPK2

Abstract

Nanog, an embryonic stem cell-specific homeodomain transcription factor, has been shown recently to play a crucial role in maintaining pluripotency of embryonic stem (ES) cells. The molecular mechanism of how Nanog regulates stem cell pluripotency, however, remains unknown. Here, we demonstrated that Nanog recruits the CtBP1 corepressor and HIPK2 to downregulate target gene expression in ES cells. We showed that Nanog can act as either a transcriptional repressor or an activator, depending on functional domains. The activation domain mapped to the C-terminal half of the protein (aa 161-305) whereas the repression domain (a.a.82-250) mapped to the middle portion of the protein that overlaps with an activation domain through the tryptophan-repeat (WR) domain. As a transcriptional repressor, Nanog physically interacted with the CtBP1 corepressor. Furthermore, the Nanog-mediated transcriptional repression of target genes was augmented by CtBP1, indicating that CtBP1 acts as a corepressor of Nanog. In cultured cells, Nanog colocalized with the HIPK2 protein kinase, and imunoprecipitation and GST-pull down assays revealed its direct interaction with HIPK2. However, we found that coexpression of the wild type HIPK2, but not of the kinase-dead HIPK2(KR), relieved repressor activity of Nanog. Interestingly, both CtBP 1 and HIPK2 interacted with Nanog via the WR domain, and deletion of the WR domain abolished Nanog interactions with CtBP1 and HIPK2, concomitant with the loss of its repressor activity. Using chromatin immunoprecipitation (CHIP) assays, we demonstrated that in ES cells, Nanog bound to GATA6 promoter and recruited the CtBP1 corepressor to downregulate GATA6 gene transcription. Upon the treatment of ES cells with retinoic acid to induce differentiation, Nanog no longer bound to the GATA6 promoter, thereby relieving transcriptional repression mediated by CtBP1. Together, our results provide the first evidence that Nanog recruits CtBP 1 and HIPK2 for its trans...