Dax1 Associates with Esrrb and Regulates Its Function in Embryonic Stem Cells

Abstract

Self-renewal capacity and pluripotency, which are controlled by the Oct3/4-centered transcriptional regulatory network, are major characteristics of embryonic stem (ES) cells. Nuclear hormone receptor Dax1 is one of the crucial factors in the network. Here, we identified an orphan nuclear receptor, Esrrb (estrogen-related receptor beta), as a Dax1-interacting protein. Interaction of Dax1 and Esrrb was mediated through LXXLL motifs of Dax1 and the activation-and ligand-binding domains of Esrrb. Furthermore, Esrrb enhanced the promoter activity of the Dax1 gene via direct binding to Esrrb-binding site 1 (ERRE1, where "ERRE" represents "Esrrb-responsive element") of the promoter. Expression of Dax1 was suppressed followed by Oct3/4 repression; however, overexpression of Esrrb maintained expression of Dax1 even in the absence of Oct3/4, indicating that Dax1 is a direct downstream target of Esrrb and that Esrrb can regulate Dax1 expression in an Oct3/4-independent manner. We also found that the transcriptional activity of Esrrb was repressed by Dax1. Furthermore, we revealed that Oct3/4, Dax1, and Esrrb have a competitive inhibition capacity for each complex. These data, together with previous findings, suggest that Dax1 functions as a negative regulator of Esrrb and Oct3/4, and these molecules form a regulatory loop for controlling the pluripotency and self-renewal capacity of ES cells. P luripotency and self-renewal capacity are major characteristics of murine embryonic stem (ES) cells. Leukemia inhibitory factor (LIF) plays an important role for the self-renewal of ES cells, and depletion of LIF from ES cell culture medium leads to spontaneous differentiation of cells and results in a failure of self-renewal (1, 2). A large number of transcription factors function downstream of signaling by LIF, and several transcription factors, including STAT3, Oct3/4, Sox2, and Nanog, play crucial roles for pluripotency and self-renewal of ES cells (3-5). Artificial activation of STAT3, which is achieved by 4-hydroxytamoxifen stimulation of nuclear localization of the STAT3-estrogen receptor fusion protein (STAT3ER), as well as forced expression of Nanog, accelerates the self-renewal in a LIF-independent manner (6-8). Oct3/4-deficient embryos develop to the blastocyst stage, but their inner cell mass (ICM), from which ES cells are established, loses pluripotency, and the deletion of Oct3/4 expression in ES cells promotes differentiation into extraembryonic trophectodermal cells (9, 10). Sox2-deficient blastocysts form abnormal ICM and fail to achieve outgrowth (11). ES cells lacking the Sox2 gene differentiate into trophoblast cells (12).Actually, these transcription factors collaboratively regulate gene expression with other factors and contribute to maintenance of pluripotency and self-renewal of ES cells. For instance, Oct3/4 interacts with Sox2, and this complex enhances expression of ES cell-specific genes, including Fgf4, Lefty1, Nanog, UTF1, and Sox2 (13). ␤-Catenin is also a binding partner of Oct3/4, and the complex re...

Esrrb directly binds to Gata6 promoter and regulates its expression with Dax1 and Ncoa3

“…Estrogen-related receptor beta (Esrrb) is considered to be a member of the core transcriptional regulatory network in ES cells [17,18], and has been associated with Oct3/4 [13,19], Nanog [10,20], and Dax1 [21], and is reportedly required for self-renewal of ES cells [22,23]. Moreover, Esrrb has been shown to regulate the self-renewal capacity of ES cells downstream of Nanog and Gsk3-Tcf3 pathways [24,25].…”

“…Two days after transfection, cells were treated with 1 μg/mL puromycin for 5-7 days, and RNA samples for RT-PCR were isolated and examined as described previously [21]. The cell extracts for the luciferase assays were prepared 48 h after transfection, and luciferase activity was measured using a luciferase assay kit (Promega) and an AB-2200 luminometer (ATTO, Tokyo, Japan).…”

“…Previously, we clarified that an interaction of Esrrb pCAGIP-Myc-Dax1, and pCAGIP-Myc-Dax1LTm, were generated as described previously [9,21]. The Ncoa3 coding region and its truncated mutants were amplified using polymerase chain reaction (PCR) before being cloned into expression vectors.…”

“…Biotin-labeled DNA pull-down assays were performed as described previously [21]. Briefly, biotin-labeled oligonucleotides were incubated with A3-1 ES extracts or HEK293 cell extracts that had been transfected with pCAGIP-Myc-Dax1 and/or pCAGIP-Esrrb in the presence of streptavidin-agarose (Novagen, Darmstadt, Germany).…”

“…as described previously [21]. Immunoprecipitation was performed with normal murine IgG (sc-2025; Santa Cruz) and anti-Esrrb antibodies (PP-H6705-00; Perseus Proteomics, Tokyo, Japan).…”