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

Abstract

Estrogen-related receptor beta (Esrrb) is expressed in embryonic stem (ES) cells and is involved in self-renewal ability and pluripotency. Previously, we found that Dax1 is associated with Esrrb and represses its transcriptional activity. Further, the disruption of the Dax1-Esrrb interaction increases the expression of the extra-embryonic endoderm marker Gata6 in ES cells. Here, we investigated the influences of Esrrb and Dax1 on Gata6 expression. Esrrb overexpression in ES cells induced endogenous Gata6 mRNA and Gata6 promoter activity. In addition, the Gata6 promoter was found to contain the Esrrb recognition motifs ERRE1 and ERRE2, and the latter was the responsive element of Esrrb. Associations between ERRE2 and Esrrb were then confirmed by biotin DNA pulldown and chromatin immunoprecipitation assays. Subsequently, we showed that Esrrb activity at the Gata6 promoter was repressed by Dax1, and although Dax1 did not bind to ERRE2, it was associated with Esrrb, which directly binds to ERRE2. In addition, the transcriptional activity of Esrrb was enhanced by nuclear receptor co-activator 3 (Ncoa3), which has recently been shown to be a binding partner of Esrrb.Finally, we showed that Dax1 was associated with Ncoa3 and repressed its transcriptional activity. Taken together, the present study indicates that the Gata6 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.The Gata6 gene promoter region (-0.9 kb) was amplified using PCR and was cloned into pGL4.10 (Promega, Madison, WI, USA) to produce the plasmid pGL4.10-Gata6P 0.9k. To construct the Esrrb-responsive element (ERRE)-mutant pGL4.10-Gata6P 0.9k plasmids, mutant ERRE1 or ERRE2 elements were generated using PCR with specific primers. PCR products were then cloned into pGL4.10 to produce pGL4.10-Gata6P 0.9k-ERRE1 mut and -ERRE2 mut vectors. All primer sequences are listed in Supplemental Table S1. 9Plasmids were introduced into cultured cells using Lipofectamine 2000 (Life Technologies, Grand Island, NY, USA). 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). Biotin-labeled DNA pull-down and chromatin immunoprecipitation assaysBiotin-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).Subsequently, non-labeled oligonucleotide (either wild-type or mutant) was added for competition assays at a ...

Genome-wide association study identifies two risk loci for tuberculosis in Han Chinese

“…Until now, the function of ESRRB in TB infection was unknown. A previous study indicated that ESRRB directly bound to Gata6 promoter and resulted in maintenance of the undifferentiated status of embryonic stem cells 31 . Interestingly, GATA6 was previously thought to be a TB susceptible gene due to close proximity to a susceptibility locus for TB on chromosome 18q11.2 variant (SNP rs4331426) identified by GWAS 7 .…”

Cooperation between HDAC3 and DAX1 mediates lineage restriction of embryonic stem cells

“…Lack of Esrrb, in contrast, partially blocks PrE conversion of Dax1 and Hdac3 mutants. Although Esrrb interacts and shares genomic binding with Dax1 and Nr5a2 (Figs 4A and 5A, and EV5A), incomplete phenotypic rescue and lack of specificity for Dax1 suggests a role of Esrrb in PrE conversion beyond the Nr5a2/Dax1 axis, e.g., by binding (Fig 5C ) and activating the Gata6 promoter (Uranishi et al, 2016) or, indirectly, by stabilizing pluripotency (Martello et al, 2012). Taken together, we propose that Nr5a2 and Esrrb support both the EPI and the PrE fate within a coherent TF network that integrates extrinsic signals with transcriptional repressors to maintain lineage choice.…”

A bipartite function of ESRRB can integrate signaling over time to balance self-renewal and differentiation

“…Several studies have shown the importance of ESRRB in naïve pluripotency, including demonstrations that loss of ESRRB causes compromised ESC fitness, and its overexpression can substitute for NANOG function in establishing and supporting pluripotency (Festuccia et al, 2012, 2016, 2018b, 2018a). However, there is also in vitro evidence supporting an active role for ESRRB in PrE differentiation as well as direct upregulation of the PrE marker Gata6 (Wamaitha et al, 2015; Uranishi et al, 2016; Herchcovici Levy et al, 2022). Does ESRRB promote differentiation at the same time as safeguarding pluripotency, and how can these two affiliations be reconciled?…”