Dax1 Up-Regulates Oct4 Expression in Mouse Embryonic Stem Cells via LRH-1 and SRA

Program in Cellular and Molecular Biology, University of Michigan, Ann Arbor, Michigan 48109, USA.
Molecular Endocrinology (Impact Factor: 4.02). 10/2010; 24(12):2281-91. DOI: 10.1210/me.2010-0133
Source: PubMed

ABSTRACT Dax1 (Nr0b1) is an atypical orphan nuclear receptor that has recently been shown to play a role in mouse embryonic stem (mES) cell pluripotency. Here we describe a mechanism by which Dax1 maintains pluripotency. In steroidogenic cells, Dax1 protein interacts with the NR5A nuclear receptor steroidogenic factor 1 (Nr5a1) to inhibit transcription of target genes. In mES cells, liver receptor homolog 1 (LRH-1, Nr5a2), the other NR5A family member, is expressed, and LRH-1 has been shown to interact with Dax1. We demonstrate by coimmunoprecipitation that Dax1 is, indeed, able to form a complex with LRH-1 in mES cells. Because Dax1 was historically characterized as an inhibitor of steroidogenic factor 1-mediated transcriptional activation, we hypothesized that Dax1 would inhibit LRH-1 action in mES cells. Therefore, we examined the effect of Dax1 on the LRH-1-mediated activation of the critical ES cell factor Oct4 (Pou5f1). Chromatin immunoprecipitation localized Dax1 to the Oct4 promoter at the LRH-1 binding site, and luciferase assays together with Dax1 overexpression and knockdown experiments revealed that, rather than repress, Dax1 accentuated LRH-1-mediated activation of the Oct4 gene. Similar to our previously published studies that defined the RNA coactivator steroid receptor RNA activator as the critical mediator of Dax1 coactivation function, Dax1 augmentation of LRH-1-mediated Oct4 activation is dependent upon steroid receptor RNA activator. Finally, utilizing published chromatin immunoprecipitation data of whole-genome binding sites of LRH-1 and Dax1, we show that LRH-1 and Dax1 commonly colocalize at 288 genes (43% of LRH-1 target genes), many of which are involved in mES cell pluripotency. Thus, our results indicate that Dax1 plays an important role in the maintenance of pluripotency in mES cells through interaction with LRH-1 and transcriptional activation of Oct4 and other genes.

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    • "Indeed, knockdown of the DAX1 gene in embryonic stem cells results in spontaneous adrenal differentiation [16] [17]. It is thought that premature activation of DAX1 may impair normal differentiation and zonation of the adrenal gland [15]. A recurring paradox in the congenital adrenal hyperplasia (CAH) literature is the numerous case reports describing " apparent combined " 21-hydroxylase and 11í µí»½hydroxylase deficiencies [18] [19] [20] [21] [22]. "
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    ABSTRACT: We report on a patient with genetically confirmed adrenal hypoplasia congenita (AHC) whose presentation and laboratory abnormalities were consistent with the more common condition, congenital adrenal hyperplasia (CAH). The patient presented with failure to thrive and salt wasting. General appearance showed marked hyperpigmentation and normal male genitalia. He displayed mildly elevated 17-hydroxyprogesterone and markedly elevated 11-deoxycortisol levels at baseline and with ACTH stimulation testing. Results were consistent with 11 -hydroxylase deficiency. He required glucocorticoids and high doses of mineralocorticoids. The marked elevation in 11-deoxycortisol directed our clinical reasoning away from a hypoplastic condition and towards a hyperplasic adrenal condition. Sequencing of the DAX1 gene (named for dosage-sensitive sex reversal (DSS) locus and the AHC locus on the X chromosome) revealed a missense mutation. A review of the literature revealed that elevated 11-deoxycortisol levels have been noted in kindreds with DAX1 mutations, but only when measured very early in life. A mouse model has recently been described that displays elevated 11-deoxycorticosterone levels and evidence for hyperplasia of the zona glomerulosa of the adrenal gland. We conclude that DAX1 testing may be considered in patients with laboratory evidence of 11 -hydroxylase deficiency, especially in those with severe salt wasting.
    Case Reports in Endocrinology 02/2013; 2013:393584. DOI:10.1155/2013/393584
    • "The predominant SRA transcripts in normal tissue are approximately 0.7–0.9 kb while less abundant, larger, transcripts of 1.3–1.5 kb have been identified [54]. SRA RNA is a co-regulator not only of steroid and non-steroid nuclear receptors [64]–[68], but also of several other transcription factors [69], [70]. The SRA RNA folds to form multiple secondary structures, which contribute to its activity [64]. "
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    ABSTRACT: Studies of various mRNAs have revealed that changes in the abundance of transcripts, through mRNA degradation, act as a critical step in the control of various biological pathways. Similarly, the regulation of non-coding RNA (ncRNA) levels is also considered to be important for their biological functions; however, far less is known about the mechanisms and biological importance of ncRNA turnover for the regulation of ncRNA functions. The growth arrest-specific 5 (GAS5) ncRNA accumulates during growth arrest induced by serum starvation and its transcript is degraded by the well characterized nonsense-mediated RNA decay (NMD) pathway. Historically, NMD was discovered as a RNA quality control system to eliminate aberrant transcripts; however, accumulating evidence shows that NMD also regulates the abundance of physiological transcripts. Interestingly, the GAS5 transcript has the ability to bind the glucocorticoid receptor (GR), resulting in the inhibition of its ligand-dependent association with DNA. The GR binds the promoters of various glucocorticoid-responsive genes, including apoptosis-related genes. In this study, we examined whether the RNA degradation pathway can regulate this function of GAS5. We measured the steady-state abundance and the decay rate of GAS5 in UPF1-depleted human cells using the 5'-bromo-uridine immunoprecipitation chase (BRIC) method, an inhibitor-free method for directly measuring RNA stability. We found that levels of the GAS5 transcript were elevated owing to prolonged decay rates in response to UPF1 depletion, and consequently the apoptosis-related genes, cIAP2 and SGK1, were down-regulated. In addition, serum starvation also increased the transcript levels of GAS5 because of prolonged decay rates, and conversely decreased levels of cIAP2 and SGK1 mRNA. Taken together, we found that the RNA degradation pathway can regulate the function of the GAS5 ncRNA in mammalian cells.
    PLoS ONE 01/2013; 8(1):e55684. DOI:10.1371/journal.pone.0055684 · 3.23 Impact Factor
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    • "Transcription factors such as zfp206, sall4, esrrb, -myb, SF1, dax1 and lrh bind to the oct4 distal enhancer (DE)/PE/promoter regions and activate oct4 gene expression (Gu et al., 2005a; Kelly et al., 2010; Tarasov et al., 2008; Yang et al., 2007; Yu et al., 2009; Zhang et al., 2006; 2008). Other transcription factors, such as HIF, GCNF and Tcf3 bind to the oct4 DE/PE/pro-moter regions and repress oct4 gene expression (Gu et al., 2005b; Moreno-Manzano et al., 2010; Tam et al., 2008). "
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    ABSTRACT: We investigated the relationship between oct4 gene expression patterns and CpG sites methylation profiles during ES cell differentiation into neurons, and identified relevant binding factor. The oct4 gene expression level gradually declined as ES cell differentiation progressed, and the CpG sites in the oct4 proximal enhancer (PE) and promoter regions were methylated in concert with ES cell differentiation. An electro-mobility shift assay (EMSA) showed that putative proteins bind to CpG sites in the oct4 PE/promoter. We purified CpG binding proteins with DNAbinding purification method, and NonO was identified by liquid chromatography-mass spectrometry. EMSA with specific competitors revealed that NonO specifically binds to the conserved CCGGTGAC sequence in the oct4 promoter. Methylation at a specific cytosine residue (CC* GGTGAC) reduced the binding affinity of NonO for the recognition sequence. Chromatin immunoprecipitation analysis confirmed that NonO binds to the unmethylated oct4 promoter. There were no changes in the NonO mRNA and protein levels between ES cells and differentiated cells. The transcriptional role of NonO in oct4 gene expression was evaluated by luciferase assays and knockdown experiments. The luciferase activity significantly increased threefold when the NonO expression vector was cotransfected with the NonO recognition sequence, indicating that NonO has a transcription activator effect on oct4 gene expression. In accordance with this effect, when NonO expression was inhibited by siRNA treatment, oct4 expression was also significantly reduced. In summary, we purified NonO, a novel protein that binds to the CpG island of oct4 promoter, and positively regulates oct4 gene expression in ES cells.
    Moleculer Cells 11/2012; 35(1). DOI:10.1007/s10059-013-2273-1 · 2.09 Impact Factor
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