The Orphan Nuclear Receptor Ear-2 Is a Negative Coregulator for Thyroid Hormone Nuclear Receptor Function

Zhu, Xuguang; Park, Kyong Soo; Kaneshige, Masahiro; Bhat, Manoj Kumar; Zhu, Qi; Mariash, Cary N.; McPhie, Peter; Cheng, Sheue-yann

doi:10.1128/mcb.20.7.2604-2618.2000

Cited by 37 publications

(23 citation statements)

References 37 publications

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“…Given the robust transactivation function provided by the enhancer, we were initially surprised that Ear2 would act to oppose transcription. In agreement with this, however, are reports that Ear2 is a negative coregulator of thyroid hormone receptor (T3R) function 16 and inhibits transcription of the luteinizing hormone receptor and a number of other genes. 14,17 Studies of T3R function suggest that Ear2 binds to T3R, preventing its binding to the TRE.…”

Section: Discussionmentioning

confidence: 61%

“…Two different molecular weight forms of Ear2 were identified in As4.1 cells ( Figure 2B). Two forms of Ear2 were previously detected by Zhu et al 16 in transfected CV1 cells. The 45-kDa form comigrated with His-tagged Ear2 purified from bacteria.…”

Section: Resultsmentioning

confidence: 77%

“…These mutations were in the zinc finger DNA binding domain. 16 Purification, Identification, and Expression of Ear2 From Escherichia coli E coli M15 containing pQE-Ear2 plasmid was cultured, induced with IPTG (1.0 mmol/L) for 4 hours, pelleted, lysed in 50 mmol/L NaPO 4 (pH 8.0), 300 mmol/L NaCl, and 10 mmol/L imidazole, and Ear2 was purified by Ni-agarose column. The lysate were separated by electrophoresis on 10% SDS polyacrylamide gel, transferred to nitrocellulose, and proteins were detected with His-tag antisera or Ear2 antisera.…”

Section: Ear2 Constructsmentioning

confidence: 99%

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Identification of a Nuclear Orphan Receptor (Ear2) as a Negative Regulator of Renin Gene Transcription

Liu

Huang

Sigmund

2003

Circulation Research

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Abstract-A potent transcriptional enhancer was previously identified upstream of the mouse renin gene. Within the enhancer is a TGACCT direct-repeat motif, required for enhancer activity, that is the consensus sequence recognized by members of the thyroid hormone subfamily of steroid hormone receptors. We previously reported that RAR/RXR bind to this sequence and mediate the induction of renin promoter activity by retinoids. However, gel mobility shift assays clearly show that other as yet unidentified factors also bind to this motif. In order to identify some of these TGACCT binding factors, we screened a yeast one-hybrid cDNA library derived from mouse As4.1 cells. One of these encoded the orphan nuclear receptor Ear2. Recombinant Ear2 was purified from Escherichia coli and an antipeptide antisera was generated. EMSA showed that purified recombinant Ear2 specifically binds the TGACCT direct-repeat motif. Transfection assays showed that Ear2 potently decreases both baseline and retinoid-induced mouse renin promoter activity in a dose-dependent, enhancer-dependent, and sequence-specific manner. Mutations in Ear2, which abolish its binding to the TGACCT motif, also abolish transcriptional repression. Ear2 was identified as a nuclear protein in As4.

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Section: Discussionmentioning

confidence: 61%

Section: Resultsmentioning

confidence: 77%

Section: Ear2 Constructsmentioning

confidence: 99%

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Identification of a Nuclear Orphan Receptor (Ear2) as a Negative Regulator of Renin Gene Transcription

Liu

Huang

Sigmund

2003

Circulation Research

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“…Ear2 forms homodimers and heterodimers with COUP-TFI and COUP-TFII (Avram et al 1999) or thyroid hormone receptor ␤ (Zhu et al 2000), and dimers bind to enhancers present in a wide range of genes (Ladias et al 1992;Chu and Zingg 1997;Zhang and Dufau 2000). Ear2 is strongly expressed in human fetal liver and in the mouse embryo (Miyajima et al 1988;Jonk et al 1994), but there are no data available about its in vivo function, which prompted us to create a loss-offunction mouse mutant.…”

mentioning

confidence: 99%

Abnormal development of the locus coeruleus in Ear2(Nr2f6)-deficient mice impairs the functionality of the forebrain clock and affects nociception

Warnecke¹,

Oster²,

Revelli³

et al. 2005

Genes Dev.

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The orphan nuclear receptor Ear2 (Nr2f6) is transiently expressed in the rostral part of the rhombic lip in which the locus coeruleus (LC) arises. LC development, regulated by a signaling cascade (Mash1 → Phox2b → Phox2a), is disrupted in Ear2 −/− embryos as revealed by an approximately threefold reduction in the number of Phox2a-and Phox2b-expressing LC progenitor cells. Mash1 expression in the rhombic lip, however, is unaffected, placing Ear2 in between Mash1 and Phox2a/b. Dopamine-␤-hydroxylase and tyrosine hydroxylase staining demonstrate that >70% of LC neurons are absent in the adult with agenesis affecting primarily the dorsal division of the LC. Normally, this division projects noradrenergic efferents to the cortex that appear to be diminished in Ear2 −/− since the cortical concentration of noradrenaline is four times lower in these mice. The rostral region of the cortex is known to contain a circadian pacemaker regulating adaptability to light-and restricted food-driven entrainment. In situ hybridization establishes that the circadian expression pattern of the clock gene Period1 is abolished in the Ear2 −/− forebrain. Behavioral experiments reveal that Ear2 mutants have a delayed entrainment to shifted light-dark cycles and adapt less efficiently to daytime feeding schedules. We propose that neurons in the dorsal division of LC contribute to the regulation of the forebrain clock, at least in part, through targeted release of noradrenaline into the cortical area.[Keywords: Circadian rhythm; Ear2; forebrain clock; locus coeruleus; nociception; nuclear orphan receptor] Supplemental material is available at http://www.genesdev.org.

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“…Yeast Two-hybrid Assay-The human colon carcinoma RKO cDNA library in pGAD 10 vector (CLONTECH Laboratories, Inc.) was constructed according to the manufacturer's protocol (28). The full-length TR␤1 was inserted into the NdeI and EcoRI site of pAS2-1 vector to generate Gal4DNA-BD-TR␤1 as the bait.…”

Section: Methodsmentioning

confidence: 99%

An Isoform of Branched-chain Aminotransferase Is a Novel Co-repressor for Thyroid Hormone Nuclear Receptors

Lin¹,

Kaneshige²,

Zhao³

et al. 2001

Journal of Biological Chemistry

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The functions of thyroid hormone receptors (TRs) are regulated by a host of co-regulatory proteins. Tissue-specific expression of these co-regulators leads to distinct expression patterns and regulation of thyroid hormone (T3) target genes in tissues. Previously we have found that human colon carcinoma RKO cells exhibit strong T3-independent transcriptional activity. We therefore searched for co-regulatory proteins in RKO cells using a yeast two-hybrid system with the intact TR␤1 as bait. One of the three positive clones, designated as P3, was identified to be an isoform of human mitochondria branched-chain aminotransferase (BCATm). P3 was a spliced variant of BCATm with an internal 12-amino acid deletion near the carboxylterminal region and was abundantly expressed in RKO cells. The expressed protein localized both to the mitochondria and the nucleus of transfected CV1 cells. P3 physically interacted with TR␤1 in a T3-independent manner that led to the inhibition in binding of TR␤1 to thyroid hormoneresponsive element. P3 not only enhanced the repressor activity of the unliganded TR but also repressed the liganddependent activation of TR. This repression was reversed by treatment of cells with trichostatin A, suggesting that in addition to the inhibition of DNA binding, the repression activity of P3 on TR may also be mediated by histone deacetylase activity. Thus, unlike the currently known corepressors, P3 is a novel ligand-independent co-repressor for TR. Thyroid hormone receptor (TR)1 is a member of the nuclear hormone receptor superfamily that acts as ligand-dependent transcription factor to control cell proliferation, differentiation, and homeostasis (1). Two distinct genes, TR␣ and TR␤, give rise to four hormone-binding TR isoforms (␣1, ␤1, ␤2, and ␤3) by alternative splicing (1, 2). The tissue specificity and differential expression of TR isoforms indicate that they play distinct functional roles in vivo (3,4). This is demonstrated by the finding that TR␤-knockout mice show very different phenotypes from TR␣1-deficient mice. TR regulates transcription by binding to thyroid hormone response element (TRE) as a homodimer or heterodimer in the promoter region of target genes. The transcriptional activity of TR relies on the types of TRE as well as on T3. In the presence of T3, TR functions as a transcriptional activator that binds to positive TREs and as a repressor that binds to negative TREs.Modulation of gene expression by TR involves the coordination of a network of co-regulatory proteins, including co-activators and co-repressors. Binding of ligand to TR induces a conformational change that promotes dissociation of the corepressors and recruitment of the co-activators, leading to gene activation. Unlike other nuclear hormone receptors, TR and retinoic acid receptors are able to bind to their target genes in the absence of ligands and actively repress transcription (5-8). The repression is mediated by a silencing domain in the carboxyl terminus (5, 9 -11) that also harbors several other functions, includ...

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The Orphan Nuclear Receptor Ear-2 Is a Negative Coregulator for Thyroid Hormone Nuclear Receptor Function

Cited by 37 publications

References 37 publications

Identification of a Nuclear Orphan Receptor (Ear2) as a Negative Regulator of Renin Gene Transcription

Identification of a Nuclear Orphan Receptor (Ear2) as a Negative Regulator of Renin Gene Transcription

Abnormal development of the locus coeruleus in Ear2(Nr2f6)-deficient mice impairs the functionality of the forebrain clock and affects nociception

An Isoform of Branched-chain Aminotransferase Is a Novel Co-repressor for Thyroid Hormone Nuclear Receptors

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