Isolation of a cDNA clone encoding a biologically active thyroid hormone receptor.

Koenig, Ronald J.; Warne, Robert; Brent, Gregory A.; Harney, John W.; Larsen, P. Reed; Moore, David D.

doi:10.1073/pnas.85.14.5031

Cited by 204 publications

(65 citation statements)

References 28 publications

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“…Because TR␣2 inhibits transcriptional activation by TR␣1 in a dose-dependent manner (15,16), any change in the ratio of these regulatory proteins is expected to alter T 3 action. The physiological significance of a 2-3-fold change in the expression of these receptors is supported by studies demonstrating that tissues with a higher TR␣1/TR␣2 mRNA ratio are more responsive to T 3 than tissues with a 2-fold lower ratio (46).…”

Section: Figmentioning

confidence: 99%

Post-transcriptional Regulation of Thyroid Hormone Receptor Expression by cis-Acting Sequences and a Naturally Occurring Antisense RNA

Hastings¹,

Ingle²,

Lazar³

et al. 2000

Journal of Biological Chemistry

122

103

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Thyroid hormone (T 3 ) coordinates growth, differentiation, and metabolism by binding to nuclear thyroid hormone receptors (TRs). The TR␣ gene encodes T 3 -activated TR␣1 (NR1A1a) as well as an antagonistic, non-T 3 -binding alternatively spliced product, TR␣2 (NR1A1b). Thus, the TR␣1/TR␣2 ratio is a critical determinant of T 3 action. However, the mechanisms underlying this post-transcriptional regulation are unknown. We have identified a non-consensus, TR␣2-specific 5 splice site and conserved intronic sequences as key determinants of TR␣ mRNA processing. In addition to these cis-acting elements, a novel regulatory feature is the orphan receptor RevErbA␣ (NR1D1) gene, which is transcribed from the opposite direction at the same locus and overlaps the TR␣2 coding region. RevErbA␣ gene expression correlates with a high TR␣1/TR␣2 ratio in a number of tissues. Here we demonstrate that coexpression of RevErbA␣ and TR␣ regulates the TR␣1/TR␣2 ratio in intact cells. Thus, both cis-and trans-regulatory mechanisms contribute to cell-specific post-transcriptional regulation of TR gene expression and T 3 action. Thyroid hormone receptors (TRs)1 mediate the diverse physiological effects of thyroid hormone (T 3 ) (1-3). TRs are encoded by two closely related genes, erbA␣ and erbA␤, in all vertebrates (4). Additional receptor diversity is generated by alternative processing of the TR␣ and TR␤ pre-mRNAs. The alternatively spliced isoforms of TR␣, TR␣1 (NR1A1a) (5) and TR␣2 (NR1A1b), are of particular interest. Although both are widely expressed, they are functionally antagonistic. Due to its variant C terminus, TR␣2, unlike TR␣1, does not bind T 3 and lacks the major activation function present in other TRs (6, 7). The dominant negative activity of TR␣2 appears to reflect both competition for binding to TR target genes as well as altered protein-protein interactions (8 -11).Expression of the two TR␣ isoforms is highly regulated, with each mRNA expressed in a tissue-specific and developmentally regulated fashion. In some tissues TR␣2 represents a relatively minor fraction of the TR-related isoforms, while in other tissues such as brain, kidney, and testes, TR␣2 is the most abundant isoform (12, 13). A developmental increase in the T 3 responsiveness of some cells correlates with a decrease in the relative expression of TR␣2 mRNA, suggesting that TR␣2 expression modulates T 3 action (13). Thus, TR␣2 may act as a tissuespecific antagonist of T 3 -responsive gene activation.Interestingly, the alternative post-transcriptional processing of the TR␣ transcript that gives rise to TR␣2 mRNA occurs exclusively in mammals (14 -17). The mammalian TR␣ gene is also remarkable in that it partially overlaps the gene for another nuclear receptor that is encoded on the opposite DNA strand. This receptor gene, RevErbA␣ (RevErb, NR1D1), is convergently transcribed with respect to the TR␣ gene such that its 3Ј end overlaps sequences coding for TR␣2, but not TR␣1 (18,19). The unusual organization of these two genes, which code for structurally re...

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Section: Figmentioning

confidence: 99%

Post-transcriptional Regulation of Thyroid Hormone Receptor Expression by cis-Acting Sequences and a Naturally Occurring Antisense RNA

Hastings¹,

Ingle²,

Lazar³

et al. 2000

Journal of Biological Chemistry

122

103

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“…RNA (3 g) were reverse-transcribed for 50 min at 42°C using 200 ng of random hexamer primers pdN6 (Pharmacia Biotech, Orsay, France) and 200 U of Moloney Murine Leukemia Virus reverse transcriptase (Super Script II, Life Technology) in 50 l of reaction mixture containing (in mM): 50 Tris-HC l, pH 8.3, 75 KC l, 3 MgC l 2 , 10 DTT, and 0.5 dN TP. Rat TR␣1, TR␣2, TR␤1, TR␤2, and glyceraldehyde-3-phosphatedehydrogenase (GAPDH) primers (Table 1) were chosen from reported cDNA sequences (Fort et al, 1985;Thompson et al, 1987;Koenig et al, 1988;Lazar et al, 1988;Hodin et al, 1989). TR␣2 primers amplified two splice variants: TR␣2vI and TR␣2vII (Mitsuhashi et al, 1988a,b).…”

Section: In Vitro Experimentsmentioning

confidence: 99%

Regulation of Microglial Development: A Novel Role for Thyroid Hormone

et al. 2001

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The postnatal development of rat microglia is marked by an important increase in the number of microglial cells and the growth of their ramified processes. We studied the role of thyroid hormone in microglial development. The distribution and morphology of microglial cells stained with isolectin B4 or monoclonal antibody ED1 were analyzed in cortical and subcortical forebrain regions of developing rats rendered hypothyroid by prenatal and postnatal treatment with methyl-thiouracil. Microglial processes were markedly less abundant in hypothyroid pups than in age-matched normal animals, from postnatal day 4 up to the end of the third postnatal week of life. A delay in process extension and a decrease in the density of microglial cell bodies, as shown by cell counts in the developing cingulate cortex of normal and hypothyroid animals, were responsible for these differences. Conversely, neonatal rat hyperthyroidism, induced by daily injections of 3,5,3Ј-triiodothyronine (T3), accelerated the extension of microglial processes and increased the density of cortical microglial cell bodies above physiological levels during the first postnatal week of life.Reverse transcription-PCR and immunological analyses indicated that cultured cortical ameboid microglial cells expressed the ␣1 and ␤1 isoforms of nuclear thyroid hormone receptors. Consistent with the trophic and morphogenetic effects of thyroid hormone observed in situ, T3 favored the survival of cultured purified microglial cells and the growth of their processes. These results demonstrate that thyroid hormone promotes the growth and morphological differentiation of microglia during development.

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“…It is of particular significance that the probe common to c-erb-A al and c-erb-A a2 did not detect in mouse neuroblastoma cells the 6 kb mRNA species characteristic of c-erb-A fi mRNA (7,12). This probe has a 25 bp segment that is 100% homologous with mond, CA, USA) and was probed separately with two synthetic oligonucleotides radiolabeled by the end-labeling with polynucleotide kinase (28).…”

Section: Discussionmentioning

confidence: 99%

“…All of the a1 forms (8,9) and the fl form (7) encode proteins that bind thyroid hormone with high affinities and with specificities for thyroid hormone analogues appropriate for thyroid hormone receptors. The human c-erb-A fi (placenta) protein binds to a specific region of the rat growth hormone gene that is transcriptionally regulated by T, (12). However, there are contradictory data as to whether (10,11) or not (13,14) the a2 protein binds thyroid hormone.…”

mentioning

confidence: 99%

Molecular Cloning and Characterization of c‐erb‐A mRNA Species in Mouse Neuroblastoma Cells

Moeller

Rapoport

Gavin

1989

J Neuroendocrinology

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The mouse neuroblastoma cell line is an excellent model in which to study thyroid hormone action and metabolism, particularly in neural tissue. We therefore undertook the molecular cloning and characterization in these cells of putative thyroid hormone receptors related to c-erb-A. Since rat brain tissue contains multiple cell types, and because of possible subtle differences between species (mouse and rat), we therefore screened a new cDNA library constructed from mouse neuroblastoma cell mRNA with synthetic oligonucleotide probes based on the published nucleotide sequence of the c-erb-A gene in whole rat brain. Despite the fact that this rat brain cDNA sequence is now recognized to represent a c-erb-A a1 form, the cDNA clones that we isolated were all members of the newly-recognized c-erb-A a2 form. This identification was made on the basis of nucleotide sequence divergence downstream of the nucleotide corresponding to amino-acid residue 370 in the predicted coding region. The two longest mouse neuroblastoma cDNA clones, clone 29 (1796 bp) and clone 32 (1410 bp), were 93% to 94% homologous with the c-erb-A ct2 and c-erb-A a1 forms in their DNA binding and thyroid hormone binding domains (up to amino-acid residue 370 in the latter). Both clones 29 and 32 were incomplete in that they terminated at their 3' ends at an internal Eco R1 site. Fortunately this 120 bp (40 derived amino-acid) truncation was downstream of the reported thyroid hormone binding domain. The 5' untranslated end of clone 29 (446 bp) was of interest because a region of its nucleotide sequence (279 bp) revealed a high degree of homology (87%) with rat brain c-erb-A a l . This highly conserved region in clone 29 appeared to be important in the regulation of translation because only clone 32, in which this region was truncated, efficiently translated protein in a cell-free system. The protein product of clone 32 did not bind thyroid hormone. Northern blot analysis of mouse neuroblastoma mRNA with site-specific synthetic oligonucleotides revealed that the c-erb-A cr2 species was dominant (major band of 2.4 kb), with a lesser amount of the c-erb-A a1 species (major band of 1.8 kb).In conclusion: 1) We report the molecular cloning of c-erb-A variants in a specific neural tissue cell type, namely mouse neuroblastoma cells; 2) The cells contained predominantly the c-erb-A a2 subtype; 3) This c-erb-A a2 form was unique up to bp 167 at its 5 untranslated end, and was then followed, up to the ATG initiation codon, by a highly conserved region common to all c-erb-A a species; 4) The 5' untranslated region appeared to play a role in the translational efficiency of this mRNA; 5) The protein product of the c-erb-A a2 mRNA in these cells did not appear to bind thyroid hormone. In view of this finding, the physiological role of the c-erb-A a2 protein remained speculative and may involve a repressor function at the thyroid hormone responsive element.

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Isolation of a cDNA clone encoding a biologically active thyroid hormone receptor.

Cited by 204 publications

References 28 publications

Post-transcriptional Regulation of Thyroid Hormone Receptor Expression by cis-Acting Sequences and a Naturally Occurring Antisense RNA

Post-transcriptional Regulation of Thyroid Hormone Receptor Expression by cis-Acting Sequences and a Naturally Occurring Antisense RNA

Regulation of Microglial Development: A Novel Role for Thyroid Hormone

Molecular Cloning and Characterization of c‐erb‐A mRNA Species in Mouse Neuroblastoma Cells

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