Characterization of Receptor Interaction and Transcriptional Repression by the Corepressor SMRT

Abstract SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) and N-CoR (nuclear receptor corepressor) are two related transcriptional corepressors that contain separable domains capable of interacting with unliganded nuclear receptors and repressing basal transcription. To decipher the mechanisms of receptor interaction and transcriptional repression by SMRT/N-CoR, we have characterized protein-protein interacting surfaces between SMRT and nuclear receptors and defined transcriptional repression domains of both SMRT and N-CoR. Deletional analysis reveals two individual nuclear receptor domains necessary for stable association with SMRT and a C-terminal helix essential for corepressor dissociation. Coordinately, two SMRT domains are found to interact independently with the receptors. Functional analysis reveals that SMRT contains two distinct repression domains, and the corresponding regions in N-CoR also repress basal transcription. Both repression domains in SMRT and N-CoR interact weakly with mSin3A, which in turn associates with a histone deacetylase HDAC1 in a mammalian two-hybrid assay. Far-Western analysis demonstrates a direct protein-protein interaction between two N-CoR repression domains with mSin3A. Finally we demonstrate that overexpression of full-length SMRT further represses basal transcription from natural promoters. Together, these results support a role of SMRT/N-CoR in corepression through the utilization of multiple mechanisms for receptor interactions and transcriptional repression.

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Structural and Functional Organization of TRAP220, the TRAP/Mediator Subunit That Is Targeted by Nuclear Receptors

Molecular and Cellular Biology ◽

10.1128/mcb.24.18.8244-8254.2004 ◽

2004 ◽

Vol 24 (18) ◽

pp. 8244-8254 ◽

Cited By ~ 72

Author(s):

Sohail Malik ◽

Mohamed Guermah ◽

Chao-Xing Yuan ◽

Weizhen Wu ◽

Soichiro Yamamura ◽

...

Keyword(s):

Nuclear Receptors ◽

Nuclear Receptor ◽

Thyroid Hormone Receptor ◽

Functional Organization ◽

Critical Role ◽

Mediator Complex ◽

Lxxll Motif ◽

Stable Association ◽

Necessary And Sufficient

ABSTRACT The TRAP/Mediator complex serves as a coactivator for many transcriptional activators, including nuclear receptors such as the thyroid hormone receptor (TR) that targets the TRAP220 subunit. The critical but selective function of TRAP220 is evidenced by the embryonic lethal phenotype of Trap220 − / − mice and by the observation that Trap220 − / − fibroblasts (isolated before embryonic death) are impaired in specific nuclear receptor-dependent pathways. Here we have used a biochemical and genetic approach to understand the basis of specificity in TRAP220 function. We show that Trap220 − / − cells possess a TRAP/Mediator complex that is relatively intact and compromised in its ability to support TR-dependent, but not VP16-dependent, transcription in vitro. Transfection studies using TRAP220 mutants revealed that the N terminus of TRAP220 is necessary and sufficient for stable association with the TRAP/Mediator complex and, further, that TRAP220-dependent TR function in transfected cells requires both of the NR boxes that contain the LXXLL motif implicated in nuclear receptor binding. Similarly, an analysis of isolated TRAP/Mediator complexes with mutations in either or both of the two NR boxes confirmed a critical role for them in in vitro coactivator function. The implications of these observations are discussed in terms of our present understanding of coactivator function.

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A novel orphan receptor specific for a subset of thyroid hormone-responsive elements and its interaction with the retinoid/thyroid hormone receptor subfamily.

Molecular and Cellular Biology ◽

10.1128/mcb.14.10.7025 ◽

1994 ◽

Vol 14 (10) ◽

pp. 7025-7035 ◽

Cited By ~ 242

Author(s):

R Apfel ◽

D Benbrook ◽

E Lernhardt ◽

M A Ortiz ◽

G Salbert ◽

...

Keyword(s):

Amino Acid ◽

Thyroid Hormone ◽

Dna Binding ◽

Nuclear Receptors ◽

Nuclear Receptor ◽

Dna Sequences ◽

Hormone Receptor ◽

Thyroid Hormone Receptor ◽

Orphan Receptor ◽

Retinoid Receptor

The steroid/hormone nuclear receptor superfamily comprises several subfamilies of receptors that interact with overlapping DNA sequences and/or related ligands. The thyroid/retinoid hormone receptor subfamily has recently attracted much interest because of the complex network of its receptor interactions. The retinoid X receptors (RXRs), for instance, play a very central role in this subfamily, forming heterodimers with several receptors. Here we describe a novel member of this subfamily that interacts with RXR. Using a v-erbA probe, we obtained a cDNA which encodes a novel 445-amino-acid protein, RLD-1, that contains the characteristic domains of nuclear receptors. Northern (RNA) blot analysis showed that in mature rats, the receptor is highly expressed in spleen, pituitary, lung, liver, and fat. In addition, weaker expression is observed in several other tissues. Amino acid sequence alignment and DNA-binding data revealed that the DNA-binding domain of the new receptor is related to that of the thyroid/retinoid subgroup of nuclear receptors. RLD-1 preferentially binds as a heterodimer with RXR to a direct repeat of the half-site sequence 5'-G/AGGTCA-3', separated by four nucleotides (DR-4). Surprisingly, this binding is dependent to a high degree on the nature of the spacing nucleotides. None of the known nuclear receptor ligands activated RLD-1. In contrast, a DR-4-dependent constitutive transcriptional activation of a chloramphenicol acetyltransferase reporter gene by the RLD-1/RXR alpha heterodimer was observed. Our data suggest a highly specific role for this novel receptor within the network of gene regulation by the thyroid/retinoid receptor subfamily.

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Transcriptional repression by nuclear receptors: mechanisms and role in disease

Biochemical Society Transactions ◽

10.1042/bst0280390 ◽

2000 ◽

Vol 28 (4) ◽

pp. 390-396 ◽

Cited By ~ 3

Author(s):

J. D. Love ◽

J. T. Gooch ◽

L. Nagy ◽

V. K. K. Chatterjee ◽

J. W. R. Schwabe

Keyword(s):

Diabetes Mellitus ◽

Thyroid Hormone ◽

Ligand Binding ◽

Nuclear Receptors ◽

Transcriptional Repression ◽

Dominant Negative ◽

Receptor Interaction ◽

Hormone Resistance ◽

Thyroid Hormone Resistance ◽

Repressor Proteins

Co-repressor proteins mediate transcriptional repression by nuclear receptors in the absence of ligand. The identification of a co-repressor-receptor interaction motif, and the finding that compressors and co-activators compete for the same site on the receptor, suggests a simple mechanism for the switch from repression to activation upon ligand binding. Defects in this mechanism result in dominant-negative receptors that repress transcription. Such receptors have been implicated in several clinically important diseases, including thyroid hormone resistance and diabetes mellitus.

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The DRIP Complex and SRC-1/p160 Coactivators Share Similar Nuclear Receptor Binding Determinants but Constitute Functionally Distinct Complexes

Molecular and Cellular Biology ◽

10.1128/mcb.20.8.2718-2726.2000 ◽

2000 ◽

Vol 20 (8) ◽

pp. 2718-2726 ◽

Cited By ~ 153

Author(s):

Christophe Rachez ◽

Matthew Gamble ◽

Chao-Pei Betty Chang ◽

G. Brandon Atkins ◽

Mitchell A. Lazar ◽

...

Keyword(s):

Nuclear Receptors ◽

Nuclear Receptor ◽

Receptor Binding ◽

Transcriptional Activation ◽

Thyroid Hormone Receptor ◽

Nuclear Receptor Coactivators ◽

Nuclear Extracts ◽

Sequence Requirements

ABSTRACT Transcriptional activation requires both access to DNA assembled as chromatin and functional contact with components of the basal transcription machinery. Using the hormone-bound vitamin D3receptor (VDR) ligand binding domain (LBD) as an affinity matrix, we previously identified a novel multisubunit coactivator complex, DRIP (VDR-interacting proteins), required for transcriptional activation by nuclear receptors and several other transcription factors. In this report, we characterize the nuclear receptor binding features of DRIP205, a key subunit of the DRIP complex, that interacts directly with VDR and thyroid hormone receptor in response to ligand and anchors the other DRIP subunits to the nuclear receptor LBD. In common with other nuclear receptor coactivators, DRIP205 interaction occurs through one of two LXXLL motifs and requires the receptor's AF-2 subdomain. Although the second motif of DRIP205 is required only for VDR binding in vitro, both motifs are used in the context of an retinoid X receptor-VDR heterodimer on DNA and in transactivation in vivo. We demonstrate that both endogenous p160 coactivators and DRIP complexes bind to the VDR LBD from nuclear extracts through similar sequence requirements, but they do so as distinct complexes. Moreover, in contrast to the p160 family of coactivators, the DRIP complex is devoid of any histone acetyltransferase activity. The results demonstrate that different coactivator complexes with distinct functions bind to the same transactivation region of nuclear receptors, suggesting that they are both required for transcription activation by nuclear receptors.

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Identification and Characterization of a Tissue-Specific Coactivator, GT198, That Interacts with the DNA-Binding Domains of Nuclear Receptors

Molecular and Cellular Biology ◽

10.1128/mcb.22.1.357-369.2002 ◽

2002 ◽

Vol 22 (1) ◽

pp. 357-369 ◽

Cited By ~ 54

Author(s):

Lan Ko ◽

Guemalli R. Cardona ◽

Alexandra Henrion-Caude ◽

William W. Chin

Keyword(s):

Protein Kinase ◽

Dna Binding ◽

Nuclear Receptors ◽

Nuclear Receptor ◽

Thyroid Hormone Receptor ◽

Pituitary Cells ◽

Tissue Specific ◽

Dna Binding Domains ◽

Binding Domains

ABSTRACT Gene activation mediated by nuclear receptors is regulated in a tissue-specific manner and requires interactions between nuclear receptors and their cofactors. Here, we identified and characterized a tissue-specific coactivator, GT198, that interacts with the DNA-binding domains of nuclear receptors. GT198 was originally described as a genomic transcript that mapped to the human breast cancer susceptibility locus 17q12-q21 with unknown function. We show that GT198 exhibits a tissue-specific expression pattern in which its mRNA is elevated in testis, spleen, thymus, pituitary cells, and several cancer cell lines. GT198 is a 217-amino-acid nuclear protein that contains a leucine zipper required for its dimerization. In vitro binding and yeast two-hybrid assays indicated that GT198 interacted with nuclear receptors through their DNA-binding domains. GT198 potently stimulated transcription mediated by estrogen receptor α and β, thyroid hormone receptor β1, androgen receptor, glucocorticoid receptor, and progesterone receptor. However, the action of GT198 was distinguishable from that of the ligand-binding domain-interacting nuclear receptor coactivators, such as TRBP, CBP, and SRC-1, with respect to basal activation and hormone sensitivity. Furthermore, protein kinase A, protein kinase C, and mitogen-activated protein kinase can phosphorylate GT198 in vitro, and cotransfection of these kinases regulated the transcriptional activity of GT198. These data suggest that GT198 is a tissue-specific, kinase-regulated nuclear receptor coactivator that interacts with the DNA-binding domains of nuclear receptors.

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A nuclear hormone receptor corepressor mediates transcriptional silencing by receptors with distinct repression domains.

Molecular and Cellular Biology ◽

10.1128/mcb.16.10.5458 ◽

1996 ◽

Vol 16 (10) ◽

pp. 5458-5465 ◽

Cited By ~ 153

Author(s):

I Zamir ◽

H P Harding ◽

G B Atkins ◽

A Hörlein ◽

C K Glass ◽

...

Keyword(s):

Amino Acid ◽

Nuclear Receptors ◽

Hormone Receptor ◽

Transcriptional Repression ◽

Hormone Receptors ◽

Thyroid Hormone Receptor ◽

Nuclear Hormone Receptor ◽

Amino Acid Sequences ◽

Orphan Receptor ◽

Primary Amino

Ligand-independent transcriptional repression is an important function of nuclear hormone receptors. An interaction screen with the repression domain of the orphan receptor RevErb identified N-CoR, the corepressor for thyroid hormone receptor (TR) and retinoic acid receptor (RAR). N-CoR is likely to be a bona fide transcriptional corepressor for RevErb because (i) RevErb interacts with endogenous N-CoR, (ii) ectopic N-CoR potentiates RevErb-mediated repression, and (iii) transcriptional repression by RevErb correlates with its ability to bind N-CoR. Remarkably, a region homologous to the CoR box which is necessary for TR and RAR to interact with N-CoR is not required for RevErb. Rather, two short regions of RevErb separated by approximately 200 amino acids are required for interaction with N-CoR. The primary amino acid sequence of the N-terminal region of RevErb essential for N-CoR interaction is not homologous to that of TR or RAR, whereas similarities exist among the C-terminal domains of the receptors. N-CoR contains two adjacent but distinct interaction domains, one of which binds tightly to both RevErb and TR whereas the other binds more weakly and differentially interacts with the nuclear receptors. These results indicate that multiple nuclear receptors, utilizing different primary amino acid sequences, repress transcription by interacting with N-CoR.

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Alternative splicing determines the interaction of SMRT isoforms with nuclear receptor–DNA complexes

Bioscience Reports ◽

10.1042/bsr20080093 ◽

2009 ◽

Vol 29 (3) ◽

pp. 143-149 ◽

Cited By ~ 16

Author(s):

Flavie Faist ◽

Stephen Short ◽

G. Geoff Kneale ◽

Colinb R. Sharpe

Keyword(s):

Retinoic Acid ◽

Alternative Splicing ◽

Nuclear Receptors ◽

Nuclear Receptor ◽

Thyroid Hormone Receptor ◽

Retinoic Acid Receptor ◽

Class Ii ◽

Dna Complexes ◽

Response Elements

Signalling by small molecules, such as retinoic acid, is mediated by heterodimers comprising a class II nuclear receptor and an RXR (retinoid X receptor) subunit. The receptors bind to DNA response elements and act as ligand-dependent transcription factors, but, in the absence of signal, the receptors bind the co-repressors SMRT [silencing mediator for RAR (retinoic acid receptor) and TR (thyroid hormone receptor)] and NCoR (nuclear receptor co-repressor) and repress gene expression. Alternative splicing of the SMRT transcript in mammals generates six isoforms containing 1, 2 or 3 CoRNR (co-repressor for nuclear receptor) box motifs which are responsible for the interactions with nuclear receptors. We show that human cell lines express all six SMRT isoforms and then determine the binding affinity of mouse SMRT isoforms for RAR/RXR and three additional class II nuclear receptor–DNA complexes. This approach demonstrates the importance of the full complement of CoRNR boxes within each SMRT protein, rather than the identity of individual CoRNR boxes, in directing the interaction of SMRT with nuclear receptors. Each class of SMRT isoform displays a distinct feature, as the 1-box isoform discriminates between DNA response elements, the 2-box isoforms promote high-affinity binding to TR complexes and the 3-box isoforms show differential binding to nuclear receptors. Consequently, the differential deployment of SMRT isoforms observed in vivo could significantly expand the regulatory capacity of nuclear receptor signalling.

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Unorthodox Transcriptional Mechanisms of Lipid-Sensing Nuclear Receptors in Macrophages: Are We Opening a New Chapter?

Frontiers in Endocrinology ◽

10.3389/fendo.2020.609099 ◽

2020 ◽

Vol 11 ◽

Author(s):

Zsolt Czimmerer ◽

Laszlo Halasz ◽

Laszlo Nagy

Keyword(s):

Nuclear Receptors ◽

Nuclear Receptor ◽

Transcriptional Activation ◽

Chromatin Immunoprecipitation ◽

Protein Protein Interaction ◽

Lipid Sensing ◽

Transcriptional Memory ◽

Genome Level ◽

Generation Sequencing

Work over the past 30 years has shown that lipid-activated nuclear receptors form a bridge between metabolism and immunity integrating metabolic and inflammatory signaling in innate immune cells. Ligand-induced direct transcriptional activation and protein-protein interaction-based transrepression were identified as the most common mechanisms of liganded-nuclear receptor-mediated transcriptional regulation. However, the integration of different next-generation sequencing-based methodologies including chromatin immunoprecipitation followed by sequencing and global run-on sequencing allowed to investigate the DNA binding and ligand responsiveness of nuclear receptors at the whole-genome level. Surprisingly, these studies have raised the notion that a major portion of lipid-sensing nuclear receptor cistromes are not necessarily responsive to ligand activation. Although the biological role of the ligand insensitive portion of nuclear receptor cistromes is largely unknown, recent findings indicate that they may play roles in the organization of chromatin structure, in the regulation of transcriptional memory, and the epigenomic modification of responsiveness to other microenvironmental signals in macrophages. In this review, we will provide an overview and discuss recent advances of our understanding of lipid-activated nuclear receptor-mediated non-classical or unorthodox actions in macrophages.

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NRIP, a Novel Nuclear Receptor Interaction Protein, Enhances the Transcriptional Activity of Nuclear Receptors

Journal of Biological Chemistry ◽

10.1074/jbc.m412169200 ◽

2005 ◽

Vol 280 (20) ◽

pp. 20000-20009 ◽

Cited By ~ 16

Author(s):

Tzung-Chieh Tsai ◽

Yung-Lu Lee ◽

Wei-Chih Hsiao ◽

Yeou-Ping Tsao ◽

Show-Li Chen

Keyword(s):

Nuclear Receptors ◽

Nuclear Receptor ◽

Transcriptional Activity ◽

Receptor Interaction

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Cardiac PPARαProtein Expression is Constant as Alternate Nuclear Receptors and PGC-1 Coordinately Increase During the Postnatal Metabolic Transition

PPAR Research ◽

10.1155/2008/279531 ◽

2008 ◽

Vol 2008 ◽

pp. 1-8 ◽

Cited By ~ 8

Author(s):

Norman E. Buroker ◽

Xue-Han Ning ◽

Michael Portman

Keyword(s):

Thyroid Hormone ◽

Nuclear Receptors ◽

Nuclear Receptor ◽

Oxidative Metabolism ◽

Thyroid Hormone Receptor ◽

Receptor Expression ◽

Pyruvate Dehydrogenase Kinase ◽

Mouse Heart ◽

Metabolic Switch ◽

Metabolic Transition

Gene expression data obtained in mouse heart indicate that increased expression for the nuclear receptor, peroxisomal proliferator activated receptorα(PPARα), prompts the postnatal transition from predominantly carbohydrate to fatty acid oxidation preference. However, no phenotypic or proteomic data are available to confirm downstream signaling and metabolic transition in mice. We studied the hypothesis that shifts in nuclear receptor expression trigger the newborn metabolic switch in a newborn sheep. This species is well characterized with regards to developmental changes in substrate oxidative metabolism. Heart tissues from fetal (130 days gestation), newborn≤24 hours, and 30-day old lambs were evaluated for protein expression from multiple enzymes controlling oxidative metabolism as well as principal nuclear receptors and coactivators. Although muscle and liver type carnitine palmitoyl transferases I showed no significant changes to correspond to the metabolic transition, hexokinase II protein content showed a profound transient drop, and pyruvate dehydrogenase kinase steadily increased. PPARαshowed no increases preceding or during the transition, while peroxisomal proliferator activated receptor gamma coactivator-1 (PGC-1) increased approximately 20-fold transiently in newborn heart in conjunction with significant increases in thyroid hormone receptorα1 and retinoid-activated receptorα. These data challenge the paradigm that increases in PPARαprompt the postnatal metabolic switch, and suggest that other nuclear receptors play a major role. As thyroid hormone (TH) modulates PGC-1 expression in sheep during development, these data further suggest that well-characterized perinatal TH surge in sheep contributes to this metabolic switch.

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