Molecular determinants of MED1 interaction with the DNA bound VDR-RXR heterodimer

AbstractThe MED1 subunit of the Mediator complex is an essential coactivator of nuclear receptor-mediated transcriptional activation. While structural requirements for ligand-dependent binding of classical coactivator motifs of MED1 to numerous nuclear receptor ligand-binding domains have been fully elucidated, the recognition of the full-length or truncated coactivator by full nuclear receptor complexes remain unknown. Here we present structural details of the interaction between a large part of MED1 comprising its structured N-terminal and the flexible receptor-interacting domains and the mutual heterodimer of the vitamin D receptor (VDR) and the retinoid X receptor (RXR) bound to their cognate DNA response element. Using a combination of structural and biophysical methods we show that the ligand-dependent interaction between VDR and the second coactivator motif of MED1 is crucial for complex formation and we identify additional, previously unseen, interaction details. In particular, we identified RXR regions involved in the interaction with structured N-terminal domain of MED1, as well as VDR regions outside the classical coactivator binding cleft affected by coactivator recruitment. These findings highlight important roles of each receptor within the heterodimer in selective recognition of MED1 and contribute to our understanding of the nuclear receptor-coregulator complexes.

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Molecular determinants of MED1 interaction with the DNA bound VDR–RXR heterodimer

Nucleic Acids Research ◽

10.1093/nar/gkaa775 ◽

2020 ◽

Vol 48 (19) ◽

pp. 11199-11213

Author(s):

Anna Y Belorusova ◽

Maxime Bourguet ◽

Steve Hessmann ◽

Sandra Chalhoub ◽

Bruno Kieffer ◽

...

Keyword(s):

Nuclear Receptor ◽

Transcriptional Activation ◽

Retinoid X Receptor ◽

Selective Recognition ◽

Receptor Complexes ◽

Binding Domains ◽

Structural Details ◽

Molecular Determinants ◽

Binding Cleft ◽

Dependent Interaction

Abstract The MED1 subunit of the Mediator complex is an essential coactivator of nuclear receptor-mediated transcriptional activation. While structural requirements for ligand-dependent binding of classical coactivator motifs of MED1 to numerous nuclear receptor ligand-binding domains have been fully elucidated, the recognition of the full-length or truncated coactivator by full nuclear receptor complexes remain unknown. Here we present structural details of the interaction between a large part of MED1 comprising its structured N-terminal and the flexible receptor-interacting domains and the mutual heterodimer of the vitamin D receptor (VDR) and the retinoid X receptor (RXR) bound to their cognate DNA response element. Using a combination of structural and biophysical methods we show that the ligand-dependent interaction between VDR and the second coactivator motif of MED1 is crucial for complex formation and we identify additional, previously unseen, interaction details. In particular, we identified RXR regions involved in the interaction with the structured N-terminal domain of MED1, as well as VDR regions outside the classical coactivator binding cleft affected by coactivator recruitment. These findings highlight important roles of each receptor within the heterodimer in selective recognition of MED1 and contribute to our understanding of the nuclear receptor-coregulator complexes.

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Multiple Signal Input and Output Domains of the 160-Kilodalton Nuclear Receptor Coactivator Proteins

Molecular and Cellular Biology ◽

10.1128/mcb.19.9.6164 ◽

1999 ◽

Vol 19 (9) ◽

pp. 6164-6173 ◽

Cited By ~ 169

Author(s):

Han Ma ◽

Heng Hong ◽

Shih-Ming Huang ◽

Ryan A. Irvine ◽

Paul Webb ◽

...

Keyword(s):

Nuclear Receptor ◽

Transcriptional Activation ◽

Thyroid Hormone Receptor ◽

Activation Function ◽

Creb Binding Protein ◽

Activation Domain ◽

Nuclear Receptor Coactivator ◽

Interacting Protein ◽

Binding Domains ◽

Signal Input

ABSTRACT Members of the 160-kDa nuclear receptor coactivator family (p160 coactivators) bind to the conserved AF-2 activation function found in the hormone binding domains of nuclear receptors (NR) and are potent transcriptional coactivators for NRs. Here we report that the C-terminal region of p160 coactivators glucocorticoid receptor interacting protein 1 (GRIP1), steroid receptor coactivator 1 (SRC-1a), and SRC-1e binds the N-terminal AF-1 activation function of the androgen receptor (AR), and p160 coactivators can thereby enhance transcriptional activation by AR. While they all interact efficiently with AR AF-1, these same coactivators have vastly different binding strengths with and coactivator effects on AR AF-2. p160 activation domain AD1, which binds secondary coactivators CREB binding protein (CBP) and p300, was previously implicated as the principal domain for transmitting the activating signal to the transcription machinery. We identified a new highly conserved motif in the AD1 region which is important for CBP/p300 binding. Deletion of AD1 only partially reduced p160 coactivator function, due to signaling through AD2, another activation domain located at the C-terminal end of p160 coactivators. C-terminal coactivator fragments lacking AD1 but containing AD2 and the AR AF-1 binding site served as efficient coactivators for full-length AR and AR AF-1. The two signal input domains (one that binds NR AF-2 domains and one that binds AF-1 domains of some but not all NRs) and the two signal output domains (AD1 and AD2) of p160 coactivators played different relative roles for two different NRs: AR and thyroid hormone receptor.

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The Corepressor CTBP2 Is a Coactivator of Retinoic Acid Receptor/Retinoid X Receptor in Retinoic Acid Signaling

Molecular and Cellular Biology ◽

10.1128/mcb.01213-12 ◽

2013 ◽

Vol 33 (16) ◽

pp. 3343-3353 ◽

Cited By ~ 16

Author(s):

Prashanth Kumar Bajpe ◽

Guus J. J. E. Heynen ◽

Lorenza Mittempergher ◽

Wipawadee Grernrum ◽

Iris A. de Rink ◽

...

Keyword(s):

Retinoic Acid ◽

Rna Interference ◽

Nuclear Receptor ◽

Transcriptional Control ◽

Target Genes ◽

Retinoic Acid Receptor ◽

Retinoid X Receptor ◽

Gene Promoters ◽

Acid Receptor ◽

Receptor Complexes

Retinoids play key roles in development, differentiation, and homeostasis through regulation of specific target genes by the retinoic acid receptor/retinoid X receptor (RAR/RXR) nuclear receptor complex. Corepressors and coactivators contribute to its transcriptional control by creating the appropriate chromatin environment, but the precise composition of these nuclear receptor complexes remains to be elucidated. Using an RNA interference-based genetic screen in mouse F9 cells, we identified the transcriptional corepressor CTBP2 (C-terminal binding protein 2) as a coactivator critically required for retinoic acid (RA)-induced transcription.CTBP2suppression by RNA interference confers resistance to RA-induced differentiation in diverse murine and human cells. Mechanistically, we find that CTBP2 associates with RAR/RXR at RA target gene promoters and is essential for their transactivation in response to RA. We show that CTBP2 is indispensable to create a chromatin environment conducive for RAR/RXR-mediated transcription by recruiting the histone acetyltransferase p300. Our data reveal an unexpected function of the corepressor CTBP2 as a coactivator for RAR/RXR in RA signaling.

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Natural vitamin D3 response elements formed by inverted palindromes: polarity-directed ligand sensitivity of vitamin D3 receptor-retinoid X receptor heterodimer-mediated transactivation.

Molecular and Cellular Biology ◽

10.1128/mcb.15.3.1154 ◽

1995 ◽

Vol 15 (3) ◽

pp. 1154-1161 ◽

Cited By ~ 99

Author(s):

M Schräder ◽

S Nayeri ◽

J P Kahlen ◽

K M Müller ◽

C Carlberg

Keyword(s):

Retinoic Acid ◽

Nuclear Receptor ◽

Vitamin D3 ◽

Transcriptional Activation ◽

Thyroid Hormone Receptor ◽

Retinoid X Receptor ◽

Direct Repeats ◽

Response Element ◽

Response Elements ◽

Ligand Sensitivity

VDR, the nuclear receptor for 1,25-dihydroxyvitamin D3 (VD), is a member of the superfamily of nuclear hormone receptors and controls multiple aspects of homeostasis, cell growth, and differentiation. VDR can function as a homodimer, but heterodimerization with the retinoid X receptor (RXR), retinoic acid receptor, or thyroid hormone receptor increases its affinity for response elements in the promoter of target genes. All natural VD response elements identified so far consist of direct repeats of a variety of hexameric core binding motifs with a preferential spacing of three nucleotides (DR3s). However, all four VD signalling pathways function also on response elements formed by inverted palindromes, although these sequences were not of natural origin. Here, we report the identification of two VD response elements consisting of inverted palindromes spaced by nine nucleotides (IP9s) in the promoters of the human calbindin D9k gene and the rat osteocalcin gene. Like most DR3-type VD response elements, both IP9s are preferentially bound by VDR-RXR heterodimers with a 5'-RXR-VDR-3' polarity, whose transcriptional activity can be enhanced by costimulation with 9-cis retinoic acid. We demonstrate that changing the response element orientation relatively to the basal promoter decreases the sensitivity of transcriptional activation by VD by about 10-fold. Our findings indicate that inverted palindromes are as functional as direct repeats. Furthermore, we suggest that the orientation of a nuclear receptor complex in relation to the basic transcriptional machinery, which is directed by heterodimer polarity and response element orientation, influences the ligand sensitivity of the respective target gene expression.

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Heterodimeric interaction between retinoid X receptor alpha and orphan nuclear receptor OR1 reveals dimerization-induced activation as a novel mechanism of nuclear receptor activation.

Molecular and Cellular Biology ◽

10.1128/mcb.17.7.3977 ◽

1997 ◽

Vol 17 (7) ◽

pp. 3977-3986 ◽

Cited By ~ 38

Author(s):

F F Wiebel ◽

J A Gustafsson

Keyword(s):

Ligand Binding ◽

Nuclear Receptors ◽

Nuclear Receptor ◽

Transcriptional Activation ◽

Receptor Activation ◽

Binding Domain ◽

Retinoid X Receptor ◽

Transcriptional Responses ◽

Receptor Alpha ◽

Retinoid X Receptor Alpha

OR1 is a member of the steroid/thyroid hormone nuclear receptor superfamily which has been described to mediate transcriptional responses to retinoids and oxysterols. On a DR4 response element, an OR1 heterodimer with the nuclear receptor retinoid X receptor alpha (RXR alpha) has been described to convey transcriptional activation in both the absence and presence of the RXR ligand 9-cis retinoic acid, the mechanisms of which have remained unclear. Here, we dissect the effects of RXR alpha and OR1 ligand-binding domain interaction on transcriptional regulation and the role of the respective carboxy-terminal activation domains (AF-2s) in the absence and presence of the RXR ligand, employing chimeras of the nuclear receptors containing the heterologous GAL4 DNA-binding domain as well as natural receptors. The results show that the interaction of the RXR and OR1 ligand-binding domains unleashes a transcription activation potential that is mainly dependent on the AF-2 of OR1, indicating that interaction with RXR activates OR1. This defines dimerization-induced activation as a novel function of heterodimeric interaction and mechanism of receptor activation not previously described for nuclear receptors. Moreover, we present evidence that activation of OR1 occurs by a conformational change induced upon heterodimerization with RXR.

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Role of molecular chaperones in steroid receptor action

Essays in Biochemistry ◽

10.1042/bse0400041 ◽

2004 ◽

Vol 40 ◽

pp. 41-58 ◽

Cited By ~ 149

Author(s):

William B Pratt ◽

Mario D Galigniana ◽

Yoshihiro Morishima ◽

Patrick J M Murphy

Keyword(s):

Transcriptional Activation ◽

Protein Trafficking ◽

Steroid Receptors ◽

Transcriptional Regulatory ◽

Ubiquitin Proteasome ◽

Proteasome Pathway ◽

Receptor Action ◽

Binding Cleft ◽

Hsp 90

Unliganded steroid receptors are assembled into heterocomplexes with heat-shock protein (hsp) 90 by a multiprotein chaperone machinery. In addition to binding the receptors at the chaperone site, hsp90 binds cofactors at other sites that are part of the assembly machinery, as well as immunophilins that connect the assembled receptor-hsp90 heterocomplexes to a protein trafficking pathway. The hsp90-/hsp70-based chaperone machinery interacts with the unliganded glucocorticoid receptor to open the steroid-binding cleft to access by a steroid, and the machinery interacts in very dynamic fashion with the liganded, transformed receptor to facilitate its translocation along microtubular highways to the nucleus. In the nucleus, the chaperone machinery interacts with the receptor in transcriptional regulatory complexes after hormone dissociation to release the receptor and terminate transcriptional activation. By forming heterocomplexes with hsp90, the chaperone machinery stabilizes the receptor to degradation by the ubiquitin-proteasome pathway of proteolysis.

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Faculty Opinions recommendation of Two functional modes of a nuclear receptor-recruited arginine methyltransferase in transcriptional activation.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1051506.503411 ◽

2006 ◽

Author(s):

Paul Webb

Keyword(s):

Nuclear Receptor ◽

Transcriptional Activation ◽

Arginine Methyltransferase

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Spanning the gap: unraveling RSC dynamics in vivo

Current Genetics ◽

10.1007/s00294-020-01144-1 ◽

2021 ◽

Author(s):

Heinz Neumann ◽

Bryan J. Wilkins

Keyword(s):

Cell Cycle ◽

Posttranslational Modifications ◽

Transcriptional Activation ◽

Binding Mode ◽

Binding Modes ◽

Binding Domains ◽

Binding Interface ◽

The Many ◽

And Function

AbstractMultiple reports over the past 2 years have provided the first complete structural analyses for the essential yeast chromatin remodeler, RSC, providing elaborate molecular details for its engagement with the nucleosome. However, there still remain gaps in resolution, particularly within the many RSC subunits that harbor histone binding domains.Solving contacts at these interfaces is crucial because they are regulated by posttranslational modifications that control remodeler binding modes and function. Modifications are dynamic in nature often corresponding to transcriptional activation states and cell cycle stage, highlighting not only a need for enriched spatial resolution but also temporal understanding of remodeler engagement with the nucleosome. Our recent work sheds light on some of those gaps by exploring the binding interface between the RSC catalytic motor protein, Sth1, and the nucleosome, in the living nucleus. Using genetically encoded photo-activatable amino acids incorporated into histones of living yeast we are able to monitor the nucleosomal binding of RSC, emphasizing the regulatory roles of histone modifications in a spatiotemporal manner. We observe that RSC prefers to bind H2B SUMOylated nucleosomes in vivo and interacts with neighboring nucleosomes via H3K14ac. Additionally, we establish that RSC is constitutively bound to the nucleosome and is not ejected during mitotic chromatin compaction but alters its binding mode as it progresses through the cell cycle. Our data offer a renewed perspective on RSC mechanics under true physiological conditions.

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