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The structural basis of direct glucocorticoid-mediated transrepression.

Hudson WH, Youn C, Ortlund EA - Nat. Struct. Mol. Biol. (2012)

Bottom Line: The nGRE differs dramatically from activating response elements, and the mechanism driving GR binding and transrepression is unknown.To unravel the mechanism of nGRE-mediated transrepression by the GR, we characterized the interaction between GR and an nGRE in the thymic stromal lymphopoietin (TSLP) promoter.We show using structural and mechanistic approaches that nGRE binding is a new mode of sequence recognition by human GR and that nGREs prevent receptor dimerization through a unique GR-binding orientation and strong negative cooperativity, ensuring the presence of monomeric GR at repressive elements.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia, USA.

ABSTRACT
A newly discovered negative glucocorticoid response element (nGRE) mediates DNA-dependent transrepression by the glucocorticoid receptor (GR) across the genome and has a major role in immunosuppressive therapy. The nGRE differs dramatically from activating response elements, and the mechanism driving GR binding and transrepression is unknown. To unravel the mechanism of nGRE-mediated transrepression by the GR, we characterized the interaction between GR and an nGRE in the thymic stromal lymphopoietin (TSLP) promoter. We show using structural and mechanistic approaches that nGRE binding is a new mode of sequence recognition by human GR and that nGREs prevent receptor dimerization through a unique GR-binding orientation and strong negative cooperativity, ensuring the presence of monomeric GR at repressive elements.

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The R460D D462R mutant reduces receptor dimerization, enhancing GR-mediated transrepression at theTSLP nGRE(a) Transfection of the R460D D462R mutant in HeLa cells potentiates downregulation of a constitutively active TSLP promoter compared to WT GR (5 ng each). Data are represented as the mean ± s.e.m of two independent experiments with five internal replicates each. (b) Superposition of GR (blue) and the GR R460D D462R mutant (magenta) bound to the TSLP nGRE (gray). (c) When bound to a (+)GRE element, Arg460 and Asp462 form two intermolecular salt bridges (red dashes) across the homodimer interface (PDB 3FYL). In the GR nGRE structure, crystal-packing interactions require the formation of a pseudo-continuous DNA helix and promote the formation of a pseudo-GR dimer across a two-fold symmetry axis (d). These interactions are necessary for crystal formation, but are not possible in solution-based binding assays. The R460D D462R mutation (e) ablates key dimerization contacts between GR monomers, disrupting symmetry-imposed dimerization contacts.
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Figure 3: The R460D D462R mutant reduces receptor dimerization, enhancing GR-mediated transrepression at theTSLP nGRE(a) Transfection of the R460D D462R mutant in HeLa cells potentiates downregulation of a constitutively active TSLP promoter compared to WT GR (5 ng each). Data are represented as the mean ± s.e.m of two independent experiments with five internal replicates each. (b) Superposition of GR (blue) and the GR R460D D462R mutant (magenta) bound to the TSLP nGRE (gray). (c) When bound to a (+)GRE element, Arg460 and Asp462 form two intermolecular salt bridges (red dashes) across the homodimer interface (PDB 3FYL). In the GR nGRE structure, crystal-packing interactions require the formation of a pseudo-continuous DNA helix and promote the formation of a pseudo-GR dimer across a two-fold symmetry axis (d). These interactions are necessary for crystal formation, but are not possible in solution-based binding assays. The R460D D462R mutation (e) ablates key dimerization contacts between GR monomers, disrupting symmetry-imposed dimerization contacts.

Mentions: The A458T mutant bound to a (+)GRE in a clear 2-site binding event (Table 1), indicating a loss of cooperativity on this element. In this way, binding of the A458T mutant to (+)GREs strongly resembles binding of WT GR to nGREs. The R460D D462R mutant showed similar DNA binding as WT to (+)GRE sequences. Next, we tested each of these mutants for binding to nGREs. The A458T mutation differentially affected binding to each of the GR binding sites on the TSLP nGRE, improving low-affinity site binding but decreasing high-affinity site binding (Table 1). The net effect of this mutation is to decrease the affinity of GR for nGREs by 500 %, to nearly 3 μM. In contrast, the R460D D462R mutation improved binding at both sites on the TSLP nGRE. We then tested the ability of each variant to repress a reporter containing a constitutively active luciferase gene preceded by the nGRE-containing region of the TSLP promoter, as performed previously10. In line with our in vitro binding data, the A458T showed a modest ability to repress luciferase expression (Supplementary Fig. 5). Strikingly, the R460D D462R mutation was a more potent repressor of luciferase activity than WT GR (Fig. 3a). To observe the effects of this mutant on the GR dimerization interface, we solved the crystal structure of the GR R460D D462R mutant bound to the TSLP nGRE (Fig. 3b, Table 2). The structure of the R460D D462R mutant shows a less favorable dimerization interface (Fig. 3c–e), suggesting that the superior binding and repressive potential of the GR R460D D462R mutant is indeed due to decreased dimerization efficiency.


The structural basis of direct glucocorticoid-mediated transrepression.

Hudson WH, Youn C, Ortlund EA - Nat. Struct. Mol. Biol. (2012)

The R460D D462R mutant reduces receptor dimerization, enhancing GR-mediated transrepression at theTSLP nGRE(a) Transfection of the R460D D462R mutant in HeLa cells potentiates downregulation of a constitutively active TSLP promoter compared to WT GR (5 ng each). Data are represented as the mean ± s.e.m of two independent experiments with five internal replicates each. (b) Superposition of GR (blue) and the GR R460D D462R mutant (magenta) bound to the TSLP nGRE (gray). (c) When bound to a (+)GRE element, Arg460 and Asp462 form two intermolecular salt bridges (red dashes) across the homodimer interface (PDB 3FYL). In the GR nGRE structure, crystal-packing interactions require the formation of a pseudo-continuous DNA helix and promote the formation of a pseudo-GR dimer across a two-fold symmetry axis (d). These interactions are necessary for crystal formation, but are not possible in solution-based binding assays. The R460D D462R mutation (e) ablates key dimerization contacts between GR monomers, disrupting symmetry-imposed dimerization contacts.
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Related In: Results  -  Collection

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Figure 3: The R460D D462R mutant reduces receptor dimerization, enhancing GR-mediated transrepression at theTSLP nGRE(a) Transfection of the R460D D462R mutant in HeLa cells potentiates downregulation of a constitutively active TSLP promoter compared to WT GR (5 ng each). Data are represented as the mean ± s.e.m of two independent experiments with five internal replicates each. (b) Superposition of GR (blue) and the GR R460D D462R mutant (magenta) bound to the TSLP nGRE (gray). (c) When bound to a (+)GRE element, Arg460 and Asp462 form two intermolecular salt bridges (red dashes) across the homodimer interface (PDB 3FYL). In the GR nGRE structure, crystal-packing interactions require the formation of a pseudo-continuous DNA helix and promote the formation of a pseudo-GR dimer across a two-fold symmetry axis (d). These interactions are necessary for crystal formation, but are not possible in solution-based binding assays. The R460D D462R mutation (e) ablates key dimerization contacts between GR monomers, disrupting symmetry-imposed dimerization contacts.
Mentions: The A458T mutant bound to a (+)GRE in a clear 2-site binding event (Table 1), indicating a loss of cooperativity on this element. In this way, binding of the A458T mutant to (+)GREs strongly resembles binding of WT GR to nGREs. The R460D D462R mutant showed similar DNA binding as WT to (+)GRE sequences. Next, we tested each of these mutants for binding to nGREs. The A458T mutation differentially affected binding to each of the GR binding sites on the TSLP nGRE, improving low-affinity site binding but decreasing high-affinity site binding (Table 1). The net effect of this mutation is to decrease the affinity of GR for nGREs by 500 %, to nearly 3 μM. In contrast, the R460D D462R mutation improved binding at both sites on the TSLP nGRE. We then tested the ability of each variant to repress a reporter containing a constitutively active luciferase gene preceded by the nGRE-containing region of the TSLP promoter, as performed previously10. In line with our in vitro binding data, the A458T showed a modest ability to repress luciferase expression (Supplementary Fig. 5). Strikingly, the R460D D462R mutation was a more potent repressor of luciferase activity than WT GR (Fig. 3a). To observe the effects of this mutant on the GR dimerization interface, we solved the crystal structure of the GR R460D D462R mutant bound to the TSLP nGRE (Fig. 3b, Table 2). The structure of the R460D D462R mutant shows a less favorable dimerization interface (Fig. 3c–e), suggesting that the superior binding and repressive potential of the GR R460D D462R mutant is indeed due to decreased dimerization efficiency.

Bottom Line: The nGRE differs dramatically from activating response elements, and the mechanism driving GR binding and transrepression is unknown.To unravel the mechanism of nGRE-mediated transrepression by the GR, we characterized the interaction between GR and an nGRE in the thymic stromal lymphopoietin (TSLP) promoter.We show using structural and mechanistic approaches that nGRE binding is a new mode of sequence recognition by human GR and that nGREs prevent receptor dimerization through a unique GR-binding orientation and strong negative cooperativity, ensuring the presence of monomeric GR at repressive elements.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia, USA.

ABSTRACT
A newly discovered negative glucocorticoid response element (nGRE) mediates DNA-dependent transrepression by the glucocorticoid receptor (GR) across the genome and has a major role in immunosuppressive therapy. The nGRE differs dramatically from activating response elements, and the mechanism driving GR binding and transrepression is unknown. To unravel the mechanism of nGRE-mediated transrepression by the GR, we characterized the interaction between GR and an nGRE in the thymic stromal lymphopoietin (TSLP) promoter. We show using structural and mechanistic approaches that nGRE binding is a new mode of sequence recognition by human GR and that nGREs prevent receptor dimerization through a unique GR-binding orientation and strong negative cooperativity, ensuring the presence of monomeric GR at repressive elements.

Show MeSH
Related in: MedlinePlus