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Structure of p300 bound to MEF2 on DNA reveals a mechanism of enhanceosome assembly.

He J, Ye J, Cai Y, Riquelme C, Liu JO, Liu X, Han A, Chen L - Nucleic Acids Res. (2011)

Bottom Line: Using structure-guided mutations and a mammalian two-hybrid assay, we show that all three interfaces contribute to the binding of MEF2 to p300, suggesting that p300 may use one of the three interfaces to interact with MEF2 in different cellular contexts and that one p300 can bind three MEF2:DNA complexes simultaneously.These studies, together with previously characterized TAZ2 complexes bound to different transcription factors, demonstrate the potency and versatility of TAZ2 in protein-protein interactions.Our results also support a model wherein p300 promotes the assembly of a higher-order enhanceosome by simultaneous interactions with multiple DNA-bound transcription factors.

View Article: PubMed Central - PubMed

Affiliation: MOE Key Laboratory for Cell Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China.

ABSTRACT
Transcription co-activators CBP and p300 are recruited by sequence-specific transcription factors to specific genomic loci to control gene expression. A highly conserved domain in CBP/p300, the TAZ2 domain, mediates direct interaction with a variety of transcription factors including the myocyte enhancer factor 2 (MEF2). Here we report the crystal structure of a ternary complex of the p300 TAZ2 domain bound to MEF2 on DNA at 2.2Å resolution. The structure reveals three MEF2:DNA complexes binding to different sites of the TAZ2 domain. Using structure-guided mutations and a mammalian two-hybrid assay, we show that all three interfaces contribute to the binding of MEF2 to p300, suggesting that p300 may use one of the three interfaces to interact with MEF2 in different cellular contexts and that one p300 can bind three MEF2:DNA complexes simultaneously. These studies, together with previously characterized TAZ2 complexes bound to different transcription factors, demonstrate the potency and versatility of TAZ2 in protein-protein interactions. Our results also support a model wherein p300 promotes the assembly of a higher-order enhanceosome by simultaneous interactions with multiple DNA-bound transcription factors.

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Related in: MedlinePlus

Structure-guided biochemical analyses of p300:MEF2 interactions. (A) Diagram of the biochemical assay. (B) Luciferase reporter assay of p300 TAZ2 mutants. Columns 1–3 are controls. Column 4 is from wild-type p300 and MEF2 interaction. Columns 5–8 are IF-I mutants, 10–14 are IF-II mutants and 16–19 are IF-III mutants. Column 6 is a combined mutant of IF-I and IF-II.
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Figure 5: Structure-guided biochemical analyses of p300:MEF2 interactions. (A) Diagram of the biochemical assay. (B) Luciferase reporter assay of p300 TAZ2 mutants. Columns 1–3 are controls. Column 4 is from wild-type p300 and MEF2 interaction. Columns 5–8 are IF-I mutants, 10–14 are IF-II mutants and 16–19 are IF-III mutants. Column 6 is a combined mutant of IF-I and IF-II.

Mentions: A plasmid was constructed to express the p300 TAZ2 domain (1721–1837) fused to the N terminal end of VP16 (Figure 5A). MEF2-Gal4 and Gal4 reporter plasmids were described by Gregoire and Yang (68). All these expression constructs were co-transfected to HeLa cells using lipofectamine 2000 or calcium phosphate. The experiments were performed in triplicate, and luciferases were measured using the dual-luciferase reporter assay kit according to manufacturer’s instructions. Briefly, 20 µl of cell lysate was transferred to a luminometer tube predispensed with 100 µl of Luciferase Assay Reagent II (LARII), and luminescence of firefly luciferase was measured by a Luminometer (Berthold). Then, 100 µl of Stop & Glo Reagent was added to the same tube and the light emission of renilla luciferase was recorded. Results are presented as activity ratios of firefly over renilla luciferases.


Structure of p300 bound to MEF2 on DNA reveals a mechanism of enhanceosome assembly.

He J, Ye J, Cai Y, Riquelme C, Liu JO, Liu X, Han A, Chen L - Nucleic Acids Res. (2011)

Structure-guided biochemical analyses of p300:MEF2 interactions. (A) Diagram of the biochemical assay. (B) Luciferase reporter assay of p300 TAZ2 mutants. Columns 1–3 are controls. Column 4 is from wild-type p300 and MEF2 interaction. Columns 5–8 are IF-I mutants, 10–14 are IF-II mutants and 16–19 are IF-III mutants. Column 6 is a combined mutant of IF-I and IF-II.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105382&req=5

Figure 5: Structure-guided biochemical analyses of p300:MEF2 interactions. (A) Diagram of the biochemical assay. (B) Luciferase reporter assay of p300 TAZ2 mutants. Columns 1–3 are controls. Column 4 is from wild-type p300 and MEF2 interaction. Columns 5–8 are IF-I mutants, 10–14 are IF-II mutants and 16–19 are IF-III mutants. Column 6 is a combined mutant of IF-I and IF-II.
Mentions: A plasmid was constructed to express the p300 TAZ2 domain (1721–1837) fused to the N terminal end of VP16 (Figure 5A). MEF2-Gal4 and Gal4 reporter plasmids were described by Gregoire and Yang (68). All these expression constructs were co-transfected to HeLa cells using lipofectamine 2000 or calcium phosphate. The experiments were performed in triplicate, and luciferases were measured using the dual-luciferase reporter assay kit according to manufacturer’s instructions. Briefly, 20 µl of cell lysate was transferred to a luminometer tube predispensed with 100 µl of Luciferase Assay Reagent II (LARII), and luminescence of firefly luciferase was measured by a Luminometer (Berthold). Then, 100 µl of Stop & Glo Reagent was added to the same tube and the light emission of renilla luciferase was recorded. Results are presented as activity ratios of firefly over renilla luciferases.

Bottom Line: Using structure-guided mutations and a mammalian two-hybrid assay, we show that all three interfaces contribute to the binding of MEF2 to p300, suggesting that p300 may use one of the three interfaces to interact with MEF2 in different cellular contexts and that one p300 can bind three MEF2:DNA complexes simultaneously.These studies, together with previously characterized TAZ2 complexes bound to different transcription factors, demonstrate the potency and versatility of TAZ2 in protein-protein interactions.Our results also support a model wherein p300 promotes the assembly of a higher-order enhanceosome by simultaneous interactions with multiple DNA-bound transcription factors.

View Article: PubMed Central - PubMed

Affiliation: MOE Key Laboratory for Cell Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China.

ABSTRACT
Transcription co-activators CBP and p300 are recruited by sequence-specific transcription factors to specific genomic loci to control gene expression. A highly conserved domain in CBP/p300, the TAZ2 domain, mediates direct interaction with a variety of transcription factors including the myocyte enhancer factor 2 (MEF2). Here we report the crystal structure of a ternary complex of the p300 TAZ2 domain bound to MEF2 on DNA at 2.2Å resolution. The structure reveals three MEF2:DNA complexes binding to different sites of the TAZ2 domain. Using structure-guided mutations and a mammalian two-hybrid assay, we show that all three interfaces contribute to the binding of MEF2 to p300, suggesting that p300 may use one of the three interfaces to interact with MEF2 in different cellular contexts and that one p300 can bind three MEF2:DNA complexes simultaneously. These studies, together with previously characterized TAZ2 complexes bound to different transcription factors, demonstrate the potency and versatility of TAZ2 in protein-protein interactions. Our results also support a model wherein p300 promotes the assembly of a higher-order enhanceosome by simultaneous interactions with multiple DNA-bound transcription factors.

Show MeSH
Related in: MedlinePlus