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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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In vitro binding assay of the interaction between the p300 TAZ2 domain and MEF2A. Top panel: binding of the 35S-labeled MEF2A to various GST-TAZ2 fusion proteins. Lane 2 was MEF2A with beads only. Lane 3 was GST only protein. Bottom panel: GST-TAZ2 fusion proteins used in the top panel were analyzed by SDS–PAGE. The non-degraded fusion protein bands are indicated by stars.
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Figure 1: In vitro binding assay of the interaction between the p300 TAZ2 domain and MEF2A. Top panel: binding of the 35S-labeled MEF2A to various GST-TAZ2 fusion proteins. Lane 2 was MEF2A with beads only. Lane 3 was GST only protein. Bottom panel: GST-TAZ2 fusion proteins used in the top panel were analyzed by SDS–PAGE. The non-degraded fusion protein bands are indicated by stars.

Mentions: The interaction between MEF2 and p300 was analyzed previously by several reports (25,26,69). These studies revealed that the MADS-box/MEF2 domain of MEF2 (amino acids 1–95) and a C-terminal region of p300 (amino acids 1572–1868) were necessary and sufficient to mediate p300 and MEF2 interaction. We confirmed this result by GST pull-down assay using individually purified proteins. By deletion analyses, we narrowed down the MEF2-binding region in p300 to a fragment that encompasses residues 1721–1837 (Figure 1, lane 4). This fragment corresponds to the TAZ2 domain of the CH3 region (amino acids 1725–1812) and a C-terminal extension (amino acids 1813–1837) with unknown function. We will refer this fragment (amino acids 1721–1837) as the TAZ2 domain hereafter. We previously hypothesized that the C-terminal extension might bind MEF2 analogously to class IIa HDACs and Cabin1 (58). However, further deletion studies revealed that while p300 (1805–1837) did bind MEF2 (Figure 1, lane 5), its affinity for MEF2 appeared to be much lower than that of p300 (1721–1837) (Figure 1, lanes 4 and 5). Moreover, the core TAZ2 domain of p300 (1721–1812) retained partial binding activity for MEF2 (Figure 1, lane 6). We also analyzed the interaction between p300 (1721–1837) and MEF2A (1–95) using electrophoresis mobility shift assay (EMSA), surface plasmon resonance (SPR), multi-angle light scattering (MALS), mass spectrometry and fluorescence anisotropy. While these experiments confirmed the binding of p300 (1721–1837) and MEF2A (1–95) in solution, quantitative analysis of binding constant and stoichiometry was complicated by the fact that the complex appears to be in equilibrium between several species (data not shown). Moreover, the p300 TAZ2 domain alone showed high background binding to DNA, making the full titration of the MEF2:DNA complex by high concentration of TAZ2 difficult. Nevertheless, our biochemical analyses suggest that p300 binds MEF2 significantly differently from Cabin1 and class IIa HDACs in that p300 uses a larger domain instead of a short helix.Figure 1.


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)

In vitro binding assay of the interaction between the p300 TAZ2 domain and MEF2A. Top panel: binding of the 35S-labeled MEF2A to various GST-TAZ2 fusion proteins. Lane 2 was MEF2A with beads only. Lane 3 was GST only protein. Bottom panel: GST-TAZ2 fusion proteins used in the top panel were analyzed by SDS–PAGE. The non-degraded fusion protein bands are indicated by stars.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: In vitro binding assay of the interaction between the p300 TAZ2 domain and MEF2A. Top panel: binding of the 35S-labeled MEF2A to various GST-TAZ2 fusion proteins. Lane 2 was MEF2A with beads only. Lane 3 was GST only protein. Bottom panel: GST-TAZ2 fusion proteins used in the top panel were analyzed by SDS–PAGE. The non-degraded fusion protein bands are indicated by stars.
Mentions: The interaction between MEF2 and p300 was analyzed previously by several reports (25,26,69). These studies revealed that the MADS-box/MEF2 domain of MEF2 (amino acids 1–95) and a C-terminal region of p300 (amino acids 1572–1868) were necessary and sufficient to mediate p300 and MEF2 interaction. We confirmed this result by GST pull-down assay using individually purified proteins. By deletion analyses, we narrowed down the MEF2-binding region in p300 to a fragment that encompasses residues 1721–1837 (Figure 1, lane 4). This fragment corresponds to the TAZ2 domain of the CH3 region (amino acids 1725–1812) and a C-terminal extension (amino acids 1813–1837) with unknown function. We will refer this fragment (amino acids 1721–1837) as the TAZ2 domain hereafter. We previously hypothesized that the C-terminal extension might bind MEF2 analogously to class IIa HDACs and Cabin1 (58). However, further deletion studies revealed that while p300 (1805–1837) did bind MEF2 (Figure 1, lane 5), its affinity for MEF2 appeared to be much lower than that of p300 (1721–1837) (Figure 1, lanes 4 and 5). Moreover, the core TAZ2 domain of p300 (1721–1812) retained partial binding activity for MEF2 (Figure 1, lane 6). We also analyzed the interaction between p300 (1721–1837) and MEF2A (1–95) using electrophoresis mobility shift assay (EMSA), surface plasmon resonance (SPR), multi-angle light scattering (MALS), mass spectrometry and fluorescence anisotropy. While these experiments confirmed the binding of p300 (1721–1837) and MEF2A (1–95) in solution, quantitative analysis of binding constant and stoichiometry was complicated by the fact that the complex appears to be in equilibrium between several species (data not shown). Moreover, the p300 TAZ2 domain alone showed high background binding to DNA, making the full titration of the MEF2:DNA complex by high concentration of TAZ2 difficult. Nevertheless, our biochemical analyses suggest that p300 binds MEF2 significantly differently from Cabin1 and class IIa HDACs in that p300 uses a larger domain instead of a short helix.Figure 1.

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