Highly cooperative recruitment of Ets-1 and release of autoinhibition by Pax5

Abstract

Pax5 (paired box binding factor 5) is a critical regulator of transcription and lineage commitment in B lymphocytes. In B cells, mb-1 (Ig-alpha/immunoglobulin-associated alpha) promoter transcription is activated by Pax5 through its recruitment of E74-like transforming sequence (Ets) family proteins to a composite site, the P5-EBS (Pax5-Ets binding site). Previously, X-ray crystallographic analysis revealed a network of contacts between the DNA-binding domains of Pax5 and Ets-1 while bound to the P5-EBS. Here, we report that Pax5 assembles these ternary complexes via highly cooperative interactions that overcome the autoinhibition of Ets-1. Using recombinant proteins, we calculated K(d(app)) values for the binding of Pax5, Ets-1, and GA-binding proteins, separately or together, to the P5-EBS. By itself, Pax5 binds the P5-EBS with high affinity (K(d) approximately equal 2 nM). Ets-1(331-440) bound the P5-EBS by itself with low affinity (K(d)=136 nM). However, autoinhibited Ets-1(280-440) alone does not bind detectably to the suboptimal sequences of the P5-EBS. Recruitment of Ets-1(331-440) or Ets-1(280-440) resulted in highly efficient ternary complex assembly with Pax5. Pax5 counteracts autoinhibition and increases binding of Ets-1 of the mb-1 promoter by >1000-fold. Mutation of Pax5 Gln22 to alanine (Q22A) enhances promoter binding by Pax5; however, Q22A greatly reduces recruitment of Ets-1(331-440) and Ets-1(280-440) by Pax5 (8.9- or >300-fold, respectively). Thus, Gln22 of Pax5 is essential for overcoming Ets-1 autoinhibition. Pax5 wild type and Q22A each recruited GA-binding protein alpha/beta1 to the mb-1 promoter with similar affinities, but recruitment was less efficient than that of Ets-1 (reduced by approximately 8-fold). Our results suggest a mechanism that allows Pax5 to overcome autoinhibition of Ets-1 DNA binding. In summary, these data illustrate requirements for partnerships between Ets proteins and Pax5.

Fig. 1

Recombinant proteins used in these studies. Proteins utilized in these studies include human Pax5(1–149) and murine Ets-1(280–440), Ets-1(331–440), GABPα (311–420) and GABPβ1(1–157). Open boxes representα–helices. Arrows represent β-sheets. Filled boxes denote the Notch-like/ankyrin repeats of GABPβ1. Boundaries of the paired domain of Pax5 (15–143) and ETS domains of Ets-1 and GABPα are indicated. The autoinhibitory domains of Ets-1 (shaded) and residues that are highlighted in the text are also indicated.

Fig. 2

Pax5(1–149)WT or Q22A bind similarly to the P5-EBS probe. (a) Representative equilibrium DNA binding analysis by EMSA with increasing amounts of Pax5 (1–149) WT (upper panel) or Q22A (lower panel) and a constant amount of the P5-EBS probe. (b) Non-linear least squares analysis of DNA binding complexes in (a). Protein concentrations shown in the graph were corrected for relative percent functional activities.

Fig. 2

Pax5(1–149)WT or Q22A bind similarly to the P5-EBS probe. (a) Representative equilibrium DNA binding analysis by EMSA with increasing amounts of Pax5 (1–149) WT (upper panel) or Q22A (lower panel) and a constant amount of the P5-EBS probe. (b) Non-linear least squares analysis of DNA binding complexes in (a). Protein concentrations shown in the graph were corrected for relative percent functional activities.

Fig. 3

Ets-1(331–440), but not Ets-1(280–440), binds to the P5-EBS probe. (a) Representative equilibrium DNA binding analysis by EMSA with increasing amounts of Ets-1(331–440) (upper panel) or (280–440) (lower panel) and a constant amount of the P5-EBS probe. (b) Non-linear least squares analysis of DNA binding complexes in (a). Graphs were adjusted for active protein concentrations as in Fig. 2b.

Fig. 3

Ets-1(331–440), but not Ets-1(280–440), binds to the P5-EBS probe. (a) Representative equilibrium DNA binding analysis by EMSA with increasing amounts of Ets-1(331–440) (upper panel) or (280–440) (lower panel) and a constant amount of the P5-EBS probe. (b) Non-linear least squares analysis of DNA binding complexes in (a). Graphs were adjusted for active protein concentrations as in Fig. 2b.

Fig. 4

Pax5 recruitment of Ets-1(331–440) or Ets-1(280–440) to bind the P5-EBS probe is highly cooperative and requires Gln22 of Pax5. (a) Representative equilibrium DNA binding analysis by EMSA with a constant amount of Pax5(1–149) WT (upper panel) or Q22A (lower panel), increasing amounts of Ets-1(331–440) and a constant amount of the P5-EBS probe. (b) Non-linear least squares analysis of DNA binding complexes in (A). Graphs were adjusted for active protein concentrations as in Fig. 2b. (c) Representative equilibrium DNA binding analysis by EMSA with a constant amount of Pax5(1–149) WT (upper panel) or Q22A (lower panel), increasing amounts of Ets-1(280–440) and a constant amount of the P5-EBS probe. (d) Nonlinear least squares analysis of DNA binding complexes in (c). Graphs were adjusted for active protein concentrations as in Fig. 2b.

Fig. 4

Pax5 recruitment of Ets-1(331–440) or Ets-1(280–440) to bind the P5-EBS probe is highly cooperative and requires Gln22 of Pax5. (a) Representative equilibrium DNA binding analysis by EMSA with a constant amount of Pax5(1–149) WT (upper panel) or Q22A (lower panel), increasing amounts of Ets-1(331–440) and a constant amount of the P5-EBS probe. (b) Non-linear least squares analysis of DNA binding complexes in (A). Graphs were adjusted for active protein concentrations as in Fig. 2b. (c) Representative equilibrium DNA binding analysis by EMSA with a constant amount of Pax5(1–149) WT (upper panel) or Q22A (lower panel), increasing amounts of Ets-1(280–440) and a constant amount of the P5-EBS probe. (d) Nonlinear least squares analysis of DNA binding complexes in (c). Graphs were adjusted for active protein concentrations as in Fig. 2b.

Fig. 4

Pax5 recruitment of Ets-1(331–440) or Ets-1(280–440) to bind the P5-EBS probe is highly cooperative and requires Gln22 of Pax5. (a) Representative equilibrium DNA binding analysis by EMSA with a constant amount of Pax5(1–149) WT (upper panel) or Q22A (lower panel), increasing amounts of Ets-1(331–440) and a constant amount of the P5-EBS probe. (b) Non-linear least squares analysis of DNA binding complexes in (A). Graphs were adjusted for active protein concentrations as in Fig. 2b. (c) Representative equilibrium DNA binding analysis by EMSA with a constant amount of Pax5(1–149) WT (upper panel) or Q22A (lower panel), increasing amounts of Ets-1(280–440) and a constant amount of the P5-EBS probe. (d) Nonlinear least squares analysis of DNA binding complexes in (c). Graphs were adjusted for active protein concentrations as in Fig. 2b.

Fig. 4

Pax5 recruitment of Ets-1(331–440) or Ets-1(280–440) to bind the P5-EBS probe is highly cooperative and requires Gln22 of Pax5. (a) Representative equilibrium DNA binding analysis by EMSA with a constant amount of Pax5(1–149) WT (upper panel) or Q22A (lower panel), increasing amounts of Ets-1(331–440) and a constant amount of the P5-EBS probe. (b) Non-linear least squares analysis of DNA binding complexes in (A). Graphs were adjusted for active protein concentrations as in Fig. 2b. (c) Representative equilibrium DNA binding analysis by EMSA with a constant amount of Pax5(1–149) WT (upper panel) or Q22A (lower panel), increasing amounts of Ets-1(280–440) and a constant amount of the P5-EBS probe. (d) Nonlinear least squares analysis of DNA binding complexes in (c). Graphs were adjusted for active protein concentrations as in Fig. 2b.

Fig. 5

Binding of the P5-EBS by GABPα is dependent on GABPβ1. (a) Representative equilibrium DNA binding analysis by EMSA with increasing amounts of GABPα/β1 (upper panel) or GABPα (lower panel) and a constant amount of the P5-EBS probe. (b) Non-linear least squares analysis of DNA binding complexes in (a). Graphs were adjusted for active protein concentrations as in Fig. 2b.

Fig. 5

Binding of the P5-EBS by GABPα is dependent on GABPβ1. (a) Representative equilibrium DNA binding analysis by EMSA with increasing amounts of GABPα/β1 (upper panel) or GABPα (lower panel) and a constant amount of the P5-EBS probe. (b) Non-linear least squares analysis of DNA binding complexes in (a). Graphs were adjusted for active protein concentrations as in Fig. 2b.

Fig. 6

Cooperative binding of the P5-EBS by Pax5 and GABPα or GABPα/β1 does not require Gln22 of Pax5. (a) Representative equilibrium DNA binding analysis by EMSA with a constant amount of Pax5(1–149) WT (upper panel) or Pax5 Q22A (lower panel), increasing amounts of GABPα and a constant amount of the P5-EBS probe. (b) Non-linear least squares analysis of DNA binding complexes in (a). Graphs were adjusted for active protein concentrations as in Fig. 2b. (c) Representative EMSA with a constant amount of Pax5(1–149) WT (upper panel) or Q22A (lower panel), increasing amounts of GABPα/β1 complexes and a constant amount of the P5-EBS probe. (d) Non-linear least squares analysis of DNA binding complexes in (c). Graphs were adjusted for active protein concentrations as in Fig. 2b.

Fig. 6

Cooperative binding of the P5-EBS by Pax5 and GABPα or GABPα/β1 does not require Gln22 of Pax5. (a) Representative equilibrium DNA binding analysis by EMSA with a constant amount of Pax5(1–149) WT (upper panel) or Pax5 Q22A (lower panel), increasing amounts of GABPα and a constant amount of the P5-EBS probe. (b) Non-linear least squares analysis of DNA binding complexes in (a). Graphs were adjusted for active protein concentrations as in Fig. 2b. (c) Representative EMSA with a constant amount of Pax5(1–149) WT (upper panel) or Q22A (lower panel), increasing amounts of GABPα/β1 complexes and a constant amount of the P5-EBS probe. (d) Non-linear least squares analysis of DNA binding complexes in (c). Graphs were adjusted for active protein concentrations as in Fig. 2b.

Fig. 6

Cooperative binding of the P5-EBS by Pax5 and GABPα or GABPα/β1 does not require Gln22 of Pax5. (a) Representative equilibrium DNA binding analysis by EMSA with a constant amount of Pax5(1–149) WT (upper panel) or Pax5 Q22A (lower panel), increasing amounts of GABPα and a constant amount of the P5-EBS probe. (b) Non-linear least squares analysis of DNA binding complexes in (a). Graphs were adjusted for active protein concentrations as in Fig. 2b. (c) Representative EMSA with a constant amount of Pax5(1–149) WT (upper panel) or Q22A (lower panel), increasing amounts of GABPα/β1 complexes and a constant amount of the P5-EBS probe. (d) Non-linear least squares analysis of DNA binding complexes in (c). Graphs were adjusted for active protein concentrations as in Fig. 2b.

Fig. 6

Cooperative binding of the P5-EBS by Pax5 and GABPα or GABPα/β1 does not require Gln22 of Pax5. (a) Representative equilibrium DNA binding analysis by EMSA with a constant amount of Pax5(1–149) WT (upper panel) or Pax5 Q22A (lower panel), increasing amounts of GABPα and a constant amount of the P5-EBS probe. (b) Non-linear least squares analysis of DNA binding complexes in (a). Graphs were adjusted for active protein concentrations as in Fig. 2b. (c) Representative EMSA with a constant amount of Pax5(1–149) WT (upper panel) or Q22A (lower panel), increasing amounts of GABPα/β1 complexes and a constant amount of the P5-EBS probe. (d) Non-linear least squares analysis of DNA binding complexes in (c). Graphs were adjusted for active protein concentrations as in Fig. 2b.

Fig. 7

Model of interaction between the paired domain of Pax5 and Ets-1(280–440) on mb-1 promoter DNA. (a) Overview of the ternary complex. The paired domain of Pax5 is represented in blue, the ETS domain of Ets-1 in magenta and the inhibitory regions of Ets-1 in green. Helix HI-1 of Ets-1 is shown in its unfolded state. (b) Enlarged view of the interface between Pax5 and Ets-1 on mb-1 promoter DNA. Hydrogen bonds between Gln22 of Pax5 and Gln336 or Tyr395 are indicated by dashed lines. A dashed line also indicates proposed interactions between H1 of Ets-1 and the sugar-phosphate backbone of DNA. The figure was made using VMD.

Fig. 7

Model of interaction between the paired domain of Pax5 and Ets-1(280–440) on mb-1 promoter DNA. (a) Overview of the ternary complex. The paired domain of Pax5 is represented in blue, the ETS domain of Ets-1 in magenta and the inhibitory regions of Ets-1 in green. Helix HI-1 of Ets-1 is shown in its unfolded state. (b) Enlarged view of the interface between Pax5 and Ets-1 on mb-1 promoter DNA. Hydrogen bonds between Gln22 of Pax5 and Gln336 or Tyr395 are indicated by dashed lines. A dashed line also indicates proposed interactions between H1 of Ets-1 and the sugar-phosphate backbone of DNA. The figure was made using VMD.