Structure and E3-ligase activity of the Ring-Ring complex of polycomb proteins Bmi1 and Ring1b

Abstract

Polycomb group proteins Ring1b and Bmi1 (B-cell-specific Moloney murine leukaemia virus integration site 1) are critical components of the chromatin modulating PRC1 complex. Histone H2A ubiquitination by the PRC1 complex strongly depends on the Ring1b protein. Here we show that the E3-ligase activity of Ring1b on histone H2A is enhanced by Bmi1 in vitro. The N-terminal Ring-domains are sufficient for this activity and Ring1a can replace Ring1b. E2 enzymes UbcH5a, b, c or UbcH6 support this activity with varying processivity and selectivity. All four E2s promote autoubiquitination of Ring1b without affecting E3-ligase activity. We solved the crystal structure of the Ring-Ring heterodimeric complex of Ring1b and Bmi1. In the structure the arrangement of the Ring-domains is similar to another H2A E3 ligase, the BRCA1/BARD1 complex, but complex formation depends on an N-terminal arm of Ring1b that embraces the Bmi1 Ring-domain. Mutation of a critical residue in the E2/E3 interface shows that catalytic activity resides in Ring1b and not in Bmi1. These data provide a foundation for understanding the critical enzymatic activity at the core of the PRC1 polycomb complex, which is implicated in stem cell maintenance and cancer.

Figure 1

Ring1b/Bmi1 and the minimal Ring1b/Bmi1 complex are catalytically active on histone H2A. GST-Ring1b, full-length Ring1b/Bmi1 (2 μM) or different Ring-domain constructs of Ring1b and Ring1a in complex with Bmi1₁₀₉ (2 μM) were incubated in ligase buffer in the presence of 28 nM human E1, 1.5 μM UbcH5c, 22 μM ubiquitin, 3 mM ATP and 4.8 μM H2A (18 μg nucleosomes). The reaction mixture was incubated at 30°C for 1 h. Samples were analysed for ubiquitination by SDS–PAGE and Western blot as described in Materials and methods. (A) Proteins and complexes used in this study. (B) Bmi1 enhances ligase activity of GST-Ring1b in vitro and is not active by itself. Upper panel: Bmi1 was titrated to the indicated final concentration into 2 μM GST-Ring1b, E1, MgATP, UbcH5c and nucleosomes. Lower panel: titration of Bmi1 with the indicated final concentrations in the presence of E1, MgATP, UbcH5c and nucleosomes and in the presence of 2 μM GST-Ring1b in lane 7 and 8, respectively. Western blots against ubiquitin. (C, D) The short Ring–Ring complex, Ring1b₁₅₉/Bmi1₁₀₉, is catalytically active in the presence of human E1 and UbcH5c. (C) Western blot against ubiquitinated histone H2A (uH2A) and histone H2A (H2A). (D) Western blot against ubiquitin. Ubiquitinated H2A, ubiquitinated Ring1b and Bmi1 are indicated. ^*Indicates diubiquitin. (E) Ring1a can substitute for Ring1b and the Ring1a₁₁₁/Bmi1₁₀₉ complex is equally active as the minimal Ring1b₁₁₄/Bmi1₁₀₉ complex against H2A; Western blot against ubiquitin. ^*Indicates diubiquitin.

Figure 2

Specific E2 requirement for histone H2A ubiquitination by the Ring1b₁₅₉/Bmi1₁₀₉ complex. Western blots against ubiquitin showing the effect of different E2 enzymes on H2A ubiquitination and poly-ubiquitin chain formation. For conditions, see Figure 1 and Materials and methods.

Figure 3

The crystal structure of the Ring1b/Bmi1 Ring–Ring complex. (A) Stereo ribbon representation of the crystal structure of the Ring1b₁₅₉/Bmi1₁₀₉ complex, with Ring1b in orange, Bmi1 in green. The Zn²⁺-ions and ligands are shown in grey. Site A is formed by Cys 51, 54, 72 and 75 in Ring1b and by Cys 18, 21, 39 and 42 in Bmi1. Site B is formed by Cys 67, 87, 90 and His 69 in Ring1b and by Cys 34, 53, 56 and His 36 in Bmi1. Transparent segments indicate disordered regions. (B) Both Ring motifs are very similar. Superposition of the Ring1b and Bmi1 Ring-domains. A three-residue insertion in the Bmi1 Ring-domain is indicated. (C, D) Details of the interface between Ring1b and Bmi1 (C) formed by the embracing arm of Ring1b and Bmi1's Ring-domain and kinked C-terminal helix, with important interactions indicated. (D) Close up view of the interface region formed by the helical bundle flanking the Ring-domains. Side chains of residues involved in the interaction according to analysis with PISA are shown. Arg 70 in Ring1b forms a salt bridge with Asp 72 in Bmi1, which is stabilised by lateral interactions with Phe 106 and Leu 75. (E) Sequence alignment of Ring-domain proteins in PRC1 with secondary structure indicated. Zn²⁺ binding site I is highlighted in blue and Zn²⁺ binding site II is highlighted in cyan. The autoubiquitination site in Ring1b is marked with a filled triangle. Sequence visible in the structure is underlined.

Figure 4

Structural comparison of Ring1b/Bmi1 with Ring and U-box dimers. (A) Superposition of Ring1b/Bmi1 onto the four-helix bundle of BRCA1/BARD1 (blue). The Zn²⁺ atoms in Ring1b/Bmi1 (grey) and BRCA1/BARD1 (magenta) are also depicted. (B) Superposition of Ring1b/Bmi1 onto the U-box domain of the ChIP homodimer (blue). The interface in these dimers is made by the regions that flank the Ring-domains.

Figure 5

Role of the N-terminus of Ring1a and Ring1b. (A) Assays performed with isolated Ring1a_30–111 and Bmi1 and complexes of Ring1b/Bmi1, Ring1b_16–114/Bmi1₁₀₉ and Ring1a₁₁₁/Bmi1₁₀₉ using conditions as described in the legend of Figure 1 and Materials and methods; Western blot against ubiquitin. (B) Bmi1 was titrated to the indicated final concentration into 2 μM Ring1a_30–111, E1, MgATP, UbcH5c and nucleosomes; Western blot against ubiquitin. (C) Titration of Bmi1 to the indicated final concentrations into 2 μM Ring1a_30–111 or Ring1a₁₁₁/Bmi1₁₀₉, respectively, using the same conditions as described in (B); Western blot against ubiquitin.

Figure 6

The E2 interaction of Ring1b/Bmi1. (A) Model of the E2/E3 interaction of Ring1b/Bmi1 with UbcH5c, based on the crystal structure of the ChIP U-box complexed to UbcH13. The Ser-Pro-Ala motif important for interaction with the U-box is indicated in UbcH5 (cyan). (B) Mutation of E2-interacting residues in Ring1b and in Bmi1 only shows effect on activity in Ring1b. Ubiquitination assay (Western blot against ubiquitin) with Ring1b₁₅₉/Bmi1₁₀₉ (wt/wt), Ring1b₁₅₉ I53A/Bmi1₁₀₉ (mt/wt) or Ring1b₁₅₉/Bmi1₁₀₉ L20A (wt/mt), as indicated with UbcH5c or UbcH6 as E2. For conditions, see Figure 1 and Materials and methods.