RING tetramerization is required for nuclear body biogenesis and PML sumoylation

Abstract

ProMyelocyticLeukemia nuclear bodies (PML NBs) are stress-regulated domains directly implicated in acute promyelocytic leukemia eradication. Most TRIM family members bind ubiquitin E2s and many acquire ligase activity upon RING dimerization. In contrast, PML binds UBC9, the SUMO E2 enzyme. Here, using X-ray crystallography and SAXS characterization, we demonstrate that PML RING tetramerizes through highly conserved PML-specific sequences, which are required for NB assembly and PML sumoylation. Conserved residues implicated in RING dimerization of other TRIMs also contribute to PML tetramer stability. Wild-type PML rescues the ability of some RING mutants to form NBs as well as their sumoylation. Impaired RING tetramerization abolishes PML/RARA-driven leukemogenesis in vivo and arsenic-induced differentiation ex vivo. Our studies thus identify RING tetramerization as a key step in the NB macro-molecular scaffolding. They suggest that higher order RING interactions allow efficient UBC9 recruitment and thus change the biochemical nature of TRIM-facilitated post-translational modifications.

Fig. 1

Crystal structure of PML RING tetramer. a Crystal structure of PML RING tetramer. The residues 51–97 of crystallized PML RING49–104 are visible in the electron density map. Four PML monomers are colored in green, magenta, blue and yellow, respectively. The contact residues (F52, F54 and L73) are shown in stick representation. Zn ions are shown in sphere representation. Sub-domain 1 (SD1_FQF) and sub-domain 2 (SD2) are bracketed. b Sequence alignment of the PML RING domains from different species. The conserved residues lying in the F52/54-interfaces and L73-interfaces are highlighted in red, while the conserved Zn-binding residues are colored in cyan. The deep purple boxes underneath the sequences are used to highlight the conserved (greater than 5 out of 6) residues among PML RING. c, d Enlarged views of PML RING dimeric interfaces. The residues involving PML oligomerization are shown in stick representation. e Structural superimposition between different PML RINGs. The NMR and crystallographic PML RINGs are colored in yellow and green, respectively. L73 positions are labeled with “Asterisk”. The internal Zn–Zn distances and the putative F₅₂Q₅₃F₅₄ swing are highlighted with dash lines. f The buried areas of the SD1_FQF/SD2 and SD2/SD2 interfaces are shown in gray

Fig. 2

Characterization of PML RING tetramers. a Analytical ultracentrifugation analysis of tetramerization of wild type PML RING, at 1 and 5 mg ml⁻¹ concentrations, respectively. b Analytical ultracentrifugation analysis of 1 mg ml⁻¹ PML RING, PML RING_F52/54E and PML RING_L73E. c SAXS characterization. Black dots, experimental data. Red line, the theoretical scattering pattern derived from mixs of PML RING multimers. d Oligomeric distribution estimated by SAXS analysis. The distribution of PML RING monomer (60.1%), dimer (26.1%) and tetramer (13.8%) was estimated using the OLIGOMER algorithm

Fig. 3

Biochemical evidence for RING tetramer formation. a Consensus sequence among TRIM RINGs (Top). PML conserved sequences are highlighted with purple boxes (Bottom). The conserved TRIM dimeric interface is highlighted with a red line. The Asn and Ile/Leu residues (red arrows) mediate dimeric assembly of other TRIMs. b Gel filtration analysis of recombinant PML RING_1–119. Two elution peaks and a last fraction, designated as Peak 1, 2 and fraction 3, respectively. c–e SEC-MALS reanalysis of Peak 1 (c), Peak 2 (d) and fraction 3 (e) derived from previous gel filtration (b). Black curve, the elution profile in UV. Orange curve, the estimated molecular weight. The theoretical molecular weight of PML RING_1–119 is 13.1 kDa. Peak 1 sample displayed distinct peaks that correspond to the tetramer (54 kDa), dimer and monomer in the solution. Peak 2 and fraction 3 samples primarily exhibit RING dimer (24 kDa). f Gel filtration of WT RING_1–119 and its mutants. g Recombinant PML RING_1–119 was subjected to chymotrypsin digestion (Supplementary Figure 5). Arrows indicate the chymotrypsin sites and amino acids indicated in red correspond to the protected fragment, as determined by mass spectrometry and N-terminal sequencing (Box). RING_49–104 sequences analyzed by X-ray crystallography are underlined with a blue line

Fig. 4

PML tetramerization in NB assembly and PML sumoylation in cellulo. a Pml^−/− MEFs stably expressing wild type HA-PML, HA-PML_F52/54E and HA-PML_L73E were analyzed by immuno-fluorescence using anti-HA antibodies (left, green). Scale bar is 5 μm. Statistical analyses (right). All experiments have been done in five independent replicates, NB count was from 10 to 20 nuclei. Values are means ± S.D. and One-way ANOVA (p-values) are indicated. b Western blot analysis demonstrating loss of sumoylation of these PML mutants, as detected by anti-HA antibodies. Uncropped images of all Western blots are shown in Supplementary Figures 9–12

Fig. 5

PML tetramerization controls NB formation and PML sumoylation. a Immortalized Pml^−/− MEFs were transduced with MSCV virus expressing PML or its mutants and subsequently transduced or not with CFP-PML. PML NBs were monitored by immuno-fluorescence using anti-HA antibody (red). DAPI is in blue. Scale bar is 5 μm. b Immortalized Pml^−/− MEFs obtained in a were treated with As₂O₃ (10^–6M, 1 h) and extracts were analyzed by Western blot using anti-HA or anti-CFP. PML and its sumoylated forms are indicated. c Co-localization of stably expressed HA-PML (red) and UBC9-GFP (green) basal condition (left) or upon 10⁻⁶M As₂O₃ for 1h (right). DAPI is in blue. Middle and bottom: Visualization of PML and UBC9 localization with/without arsenic. Scale bars are 5 μm (top) and 0.5 μm (middle and bottom). d Mammalian two-hybrid: relative luciferase activities (RLU) were used to estimate the interaction between UBC9 and PML/mutants. Statistical significance is indicated. All experiments have been done at least with three independent replicates. Values are means ± S.E. ***p < 0.001 are used to show statistically significant between recombinant derivatives. pACT-PML and pBIND-PML interaction is shown as a positive control

Fig. 6

PML RING tetramerization is important for APL development and arsenic targeting of PML/RARA-transformed cells. a Survival data of MRP8-PML/RARA or -PML/RARA_L73E transgenic mice. b Pml^−/− MEFs expressing HA-PML/RARA, HA-PML/RARA_F52/54E or HA-PML/RARA_L73E were treated with As₂O₃ (10^–6M) prior to immunofluorescence analysis. Scale bar is 5 μm. c Sumoylation of HA-PML/RARA after 1 h of As₂O₃ exposure was monitored by Western blot using anti-RARA antibody. d MGG staining of mouse hematopoietic progenitors transformed by PML/RARA and the indicated mutants after arsenic treatment (10^–7M, 7 days). e Proposed model for NB assembly and PML sumoylation integrating the PML RING tetramer formation