Abstract
ProMyelocyticLeukemia (PML) protein can polymerize into a mega-Dalton nuclear assembly of 0.1–2 μm in diameter. The mechanism of PML nuclear body biogenesis remains elusive. Here, PMLRBCC is successfully purified. The gel filtration and ultracentrifugation analysis suggest a previously unrecognized sequential oligomerization mechanism via PML monomer, dimer, tetramer and N-mer. Consistently, PML B1-box structure (2.0 Å) and SAXS characterization reveal an unexpected networking by W157-, F158- and SD1-interfaces. Structure-based perturbations in these B1 interfaces not only impair oligomerization in vitro but also abolish PML sumoylation and nuclear body biogenesis in HeLaPml-/- cell. More importantly, as demonstrated by in vivo study using transgenic mice, PML-RARα (PR) F158E precludes leukemogenesis. In addition, single cell RNA sequencing analysis shows that B1 oligomerization is an important regulator in PML-RARα-driven transactivation. Altogether, these results not only define a previously unrecognized B1-box oligomerization in PML, but also highlight oligomerization as an important factor in carcinogenesis.
Highlights
ProMyelocyticLeukemia (PML) protein can polymerize into a mega-Dalton nuclear assembly of 0.1–2 μm in diameter
The ProMyelocyticLeukemia (PML) protein, known as TRIM19, is the core component of PML nuclear bodies (NBs) that are tightly associated with nuclear matrix
In order to investigate the RBCC oligomerization, the purified PML1–256 was subjected to gel filtration (GF) analysis using a Superdex 200 column (Fig. 1c)
Summary
ProMyelocyticLeukemia (PML) protein can polymerize into a mega-Dalton nuclear assembly of 0.1–2 μm in diameter. PML B1-box structure (2.0 Å) and SAXS characterization reveal an unexpected networking by W157-, F158- and SD1-interfaces Structurebased perturbations in these B1 interfaces impair oligomerization in vitro and abolish PML sumoylation and nuclear body biogenesis in HeLaPml-/- cell. Single cell RNA sequencing analysis shows that B1 oligomerization is an important regulator in PML-RARα-driven transactivation. We report the largest RBCC fragment ever purified and the 2.0 Å crystal structure of B1 multimers. These results have prompted the in vitro and in vivo characterizations of PML and its oncogenic fusion PML-RARα (PR) in atomic detail
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