Electron microscopic study of the non-canonical polycomb repressive complex 1.6

Objective·To analyse the structure of non-canonical polycomb repressive complex 1.6 (PRC1.6) by negative staining and transmission electron microscopy (TEM), and obtain the three-dimensional (3D) profile information of human PRC1.6 heptameric complex.Methods·Seven PRC1.6 components, RNF2, PCGF6, RYBP, L3MBTL2, CBX3, E2F6, and TFDP1, were cloned into the pMLink vector with a 6×His-3×Flag tag at the N-terminus, respectively. The proteins were expressed in Expi293F cells grown in suspension cultures by using transfection with polyethylenimine. The tagged proteins were isolated via affinity purification with anti-DYKDDDDK G1 affinity resin, followed by gel filtration chromatography with Superdex 200 Increase 10/300 GL and glycerol density gradient centrifugation. The components of the PRC1.6 heptameric complex were confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The in vitro ubiquitination activity and nucleosome-binding affinity of the purified heptameric complex were verified by the ubiquitination activity assay and the electrophoretic mobility shift assay (EMSA). The protein samples were stained by uranyl acetate and observed by TEM. The 3D information of the PRC1.6 complex was studied by single particle analysis. To predict the localization of the seven components within the structure model of PRC1.6 complex, the structure models of proteins in Protein Data Bank (PDB) were docked into the electron density map of PRC1.6 complex by using UCSF Chimera software.Results·The PRC1.6 complex with high purity and good homogeneity was obtained by eukaryotic expression, affinity purification, gel filtration chromatography and glycerol density gradient centrifugation, and confirmed as the heptameric complex by LC-MS/MS. The purified proteins showed ubiquitination activity and nucleosome-binding affinity in vitro. The 3D structure of the PRC1.6 heptameric complex with a resolution of 15.2 Å (1 Å=10-10 m) was preliminarily resolved by negative staining, TEM, and single particle analysis. The available structure models of RNF2, PCGF6, RYBP, L3MBTL2, CBX3, and DP1 proteins, as well as the predicted E2F6 structure by AlphaFold2, were docked into the reconstructed density map of PRC1.6 complex. The position of each component in the complex was preliminarily confirmed.Conclusion·The 3D structural model of the human PRC1.6 heptameric complex is obtained by negative staining, TEM, and single particle analysis.