Journal of Shanghai Jiao Tong University (Medical Science) >

2024 , Vol. 44 >Issue 9: 1136 - 1145

DOI: https://doi.org/10.3969/j.issn.1674-8115.2024.09.008

Basic research

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

  • Dan CAI ,
  • Jing HUANG
Expand
  • Shanghai Institute of Precision Medicine, Shanghai Ninth People′s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200125, China
HUANG Jing, E-mail: huangjing@shsmu.edu.cn.

Received date: 2024-01-29

  Accepted date: 2024-04-12

  Online published: 2024-09-28

Supported by

“Two-Hundred Talents” Program of Shanghai Jiao Tong University School of Medicine(20171922)

Fold

Abstract

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 ? (1 ?=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.

Key words： polycomb repressive complex 1 (PRC1); epigenetic regulation; histone ubiquitination; negative staining; transmission electron microscopy (TEM); three-dimensional protein structure

Cite this article

Dan CAI , Jing HUANG . Electron microscopic study of the non-canonical polycomb repressive complex 1.6[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024 , 44(9) : 1136 -1145 . DOI: 10.3969/j.issn.1674-8115.2024.09.008

References

1 MORGAN M A J, SHILATIFARD A. Reevaluating the roles of histone-modifying enzymes and their associated chromatin modifications in transcriptional regulation[J]. Nat Genet, 2020, 52(12): 1271-1281.
2 GAO Z H, ZHANG J, BONASIO R, et al. PCGF homologs, CBX proteins, and RYBP define functionally distinct PRC1 family complexes[J]. Mol Cell, 2012, 45(3): 344-356.
3 ARANDA S, MAS G, DI CROCE L. Regulation of gene transcription by polycomb proteins[J]. Sci Adv, 2015, 1(11): e1500737.
4 SCELFO A, FERNáNDEZ-PéREZ D, TAMBURRI S, et al. Functional landscape of PCGF proteins reveals both RING1A/B-dependent- and RING1A/B-independent-specific activities[J]. Mol Cell, 2019, 74(5): 1037-1052.e7.
5 ZDZIEBLO D, LI X, LIN Q, et al. Pcgf6, a polycomb group protein, regulates mesodermal lineage differentiation in murine ESCs and functions in iPS reprogramming[J]. Stem Cells, 2014, 32(12): 3112-3125.
6 YANG C S, CHANG K Y, DANG J, et al. Polycomb group protein Pcgf6 acts as a master regulator to maintain embryonic stem cell identity[J]. Sci Rep, 2016, 6: 26899.
7 ENDOH M, ENDO T A, SHINGA J, et al. PCGF6-PRC1 suppresses premature differentiation of mouse embryonic stem cells by regulating germ cell-related genes[J]. eLife, 2017, 6: e21064.
8 ZHU M, ZHANG R N, ZHANG H, et al. PCGF6/MAX/KDM5D facilitates MAZ/CDK4 axis expression and pRCC progression by hypomethylation of the DNA promoter[J]. Epigenetics Chromatin, 2023, 16(1): 9.
9 STORRE J, ELS?SSER H P, FUCHS M, et al. Homeotic transformations of the axial skeleton that accompany a targeted deletion of E2f6[J]. EMBO Rep, 2002, 3(7): 695-700.
10 BROWN J P, BULLWINKEL J, BARON-LüHR B, et al. HP1γ function is required for male germ cell survival and spermatogenesis[J]. Epigenetics Chromatin, 2010, 3(1): 9.
11 MENG C L, LIAO J Y, ZHAO D F, et al. L3MBTL2 regulates chromatin remodeling during spermatogenesis[J]. Cell Death Differ, 2019, 26(11): 2194-2207.
12 MCGINTY R K, HENRICI R C, TAN S. Crystal structure of the PRC1 ubiquitylation module bound to the nucleosome[J]. Nature, 2014, 514(7524): 591-596.
13 GARCíA E, MARCOS-GUTIéRREZ C, DEL MAR LORENTE M, et al. RYBP, a new repressor protein that interacts with components of the mammalian polycomb complex, and with the transcription factor YY1[J]. EMBO J, 1999, 18(12): 3404-3418.
14 WANG R J, TAYLOR A B, LEAL B Z, et al. Polycomb group targeting through different binding partners of RING1B C-terminal domain[J]. Structure, 2010, 18(8): 966-975.
15 GUO Y H, NADY N, QI C, et al. Methylation-state-specific recognition of histones by the MBT repeat protein L3MBTL2[J]. Nucleic Acids Res, 2009, 37(7): 2204-2210.
16 RUAN J B, OUYANG H, AMAYA M F, et al. Structural basis of the chromodomain of Cbx3 bound to methylated peptides from histone H1 and G9a[J]. PLoS One, 2012, 7(4): e35376.
17 LIU Y L, QIN S, LEI M, et al. Peptide recognition by heterochromatin protein 1 (HP1) chromoshadow domains revisited: plasticity in the pseudosymmetric histone binding site of human HP1[J]. J Biol Chem, 2017, 292(14): 5655-5664.
18 BANNISTER A J, ZEGERMAN P, PARTRIDGE J F, et al. Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain[J]. Nature, 2001, 410(6824): 120-124.
19 KREK W, LIVINGSTON D M, SHIRODKAR S. Binding to DNA and the retinoblastoma gene product promoted by complex formation of different E2F family members[J]. Science, 1993, 262(5139): 1557-1560.
20 TRIMARCHI J M, FAIRCHILD B, WEN J, et al. The E2F6 transcription factor is a component of the mammalian Bmi1-containing polycomb complex[J]. Proc Natl Acad Sci U S A, 2001, 98(4): 1519-1524.
21 LIBAN T J, MEDINA E M, TRIPATHI S, et al. Conservation and divergence of C-terminal domain structure in the retinoblastoma protein family[J]. Proc Natl Acad Sci U S A, 2017, 114(19): 4942-4947.
22 SCHERES S H W. RELION: implementation of a Bayesian approach to cryo-EM structure determination[J]. J Struct Biol, 2012, 180(3): 519-530.
23 ZHAO W K, TONG H, HUANG Y K, et al. Essential role for polycomb group protein Pcgf6 in embryonic stem cell maintenance and a noncanonical polycomb repressive complex 1 (PRC1) integrity[J]. J Biol Chem, 2017, 292(7): 2773-2784.
24 PETTERSEN E F, GODDARD T D, HUANG C C, et al. UCSF Chimera: a visualization system for exploratory research and analysis[J]. J Comput Chem, 2004, 25(13): 1605-1612.
25 MAEZAWA S, HASEGAWA K, YUKAWA M, et al. Polycomb directs timely activation of germline genes in spermatogenesis[J]. Genes Dev, 2017, 31(16): 1693-1703.
Options
Outlines

/

〈 〉