title: Distinct Cellular Assembly Stoichiometry of Polycomb Complexes on Chromatin Revealed by Single-molecule Chromatin Immunoprecipitation Imaging.
abstract: Epigenetic complexes play an essential role in regulating chromatin structure, but information about their assembly stoichiometry on chromatin within cells is poorly understood. The cellular assembly stoichiometry is critical for appreciating the initiation, propagation, and maintenance of epigenetic inheritance during normal development and in cancer. By combining genetic engineering, chromatin biochemistry, and single-molecule fluorescence imaging, we developed a novel and sensitive approach termed single-molecule chromatin immunoprecipitation imaging (Sm-ChIPi) to enable investigation of the cellular assembly stoichiometry of epigenetic complexes on chromatin. Sm-ChIPi was validated by using chromatin complexes with known stoichiometry. The stoichiometry of subunits within a polycomb complex and the assembly stoichiometry of polycomb complexes on chromatin have been extensively studied but reached divergent views. Moreover, the cellular assembly stoichiometry of polycomb complexes on chromatin remains unexplored. Using Sm-ChIPi, we demonstrated that within mouse embryonic stem cells, one polycomb repressive complex (PRC) 1 associates with multiple nucleosomes, whereas two PRC2s can bind to a single nucleosome. Furthermore, we obtained direct physical evidence that the nucleoplasmic PRC1 is monomeric, whereas PRC2 can dimerize in the nucleoplasm. We showed that ES cell differentiation induces selective alteration of the assembly stoichiometry of Cbx2 on chromatin but not other PRC1 components. We additionally showed that the PRC2-mediated trimethylation of H3K27 is not required for the assembly stoichiometry of PRC1 on chromatin. Thus, these findings uncover that PRC1 and PRC2 employ distinct mechanisms to assemble on chromatin, and the novel Sm-ChIPi technique could provide single-molecule insight into other epigenetic complexes.
authors.affiliation: Department of Chemistry, University of Colorado Denver, Denver, Colorado 80217-3364.
authors.forename: Roubina
authors.initials: R
authors.lastname: Tatavosian
authors.affiliation: Department of Chemistry, University of Colorado Denver, Denver, Colorado 80217-3364.
authors.forename: Chao Yu
authors.initials: CY
authors.lastname: Zhen
authors.affiliation: Department of Chemistry, University of Colorado Denver, Denver, Colorado 80217-3364.
authors.forename: Huy Nguyen
authors.initials: HN
authors.lastname: Duc
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado 80045.
authors.forename: Maggie M
authors.initials: MM
authors.lastname: Balas
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado 80045.
authors.forename: Aaron M
authors.initials: AM
authors.lastname: Johnson
authors.affiliation: Department of Chemistry, University of Colorado Denver, Denver, Colorado 80217-3364. Electronic address: stolson@uic.edu.
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Ren
mesh_terms: D000818:Animals; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation; D044127:Epigenesis, Genetic; D006801:Humans; D051379:Mice; D063146:Polycomb-Group Proteins / Q000378:metabolism*
chemical_list: D002843:Chromatin; D063146:Polycomb-Group Proteins
keywords: PRC1; PRC2; Sm-ChIPi; chromatin; chromatin immunoprecipitation (ChiP); epigenetics; histone chromosomal protein; polycomb; single-molecule biophysics; stoichiometry