Extended Data Fig. 3: RYBP-PRC1 propagates H2AK119ub1 by a positive-feedback loop.
a, Representative EM images of 12×-polynucleosome arrays (by metal-shadowing method). b, A schematic illustrating the octamers in chromatin assembly. c, Representative immunoblot of in vitro ubiquitination assay for measuring the activity of RYBP-PRC1 on mononucleosome and polynucleosome substrates out of 3 independent experiments. Triangles represent RYBP-PRC1 from low to high level, also in (e). d, Representative immunoblot of in vitro ubiquitination assay for measuring the effect of H2AK119ub1 on RYBP-PRC1 activity in trans out of 3 independent experiments. 12×-H2AKK/RR- or 12×-H2AK119ub1-polynucleosomes were mixed with 12×-H2A-polynucleosome substrates immediately prior ubiquitination reactions in substrate I or in substrate II, respectively. e, Representative immunoblot of in vitro ubiquitination assay for measuring the ubiquitin E3 ligase activity of RYBP-PRC1 on 12×-H2AKK/RR- and 12×-H2A-polynucleosomes out of 3 independent experiments. f, A schematic illustrating the strategy for generating 12×-polynucleosome substrates by sequential ligation of tetranucleosomes. g, Representative EM images of the tetranucleosomes for ligation assays. h, Representative EM images of 12×-H2A-polynucleosomes generated by sequential ligation of tetranucleosomes. Scale bar in a, g and h is 50 nm. The EM in a, g and h was performed in triplicates with three biologically independent samples (once per sample). i, Representative 1.2% agarose gel electrophoresis of 12×-H2A-polynucleosomes generated by sequential ligation of tetranucleosomes out of 3 independent experiments. j, A schematic illustrating the TetR-tetO targeting system. RING1B-TetR-HA is recruited by specific binding of TetR to 8xtetO arrays. RYBP-FLAG or RYBPTF/AA-FLAG was overexpressed to promote the spreading of H2AK119ub1 around 8xtetO sites. k, ChIP-qPCR analysis of HA (TetR-HA), FLAG and H2AK119ub1 across the 8xtetO sites in TetR-tetO targeting mESCs with TetR-HA transient transfection (n=3 biologically independent samples). Data represents mean ± s.d. ChIP enrichments are normalized to input. Unprocessed blots and statistical source data are available in Source Data.
