Fig. 7: Depletion of RING1B interferes with cell-cycle-phase-dependent ability of mESCs to differentiate.
From: Changes in PRC1 activity during interphase modulate lineage transition in pluripotent cells
a Scheme showing the experimental design to study the cell-cycle-phase-specific role of RING1B in productive cell differentiation. Ring1a−/−;AID::Ring1b;FUCCI mESCs were cell cycle sorted and plated at low density in differentiation media (0.1 µM RA in the absence of LIF) with (RING1B-depleted) or without (control UNT) auxin (IAA). After 6 h cells were cultured in standard ESCs media and colonies were fixed and analyzed after five days. b Western blots of whole cell extracts for RING1B and H2AK119ub1 proteins in Ring1a−/−;AID::Ring1b;FUCCI mESCs treated with IAA for the indicated times. Lamin B was used as loading control. Molecular weight is indicated in kDa. Two biological replicates were carried out. c Histograms showing the percentage of differentiated colonies relative to G1 obtained after five days from cell cycle sorted populations in parental control (UNT) and IAA-treated (RING1B-depleted) cells (see experimental setup in Fig. 6h). Pictures showing examples of colonies with undifferentiated (compact and 3D colonies with round cells) or differentiated (flat and spread colonies with elongated cells) morphologies are shown. Mean ± S.D of biological quadruplicates are shown. Asterisks mark statistically significant differences (**p < 0.01, ****p < 0.0001) using Mann–Whitney test. d Scheme summarizing the observations made in this manuscript. Black boxes indicate genes, red lollipops H2AK119ub1 and PRC1 complexes are represented as blue or red spheres. RNA expression is represented as dashed (leaky expression) or solid (higher expression) wavy lines. PRC1 complexes accumulate around target promoters in G2 compared to G1 phase, leading to enhanced ubiquitination of H2AK119, stronger 3D interactions and firmer gene repression. Different PRC1-target genes might display varying combinations of these features. Upon induction of cell differentiation PRC1 target genes are preferentially activated during G1 phase. This cell-cycle-phase-dependent activation is perturbed in RING1B-depleted cells. Source data are provided as a source data file.
