Fig. 6: RING1B regulates the cell-cycle-dependent induction of gene transcription during lineage transition in mESCs.
From: Changes in PRC1 activity during interphase modulate lineage transition in pluripotent cells
a Scheme representing the experimental strategy to analyze the cell-cycle-phase-specific function of RING1B during lineage. Ring1a−/−;AID::Ring1b;FUCCI mESCs were cell cycle sorted and plated in differentiation media (5 µM RA in the absence of LIF) with (IAA+) or without (control UNT) auxin (IAA) during 6 h before RNA collection. 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. LAMINB was used as loading control. Molecular weight is indicated in kDa. Two biological replicates were carried out. c Hierarchical clustering analysis of RNA expression levels (TMM) of the summation of differentially expressed (2260 genes, FC > 2, FDR < 0.05) genes (0 h vs 6 h UNT and 0 h vs 6 h IAA) at any cell cycle phase during RA induction. Cluster I highlight 871 genes responding to RA stimulation but not to RING1B depletion. Cluster II includes 915 genes that are de-repressed upon RING1B depletion. These include a group of genes that are transcriptionally induced by RA preferentially during the G1 phase (371 genes in cluster II-A). Cluster III contains 474 genes downregulated during RA differentiation. Expression relative to the geometric mean of each gene is plotted. d Boxplot showing the enrichment of indicated PRC1 proteins at the promoter region (−0.5 kb to +1.5 kb relative to TSS) of genes belonging to the three clusters identified in (c). Boxes show median and Q1–Q3 values. Whiskers denote the 1.5× the interquartile range. Asterisks (*) mark statistically significant differences (p < 0.0001) compared to the same cell cycle phase of cluster II using Mann–Whitney test. e, f Plots showing Gene Set Enrichment Analysis of differential expression in indicated categories and clusters. Normalized enrichment score (NES) and false discovery rates (FDR) are indicated. g Genome browser view of mRNA expression at indicated phases of the cell cycle at 0 and 6 h after RA stimulation in cell cycle sorted RING1B-depleted and control UNT cells at the Cdx2. Source data are provided as a source data file.
