Fig 1: RUVBL2 interacts with RPB1 CTD on chromatin.a An illustration of the Pol II-mediated clusters. RUVBL1/2 were identified by Pol II, H3K27ac, and H3K4me3 ChIP-MS. b RNA polymerase I/II/III subunits were detected in RUVBL2 ChIP-MS preparations. The lines indicate the protein-protein interactions detected in the STRING database. c Heatmap illustrating that RUVBL1 and RUVBL2 globally colocalized with Pol II on the mESC genome. d Schematic diagram showing the subcellular fractionation (cytoplasm, nucleoplasm, and chromatin) procedure, which was adopted from Damianov et al.112. e Western blotting was performed to examine the different subcellular fractions of mESCs. a-Tubulin, histone H3, and snRNP70 were used as marker proteins of the cytoplasm, chromatin, and nucleoplasm, respectively. WCE indicates whole-cell extracts. f Chromatin fractions in wild-type mESCs were separated through size exclusion chromatography, and western blotting was performed to identify RPB1, RPB7, RUVBL1, and RUVBL2 in the gradient eluents based on their different molecular weights. g Protein-protein interactions between Pol II (RPB1) and RUVBL1 or RUVBL2 were examined under different concentrations of NaCl. h The phosphorylation states of Pol II interacting with RUVBL1/2 using RUVBL1 and RUVBL2 degron mESCs were examined. RPB1-CTD or RPB1-Unp indicates the unphosphorylated RPB1 CTD, and RPB1-S2P and RPB1-S5P indicate RBP1 phosphorylated at serine 2 and serine 5 in the CTD, respectively (lower panel). i The RPB1 CTD was acutely degraded, and the native chromatin fractions were subjected to an antibody recognizing the NTD of RPB1 during immunoprecipitation. j Schematic diagram showing the method used to confirm that RUVBL1/2 interacted with RPB1 CTD on chromatin (upper panel). The ChIP-qPCR data shown represent 3 biological replications. Two-tailed Student’s t tests were performed to determine the significance of differences between groups, data are presented as mean +/- SDs (ns, not significant, *p < 0.05, ***p < 0.001, ****p < 0.0001), the exact p values can be found in the source data.