Fig. 3

RNA Pol II recruitment at the Atp1b3 promoter is regulated by FACT/TRIM33. a Atp1b3 and Rnf7 mRNA levels expressed as fold change over the WT BMDM. Mean ± SEM, n = 4. b RNA Pol II occupancy at the Atp1b3 gene. c (Left) Nascent Atp1b3 pre-mRNA levels from WT and Trim33^−/− BMDM treated with DMSO or Flavopiridol (FLAVO) for 4 h, expressed as fold change over the DMSO-treated WT BMDM. RT-qPCR were performed using total RNA and intronic positions of primers are relative to TSS. (Right) Fold decrease, expressed as the ratio between DMSO and FLAVO-treated BMDM. Mean ± SEM, n = 5. d Antisense promoter transcripts levels (450nt upstream the TSS) in WT and Trim33^−/− BMDM, relative to expression of WT BMDM. Mean ± SEM, n = 5. e ChIP-seq profiles of indicated chromatin modifications along with ChromHMM analysis at the Atp1b3 gene. f SPT16 ChIP-seq profiles at the Atp1b3 locus. g (Up) Schematic for EcoRI fragments and PCR primers positions used in Chromosome Conformation Capture (3C) at the Atp1b3/Rnf7 locus. (Down) 3C analysis of interaction between the − 35 kb region and the Atp1b3 promoter (− 35 kb/ Atp1b3 prom) in WT and Trim33^−/− BMDM (two independent replicates). Negative controls (− 35 kb/− 92 kb and Atp1b3 prom/− 92 kb) included a region located 92 kb upstream the Atp1b3 TSS and not bound by TRIM33. Positive controls (Control) included digested and ligated genomic PCR fragments containing all ligation junctions. Genomic DNA after ligation (Input) is shown as a loading control. h (Left) RNA Pol II ChIP-qPCR at the Atp1b3 promoter 4 h after treatment of WT and Trim33^−/− BMDM with the FACT inhibitor CBL0137 (CBL). (Right) Fold decrease, expressed as the ratio between DMSO and CBL-treated BMDM. Mean ± SEM, n = 3. i (Left) Atp1b3 pre-mRNA levels in WT and Trim33^−/− BMDM 4 h after CBL treatment, relative to expression of DMSO-treated WT BMDM. RT-qPCR was performed using total RNA. (Right) Fold decrease, expressed as the ratio between DMSO and CBL-treated BMDM. Mean ± SEM, n = 3