Fig 2: Effects of depleting elongation factors. (A) Diagram depicting the FKBP12F36V tagged cell lines generated and the times of treatment with dTAGV-1 (VHL) before library preparation for PRO-Seq. For each cell line, cells were plated at identical densities. The timing of treatments was such that nuclei isolation for the DMSO control and the 2 h and 48 h depletion were performed at the same time. An aliquot (equal volumes for all) from the same nuclei preparation used for PRO-Seq was utilized for western blot analysis to determine levels of NELFB ± Flavopiridol (B), PAF1 (C) and RTF1 (D) before and after depletion for the indicated times. Protein-specific antibodies were used for PAF1 and RTF1, and anti-FKBP12 antibody was used for NELFB. TBP was used as a loading control. (E) Genome Browser tracks for two example genes (GPBP1 and WAC) depicting sense spike-in normalized PRO-Seq transcript pileups for the combination of the two duplicate datasets (NELFB Flavo has only one rep). (F) Fold change heatmaps aligned by TSSs (left) or TESs (right) were generated from spike-in normalized reads (n = 11 420) for NELFB, PAF1, and RTF1 sorted by gene length. Line plots show the average fold change (depletion/control) in 1000 bp bins starting at + 1000 bp from the MaxTSS up to +100 000 bp for 2466 genes with a length of at least 100 kb. Histograms represent the transition points in 1000 bp bins determined with hidden Markov model for each gene (n = 1574).

Fig 3: TET3 interacts with RNA Pol II and proteins involved in transcriptional initiation and elongation(A) STRING analysis indicates that TET3 interacts with subunits of RNA Pol II as well as additional proteins involved in transcriptional initiation and elongation such as the mediator complex and BRD4.(B) STRING analysis of the proteins identified in A reveals the major biological functions regulated by this cluster. The RNA Pol II pre-transcription events category is the most significant category, while transcription initiation RNA Pol II promoter follows.(C) IP (left) of endogenous TET3 (+TET3) or IgG (+IgG) (negative control) from nuclear extracts of thymocytes in the presence of EtBr and benzonase followed by immunoblotting for TET3 and PAF1. Note that TET3 and PAF1 are specifically precipitated in the +TET3 condition but not in the negative control. Total nuclear extracts were used as input (right). Note that TET3 and PAF1 are detected by western blot in both samples. Histone 3 (H3) was used as loading control. One out of two representative experiments is shown.

Fig 4: Effects of depletion of PAF1 and RTF1. (A) Spike-in normalized 5′ end PRO-Seq tracks for the indicated NELFB, PAF1, and RTF1 datasets over two example genes, SRP54 and SETD5. (B) The log2 3′ fold changes (FC) of NELFB 2 h dep over NELFB control were calculated at each base in the ±500 bp region and the values in −100 to +250 were used for K-means clustering analysis (K = 10) (left). Genes containing a TBP motif in the −36 to −19 bp region (23) are shown as black horizonal bars (center right). NELFB control H3K4me3 DFF-ChIP fragment centers sorted by the same K-means cluster sort. (C) Log2 3′ fold changes (FC) of NELFC, PAF1, and RTF1 using the K-means cluster sort in (B). (D) Pol II DFF-ChIP fragment centers sorted as described in (B). Fragment lengths of 65–100, 40–65 and 165–215 were used for the PIC, free, and abutted Pol II heatmaps, respectively. Pol II (NELFB control) and Pol II (2 h NELFB dep) heatmaps can be compared directly as they have been spike-in normalized together and share an identical scale, ensuring that equivalent numerical values correspond to the same color intensities. (E) All heatmaps are sorted by the distance of the weighted average position of NELFB control DFF-ChIP H3K4me3 fragment centers in the −100 to +250 bp region to each individual MaxTSS. H3K4me3 and Pol II heatmaps show fragment centers. H3K4me3 (NELFB control) and H3K4me3 (2 h NELFB dep) heatmaps or Pol II (NELFB control) and Pol II (2 h NELFB dep) heatmaps can be compared directly as each pair has been spike-in normalized together and share an identical scale.

Fig 5: SEI1 bridges the interaction between CBP/p300 and PAF1 complex to regulate PD‐L1 transcription. A) HA‐SEI1 overexpressed IM‐9 cells were lysed, and the total protein lysate was immunoprecipitated with an agarose‐immobilized HA antibody and analysis with mass spectrometry. The proteins identified are indicated. B,C) Co‐immunoprecipitation of SEI1, PAF1, CDC73, or p300 in ARH‐77 or IM‐9 cells treated with melphalan or bortezomib. D) Co‐immunoprecipitation of SEI1 along with PAF1, CDC73, or p300 in HEK293T cells co‐transfected with SEI1 and either PAF1, CDC73, or p300 plasmid. E) Schematic of the truncations including ΔCABD, ΔSD, ΔPBID, and ΔCT fragments. F) Western blotting showing different truncations of SEI1 (full length, ΔCABD, ΔSD, ΔPBID, and ΔCT) in HEK293T cells. G–K) Pulldown of PAF1, CDC73, or p300 with different truncations of SEI1 (full length, ΔCABD, ΔSD, ΔPBID, and ΔCT) in HEK293T cells. L) Pulldown of MYC‐p300 with CDC73 or PAF1 in HEK293T cells. M) Gene tracks showing representative ATAC‐seq profiles at PD‐L1 gene loci in primary CD138+ plasma cells from two relapsed myeloma patients following treatment with bortezomib. N,O) ChIP PCR assay showing SEI1, p300, or H3K27ac enrichment on PD‐L1 promoter of ARH‐77 or IM‐9 cells treated with or without melphalan or bortezomib. P) ChIP‐seq profiles show H3K27ac signal tracks at the PD‐L1 gene loci for ARH‐77 cells treated with or without melphalan or bortezomib. Q) ChIP PCR assay showing H3K27ac enrichment on PD‐L1 promoter of ARH‐77 or IM‐9 cells treated with or without melphalan or bortezomib. R) ChIP PCR assay showing enrichment of H3K27ac on PD‐L1 promoter in shCtrl or shSEI1 ARH‐77 or IM‐9 cells. S) Expression of PAF1 in nonspecific, PAF1 shRNA‐expressing myeloma cells. T,U) The relative expression of PD‐L1 in PAF1 knockdown ARH‐77 and IM‐9 cells treated with or without melphalan or bortezomib. V,W) ChIP PCR assay showing enrichment of V) SEI1 or W) H3K27ac on PD‐L1 promoter in shCtrl or shPAF1 ARH‐77 and IM‐9 cells. X) Schematic of the SEI1 bridges the interaction between CBP/p300 and PAF1 complex to regulate PD‐L1 transcription. B–D,F–L,N,O,Q–W) Representative of three independent experiments. Data are averages ± SD. ***p < 0.001 and ****p < 0.0001. All p‐values were determined using one way ANOVA.