Fig 2: MYC-Mediated Transfer of SPT5 Is Required to Maintain Pol II Processivity(A) Schematic of 4sU sequencing. Nascent transcripts were labeled with 4sU in U2OSMYC-Tet-On cells, converted into strand-specific cDNA and sequenced. Directionality scores were calculated by dividing reads from TSS-TES by TSS-1.5 kb gene regions for all transcribed genes in U2OS cells in the absence and presence of MYC. Processivity scores were calculated by dividing distal (5–7 kb after TSS) by proximal (1-2 kb after TSS) reads.(B) Genome browser pictures of nascent RNA. Example of 4sU signal at the STAMBP gene from U2OS cells in the presence and absence of MYC.(C) Average read density of 4sU sequencing experiments (upper panel) in U2OS cells in the absence and presence of MYC. Curves show the spatial distribution of reads independently aligned to sense and antisense strands within 7.5 kb of the TSS for genes longer than 8 kb. Comparison to MYC and Pol II binding in the same region originating from ChIP-sequencing data (lower panel) is shown as average read density (Walz et al., 2014).(D) Heatmap with normalized directionality scores for three replicates calculated in the presence and absence of MYC. Negative values indicate reduced promoter directionality.(E) Heatmap with normalized processivity scores for three replicates calculated in the absence and presence of MYC. Negative values indicate reduced Pol II processivity.(F) Average read density of Pol II ChIP-sequencing experiments around transcriptional start sites (TSS, left) and transcriptional end sites (TES, right). ChIP-RX sequencing was performed with antibodies precipitating total Pol II in U2OS cells depleted of SPT5 by doxycycline-induced shRNA (orange) and control cells (blue; black line: input).(G) Heatmap of normalized processivity scores for two replicates of total Pol II ChIP sequencing in the absence (shSPT5) and presence (Ctr) of SPT5.See also Figure S5.

Fig 3: MYC Mediates Changes in Pol II Complex Composition(A) Graphic displaying the method used to identify Pol II-associated proteins. T-lymphomaMYC-Tet-Off cells were stably transfected either with a lentiviral vector expressing HA-tagged RPB3 or an empty lentiviral vector. Cells were harvested, nuclei were isolated, nuclear membranes were lysed, and chromatin-associated proteins were solubilized and used as input for HA-directed immunoprecipitation (IP). Eluted fraction was used in label-free quantitative mass spectrometry (qMS).(B) Volcano plot of the Pol II interactome with transcription elongation factors marked in orange. The x axis displays the enrichment (log2FC) of proteins in HA-RPB3-expressing cells compared to control cells (Ctr). The y axis shows the significance (p value) of enrichment calculated from five biological replicate experiments.(C) Volcano plot showing proteins changing their association with Pol II in response to MYC depletion in T-lymphomaMYC-Tet-Off cells. The x axis displays the enrichment of proteins (log2FC) between cells expressing (ON) and depleted of (OFF) MYC. Positive values indicate the protein requires MYC to associate with Pol II (e.g., TCEA3 or SPT5). The y axis shows the significance (p value) of enrichment calculated from four biological replicate experiments. Pol II-interacting proteins are shown as orange circles whose size indicates overall enrichment. The insert shows an immunoblot of MYC in T-lymphomaMYC-Tet-Off cells treated with doxycycline (MYC OFF) or ethanol (MYC ON, Vinculin: loading control).(D) Enrichment-values (FC) for SPT5 in the Pol II interactome in the absence and presence of MYC in the four experiments used for the analysis shown in (C).See also Figure S1.

Fig 4: CDK7 Activity Is Required for Transfer of SPT5 from MYC to Pol II(A and B) Immunofluorescence images of PLAs between SPT5 and MYC (A) or SPT5 and total Pol II (B) in cells treated with CDK inhibitors (DRB, THZ1, LDC4297, LDC067) or DMSO vehicle.(C) Quantification of PLAs shown in (A). The number of proximity pairs upon inhibitor treatment was quantified in independent experiments and normalized to the DMSO condition.(D) Quantification of PLAs shown in (B). The number of proximity pairs upon inhibitor treatment was quantified in independent experiments and normalized to the DMSO condition.(E) Immunoblots of endogenous SPT5 precipitated from HEK293 cells treated with LDC4297 or DMSO. Co-precipitated total Pol II and SPT4 were analyzed by immunoblotting. Beads coupled to non-specific IgG were used as controls.(F) Immunoblots of immunoprecipitation experiments. FLAG-SPT5 and HA-MYC were overexpressed by transient transfection in HEK293 cells. HA-MYC was immunoprecipitated and incubated with recombinant CDK7, and co-precipitating FLAG-SPT5 was analyzed by immunoblotting. Cells not expressing HA-MYC were used as control.(G) Immunoblot of TFIIEß depleted cells. U2OS cells were treated with an siRNA against TFIIEß or a non-targeting control (Vinculin: loading control).(H and I) Immunofluorescence images (H) and quantification (I) of PLAs between SPT5 and total Pol II. U2OS cells were treated with LDC4297 or an siRNA against CDK7 after depletion of TFIIEß and in control cells. The number of proximity pairs was quantified and its change in TFIIE-depleted cells to control cells was calculated as fold change (FC).For (A), (B), and (H): yellow dots: intensity centers of proximity pairs; blue: Hoechst stained nuclei; magenta: Phalloidin staining; scale bar: 5 µm. See also Figure S4.

Fig 5: MYC Recruits SPT5 by Binding to its N-terminal Region(A) Immunoblots of endogenous SPT5 IP from HEK293 cells and co-precipitated MYC. Beads coupled to non-specific IgG were used as control.(B) Immunofluorescence images from PLAs. FLAG-SPT5 and HA-MYC were stably co-expressed in U2OS cells by lentiviral transduction, and cells expressing only one protein were used as controls. Proximity between MYC and SPT5 was analyzed with anti-FLAG and anti-HA antibodies (yellow dots: intensity centers of proximity pairs; blue: Hoechst-stained nuclei; magenta: Phalloidin staining; scale bar: 5 µm).(C) Immunoblots of IP experiments. FLAG-SPT5 and HA-MYC were overexpressed by transient transfection in HEK293 cells. FLAG-SPT5 was precipitated and co-precipitating HA-MYC was analyzed by immunoblotting. Cells not expressing FLAG-SPT5 or beads coupled to non-specific IgG were used as controls (* indicates the antibody heavy chain).(D) Immunoblots of IP experiments. FLAG-SPT5, HA-MYC (WT), and an HA-tagged N-terminal deletion mutant of MYC144–439 (MUT) were overexpressed by transient transfection in HEK293 cells. HA-MYC was precipitated and co-precipitating FLAG-SPT5 was analyzed by immunoblotting. Cells not expressing HA-MYC were used as controls.(E) Scheme of FLAG-tagged SPT5 deletion mutants (K: KOW domain; CTR: C-terminal repeat region).(F) Immunoblots of IP experiments. HA-MYC, FLAG-SPT5, and FLAG-tagged deletion mutants of SPT5 shown in (E) were overexpressed by transient transfection in HEK293 cells. HA-MYC was precipitated and co-precipitating FLAG-SPT5 was detected by immunoblotting. Cells not expressing HA-MYC were used as controls (* indicates signal from IgG chains).(G) Recombinant proteins from pull-down assays visualized by silver staining and immunoblotting. SPT5 (together with SPT4), GST-Myc1–163, and GST were isolated from E. coli (left, silver staining). GST or GST-Myc1–163 were coupled to sepharose and incubated with SPT5. Input and eluted proteins were visualized with antibodies detecting GST or SPT5 (right, immunoblot).See also Figure S3.