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. 2024 Feb 16;15:1446. doi: 10.1038/s41467-024-45760-8

Fig. 3. Characterization of PAF1c function in transcription elongation.

Fig. 3

a (Left) Representative images of PLAs between CTR9 and pS5RNAPII in cells harboring doxycycline-inducible shRNA targeting MYC. Scale bar: 10 µm. (Right) Quantification of the nuclear PLA foci. Nuclei were stained with Hoechst. Data are presented as mean ± s.d. (n = 3 independent experiments; unpaired two-sided t-test). b Bar plot showing percentage of pKAP1-positive nuclei. siRNAs were transfected 48 h before fixation and AZD6738 (0.1 µM) was added for 24 h. Data are presented as mean ± s.d. (n = 3 independent biological replicate for all siRNAs except siNTC, n = 12). c Average density plot of MYC occupancy localized around the transcription start site (TSS) of all expressed (10920) genes analyzed by CUT&RUN (n = 2). “-CTR9/MYC” indicates samples where CTR9 or MYC depletion was induced by doxycycline (1 µg/ml) for 48 h in cells harboring doxycycline-inducible shRNA targeting CTR9, and “+CTR9/MYC” ethanol-treated cells. IgG was used as negative control. d GSEA enrichment plot of the gene set “HALLMARK MYC TARGETS V1” in cells expressing shRNA targeting luciferase versus either MYC (left) or CTR9 (right) (n = 3). e Table showing downregulated GO terms upon either CTR9, CDC73 or MYC depletion. Enrichr package in R was used for the GO term search and P values were calculated using Fisher’s exact test. False discovery rate (FDR) is calculated using the Benjamini–Höchberg procedure to adjust P values for multiple comparisons. f RT-qPCR measurement of Atr and Fancd2 mRNA levels upon depletion of either MYC, CTR9 or CDC73 relative to the control. Doxycycline addition was done for 48 h. Each data point represents an independent biological replicate (n = 2 biological replicates). g Metagene plots mapping nascent transcription marked by 4sU incorporation. Metagene plots show read density averaged over the gene bodies of all expressed genes stratified by increasing length from quartile 1 (shortest) to quartile 4 (longest), where each quartile contains 2091 or 2092 genes. Only intronic 4sU-seq reads were considered (n = 3). h Browser picture showing read distribution of RNAPII ChIP-sequencing (top) and 4sU- sequencing showing nascent transcription (bottom) at two long DNA repair (GO-0006281) genes (n = 2, for the RNAPII-ChIP-sequencing. n = 3, for the 4sU-sequencing experiment). Source data are provided as a Source Data file.