Journal: bioRxiv
Article Title: Dual Modes of Gene Regulation by CDK12
doi: 10.1101/2025.09.22.677923
Figure Lengend Snippet: A. Boxplots depict PRO-seq read density in mRNAs upregulated (N = 117) or downregulated (N = 805) upon THZ531 treatment, as defined in . Reads were counted from +250 nt downstream of the TSS to the transcript end site (TES), and normalized for gene length (reads/kb). Boxes show 25th–75th percentiles and whiskers depict 1.5 times the interquartile range. p values from Wilcoxon matched-pairs signed rank test. B. Metagene plot of average PRO-seq signal in vehicle and THZ531-treated HCC1954 cells across all active mRNA genes > 400 nt (N=11,876). Bins from TSS to TES are scaled to gene length, with 100 bins/gene, and data outside gene bodies are shown as average reads per gene in 200-nt bins. C. Cumulative distribution plot comparing the pausing indices of all genes shown in (B), comparing vehicle to THZ531 treatment. D-E. Metagene analysis of PRO-seq signal at (D) uaRNA loci (N = 9,617) or (E) eRNA loci (N=24,790) upon 2 h of indicated treatment. Data are shown as average reads per TSS in 25-nt bins. F. The distribution of the number of enhancers associated with the 13,530 active genes in HCC1954 cells. Association was based on the nearest active gene to each enhancer TSS. A subset of genes (n = 152; marked by dotted line) contains ≥ 100 associated eTSSs. G. Heat maps rank-ordered by decreasing size of each enhancer region. Shown are histone modifications (H3K27ac), and PRO-seq reads on both strands, with data centered around the dominant eTSS in each enhancer cluster (N = 7,631). H. Metagene plots of average PRO-seq signal in vehicle and THZ531-treated HCC1954 cells across genes defined as upregulated or downregulated in RNA-seq. Data are shown as in (B). I. Top cartoon: Measurements of Pol II diffusion kinetics determined by fluorescence recovery after photobleaching of a narrow strip spanning the nucleus (Strip-FRAP). (Bottom Left) FRAP was performed in untreated MRC-5 GFP-RPB1 KI cells and GFP-RPB1 fluorescence in the strip was background-corrected and normalized to pre-bleach fluorescence intensity and set at 1. The three dotted lines indicate kinetically distinct Pol II fractions. (Bottom Right) Pol II mobility in untreated cells or upon treatment with 400 nM THZ531 for 90 min, or 2 µM THZ1 for 90 min. Mean values from >13 nuclei of two independent experiments were plotted. RFI, Relative fluorescence intensity.
Article Snippet: The following primary antibodies were purchased and used for fluorescence immunoblotting: RNA polymerase II subunit B1 (phospho CTD Ser-2) Antibody, clone 3E10 (EMD Millipore, #04-1571); Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb (Cell Signaling Technology, #13499); Phospho RNA Polymerase II (S2) Antibody, (Bethyl Laboratories, A300-654A); RNA polymerase II subunit B1 (phospho-CTD Ser-5) Antibody, clone 3E8 (EMD Millipore, #04-1572); Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb (Cell Signaling Technology, #13523) RNA polymerase II subunit B1 (phospho-CTD Ser-7) Antibody, clone 4E12 (EMD Millipore, #04-1570); RNA Polymerase II Antibody (Bethyl Laboratories, A300-653A); RNA Polymerase II RPB1, clone 8WG16 (BioLegend, #664906); Rpb1 NTD (D8L4Y) Rabbit mAb (Cell Signaling Technology, #14958); c-Myc (clone Y69) Rabbit mAb (Abcam, #ab32072); c-Myc (D84C12) Rabbit mAb (Cell Signaling Technology, #5605); anti-CDK12 (Cell Signaling Technology, #11973); anti-CDK12 (proteintech, #26816-1-AP); anti-CDK12, clone 45F7-H2 (BIO-RAD, #VMA00874); anti-CDK13, clone 46B7-G7 (BIO-RAD, #VMA00875); CDK7 Recombinant Monoclonal Antibody (BL-80-5D4) (Bethyl, #A700-006); CDK7 (MO1) Mouse mAb (Cell Signaling Technology, #2916); CDK8 (D6M3J) Rabbit mAb (Cell Signaling Technology, #17395); CDK9 (C12F7) Rabbit mAb (Cell Signaling Technology, #2316); Anti-CDK19 (Sigma-Aldrich, #HPA007053); JunB Rabbit mAb, clone ARC0268 (ABclonal, #A4848); c-Jun (60A8) Rabbit mAb (Cell Signaling Technology, #9165); c-Fos (9F6) Rabbit mAb (Cell Signaling Technology, #2250); Anti-c-ErbB2/c-Neu (Ab-3) Mouse mAb (3B5) (EMD Millipore, #OP15); anti-PARP (Cell Signaling Technology, #9542); anti-β-Actin, clone AC-15 (Sigma, A5441); anti-Vinculin (Sigma, V9131); anti-β-Tubulin (BioLegend, # 903401).
Techniques: RNA Sequencing, Diffusion-based Assay, Fluorescence, Stripping Membranes
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