Journal: Nature Communications

Article Title: Structural basis of Cdk7 activation by dual T-loop phosphorylation

doi: 10.1038/s41467-024-50891-z

Figure Lengend Snippet: a SDS-PAGE analysis and immunoblotting of Cdk7/Cyclin H/Mat1 complexes. Protein (3 µg) was resolved in a 15% SDS-polyacrylamide gel and stained with Coomassie blue. Note the difference in migration behavior of Cdk7 depending on the phosphorylation status; doubly phosphorylated Cdk7 and Cyclin H migrate at the same size. For immunoblot analysis of the Cdk7 T-loop phosphorylation status, 1 µL protein at 2.6 µM was resolved by SDS-PAGE, transferred to nitrocellulose and probed with phospho-specific antibodies recognizing Cdk7 pT170 or Cdk7 pS164. b Alignment of transcriptional CDK T loops. c , Radiometric kinase assay probing the preparations for activity towards RNAPII CTD. Each Cdk7 complex (0.1 µM) was incubated with 10 µM GST-CTD [52] in the presence of 1 mM ATP containing 0.35 µCi [ 32 P]-γ-ATP for 15 min at 30 °C. Bars represent mean of duplicate measurement. d , Crystal structure of the doubly T-loop phosphorylated Cdk7/Cyclin H/Mat1/VHH RD7-04 complex at 2.15 Å resolution. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used in this study: α-pSer2 CTD, rat monoclonal, clone3E10, 1:100; α-pSer5 CTD, rat monoclonal, clone 3E8, 1:500; α-pSer7 CTD, rat monoclonal, clone 4E12, 1:100; α-pThr4 CTD, rat monoclonal, clone 1G7, 1:100 (CTD antibodies were a kind gift from Dirk Eick, Munich); rat monoclonal, clone 6D7, 1:1000 (ActiveMotif #61362); α-Cdk7 (total), mouse monoclonal, clone 31TF2-1F8, 1:1000, (Invitrogen #MA3-001); α-Cdk7 (total), mouse monoclonal, clone C4, 1:1000 (Santa Cruz Biotechnology #sc-7344); α-phospho Cdk7 (pT170), rabbit polyclonal, 1:1000 (Affinity Biosciences #CPA5749); α-phospho-Cdk7 (pT170), rabbit polyclonal, 1:1000 13 ; α-phospho-Cdk7 (pS164), rabbit polyclonal, 1:1000 (Invitrogen #PA5-105583); α-GST, mouse monoclonal, 1:1000 (Thermo Fisher Scientific # 740007M); α-HA tag, mouse monoclonal, clone F-7 (Santa Cruz Biotechnology #sc-7392); α-MBP, rabbit polyclonal, 1:10 for immobilization on SPR sensor ship (Novus Biologicals, NBP2-22462).

Techniques: SDS Page, Western Blot, Staining, Migration, Phospho-proteomics, Kinase Assay, Activity Assay, Incubation

Journal: Nature Communications

Article Title: Structural basis of Cdk7 activation by dual T-loop phosphorylation

doi: 10.1038/s41467-024-50891-z

Figure Lengend Snippet: a Close-up of the interaction network of pT170. Salt bridges are formed to Cdk7 residues R61, R136 and K160, forming the canonical triad of T-loop phosphorylation recognition that is conserved in CDKs. A water-mediated hydrogen bond is formed to the backbone carbonyl of A168. An intermolecular salt bridge between R61 and E117 of Cyclin H extends to K64, coordinating the kinase αC helix. Additional contacts are formed between R167 of Cdk7 and D116 of Cyclin H. b Close-up of the interaction network of the non-canonical pS164. An intermolecular salt bridge is formed to R165 on the second cyclin box of Cyclin H, which is continued to D299 of Mat1. The side chain of N166 of Cdk7 is coordinated by pS164. Several water molecules in proximity to the phospho-sites mediate the tripartite interaction. c Electrostatic surface display of the T-loop residues in Cdk7. The position of residues pS164, R167 and pT170 is marked. The surface charge and accessibility are visible in the upper panel with the interacting side chains shown in the transparent display of the lower panel. d Electrostatics of the ternary Cdk7/Cyclin H/Mat1 complex assembly. The open triptychon display shows charged interactions in the Cdk7/Cyclin H interface while the interactions of Mat1 with both subunits is largely hydrophobic. The electrostatic surface charge is shown from −5 k B T (red) to +5 k B T (blue).

Article Snippet: The following antibodies were used in this study: α-pSer2 CTD, rat monoclonal, clone3E10, 1:100; α-pSer5 CTD, rat monoclonal, clone 3E8, 1:500; α-pSer7 CTD, rat monoclonal, clone 4E12, 1:100; α-pThr4 CTD, rat monoclonal, clone 1G7, 1:100 (CTD antibodies were a kind gift from Dirk Eick, Munich); rat monoclonal, clone 6D7, 1:1000 (ActiveMotif #61362); α-Cdk7 (total), mouse monoclonal, clone 31TF2-1F8, 1:1000, (Invitrogen #MA3-001); α-Cdk7 (total), mouse monoclonal, clone C4, 1:1000 (Santa Cruz Biotechnology #sc-7344); α-phospho Cdk7 (pT170), rabbit polyclonal, 1:1000 (Affinity Biosciences #CPA5749); α-phospho-Cdk7 (pT170), rabbit polyclonal, 1:1000 13 ; α-phospho-Cdk7 (pS164), rabbit polyclonal, 1:1000 (Invitrogen #PA5-105583); α-GST, mouse monoclonal, 1:1000 (Thermo Fisher Scientific # 740007M); α-HA tag, mouse monoclonal, clone F-7 (Santa Cruz Biotechnology #sc-7392); α-MBP, rabbit polyclonal, 1:10 for immobilization on SPR sensor ship (Novus Biologicals, NBP2-22462).

Techniques: Phospho-proteomics

Journal: Nature Communications

Article Title: Structural basis of Cdk7 activation by dual T-loop phosphorylation

doi: 10.1038/s41467-024-50891-z

Figure Lengend Snippet: a SDS-PAGE and immunoblot analysis of GST-Cdk7/Cyclin H complexes (left panels) and MBP-Mat1 230-309 . The protein (2 µg) was resolved on a 12% SDS gel and stained with Coomassie blue. For analysis of the Cdk7 T-loop phosphorylation status, 1 µL at 2.6 µM was immunoblotted with phospho-specific antibodies recognizing Cdk7 pT170 or Cdk7 pS164. Total Cdk7 was used as loading control. b Radiometric kinase assay probing the preparations shown in a for activity towards RNAPII CTD and Cdk2 in absence and presence of Mat1. Cdk7/Cyclin H (0.1 µM) was pre-incubated with buffer or 0.4 µM Mat1, and 10 µM GST-CTD [52] or 15 µM GST-Cdk2 for 10 min at room temperature. Reaction was started by addition of 1 mM ATP containing 0.35 µCi [ 32 P]-γ-ATP and incubated for 15 min (CTD) or 30 min (Cdk2) at 30 °C. Data represent mean ± SD of triplicate measurements. c SDS-PAGE analyses of GST-Cdk7/Cyclin H and MBP-Mat1; 3 µg of each sample was resolved on a 12% SDS-Gel. Cdk7 T-loop phosphorylation status was analyzed as in a . Immunoblotting for GST was used as loading control for Cdk7. d Radiometric kinase activity assay. Activity was measured towards GST-CTD [52] as described in b (left panel). Data represent mean of duplicate measurements. Fold-change of kinase activity upon MBP-Mat1 230-309 incubation in relation to the same kinase preparation without Mat1 (right panel). Data represent mean ± SEM; wt, N = 14; S164A, N = 4; S164E, N = 9. e Molecular interaction network of R295 of Mat1 with neighboring amino acids. f Radiometric kinase activity assay was performed as in b . Cdk7, Cyclin H, and Mat1 mutations were as indicated. Data were normalized to the activity of the respective complex without Mat1. Data represent mean ± SEM from two independent experiments, each performed in triplicate. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used in this study: α-pSer2 CTD, rat monoclonal, clone3E10, 1:100; α-pSer5 CTD, rat monoclonal, clone 3E8, 1:500; α-pSer7 CTD, rat monoclonal, clone 4E12, 1:100; α-pThr4 CTD, rat monoclonal, clone 1G7, 1:100 (CTD antibodies were a kind gift from Dirk Eick, Munich); rat monoclonal, clone 6D7, 1:1000 (ActiveMotif #61362); α-Cdk7 (total), mouse monoclonal, clone 31TF2-1F8, 1:1000, (Invitrogen #MA3-001); α-Cdk7 (total), mouse monoclonal, clone C4, 1:1000 (Santa Cruz Biotechnology #sc-7344); α-phospho Cdk7 (pT170), rabbit polyclonal, 1:1000 (Affinity Biosciences #CPA5749); α-phospho-Cdk7 (pT170), rabbit polyclonal, 1:1000 13 ; α-phospho-Cdk7 (pS164), rabbit polyclonal, 1:1000 (Invitrogen #PA5-105583); α-GST, mouse monoclonal, 1:1000 (Thermo Fisher Scientific # 740007M); α-HA tag, mouse monoclonal, clone F-7 (Santa Cruz Biotechnology #sc-7392); α-MBP, rabbit polyclonal, 1:10 for immobilization on SPR sensor ship (Novus Biologicals, NBP2-22462).

Techniques: SDS Page, Western Blot, SDS-Gel, Staining, Phospho-proteomics, Control, Kinase Assay, Activity Assay, Incubation

Journal: Nature Communications

Article Title: Structural basis of Cdk7 activation by dual T-loop phosphorylation

doi: 10.1038/s41467-024-50891-z

Figure Lengend Snippet: a Time course measurement of kinase activity of Cdk7/Cyclin H/Mat1 complexes doubly phosphorylated (pS_pT), singly phosphorylated at T170 (A_pT), or non-phosphorylated (S_T), towards GST-CTD [52] . Cdk7 complex at 0.1 µM concentration was incubated with 10 µM GST-CTD [52] in the presence of 1 mM ATP containing 0.35 µCi [ 32 P]-γ-ATP for indicated times. Curves were obtained by curve fitting using GraphPad prism. b Determination of the end-point phosphorylation. The respective Cdk7/Cyclin H/Mat1 complex (1 µM) was incubated with 2.5 µM GST-CTD [52] for 120 min. Data represent mean ± SD of a sextuplicate measurement. c Immunoblot analysis of substrate site specificity. Fully phosphorylated GST-CTD [52] (100 ng) was separated by SDS-PAGE, blotted and probed with phospho-specific antibodies recognizing pSer2, pSer5, pSer7, and pThr4 respectively. Migration of hypo-phosphorylated (CTD) and hyper-phosphorylated (pCTD) forms is indicated. d Cdk7/Cyclin H/Mat1 complex (0.1 µM) was incubated with 35 µM GST-Cdk2, 10 µM GST-CTD [52] , 50 µM GST-CTD [9] , 250 µM CTD peptide (CTD [3] ), or GST-SPT5 748-1087 (SPT5 CTR). The activity of pS_pT towards each substrate was set to 100% and the other activities normalized accordingly. Data represent mean ± SEM of triplicate measurements. e Cdk7/Cyclin H/Mat1 complex (0.1 µM) was incubated with 250 µM CTD peptides bearing three heptad repeats with either no modification (cons.), consecutive phosphorylations at the indicated site in every CTD repeat (pY1, pS2, pT4, pS5, pS7) or a substitution of the serine at position 7 to lysine. Lysines were either non-modified (K7) or acetylated (K7-ac). Assays were started with 1 mM ATP containing 0.35 µCi [ 32 P]-γ-ATP and incubated for 15 min at 30 °C. Data represent mean ± SD of triplicate measurements. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used in this study: α-pSer2 CTD, rat monoclonal, clone3E10, 1:100; α-pSer5 CTD, rat monoclonal, clone 3E8, 1:500; α-pSer7 CTD, rat monoclonal, clone 4E12, 1:100; α-pThr4 CTD, rat monoclonal, clone 1G7, 1:100 (CTD antibodies were a kind gift from Dirk Eick, Munich); rat monoclonal, clone 6D7, 1:1000 (ActiveMotif #61362); α-Cdk7 (total), mouse monoclonal, clone 31TF2-1F8, 1:1000, (Invitrogen #MA3-001); α-Cdk7 (total), mouse monoclonal, clone C4, 1:1000 (Santa Cruz Biotechnology #sc-7344); α-phospho Cdk7 (pT170), rabbit polyclonal, 1:1000 (Affinity Biosciences #CPA5749); α-phospho-Cdk7 (pT170), rabbit polyclonal, 1:1000 13 ; α-phospho-Cdk7 (pS164), rabbit polyclonal, 1:1000 (Invitrogen #PA5-105583); α-GST, mouse monoclonal, 1:1000 (Thermo Fisher Scientific # 740007M); α-HA tag, mouse monoclonal, clone F-7 (Santa Cruz Biotechnology #sc-7392); α-MBP, rabbit polyclonal, 1:10 for immobilization on SPR sensor ship (Novus Biologicals, NBP2-22462).

Techniques: Activity Assay, Concentration Assay, Incubation, Phospho-proteomics, Western Blot, SDS Page, Migration, Modification

Journal: Nature Communications

Article Title: Structural basis of Cdk7 activation by dual T-loop phosphorylation

doi: 10.1038/s41467-024-50891-z

Figure Lengend Snippet: a , b Multi-cycle kinetics of SPR measurements. For analysis, Cdk7/Cyclin H complex was used as analyte in a serial 1:3 dilution ranging from 150 nM to 0.2 nM (left panels). Dissociation constants were calculated by determination of the steady-state affinity (right panels). c Thermal stability of Cdk7 complexes was determined at a protein concentration of 2.5 µM in storage buffer (20 mM Hepes pH7.6, 150 mM NaCl, 1 mM TCEP) by monitoring intrinsic fluorescence at 350 and 330 nm with a nanoDSF device (NanoTemper). The chromatogram shows the melting curves of the ternary complexes indicated. d Dotplot representation of the melting point (T m ) of binary and ternary Cdk7 preparations. Stability was monitored in triplicate (samples w/o Mat1 and with Mat1 1-309 ) or comprising twelve (S_T), nine (A_pT) and eleven (pS_pT) replicates (samples with Mat1 230-309 ). Data represent mean ± SD. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used in this study: α-pSer2 CTD, rat monoclonal, clone3E10, 1:100; α-pSer5 CTD, rat monoclonal, clone 3E8, 1:500; α-pSer7 CTD, rat monoclonal, clone 4E12, 1:100; α-pThr4 CTD, rat monoclonal, clone 1G7, 1:100 (CTD antibodies were a kind gift from Dirk Eick, Munich); rat monoclonal, clone 6D7, 1:1000 (ActiveMotif #61362); α-Cdk7 (total), mouse monoclonal, clone 31TF2-1F8, 1:1000, (Invitrogen #MA3-001); α-Cdk7 (total), mouse monoclonal, clone C4, 1:1000 (Santa Cruz Biotechnology #sc-7344); α-phospho Cdk7 (pT170), rabbit polyclonal, 1:1000 (Affinity Biosciences #CPA5749); α-phospho-Cdk7 (pT170), rabbit polyclonal, 1:1000 13 ; α-phospho-Cdk7 (pS164), rabbit polyclonal, 1:1000 (Invitrogen #PA5-105583); α-GST, mouse monoclonal, 1:1000 (Thermo Fisher Scientific # 740007M); α-HA tag, mouse monoclonal, clone F-7 (Santa Cruz Biotechnology #sc-7392); α-MBP, rabbit polyclonal, 1:10 for immobilization on SPR sensor ship (Novus Biologicals, NBP2-22462).

Techniques: Protein Concentration, Fluorescence, Nano Differential Scanning Fluorimetry

Journal: Nature Communications

Article Title: Structural basis of Cdk7 activation by dual T-loop phosphorylation

doi: 10.1038/s41467-024-50891-z

Figure Lengend Snippet: a Phos-tag-SDS-PAGE and conventional SDS-PAGE immunoblot analysis of HCT116 whole-cell lysate. Membranes were probed with α-Cdk7 antibody to visualize total Cdk7 and in parallel with phospho-specific antibodies recognizing Cdk7 pT170 or Cdk7 pS164. Unspecific bands are marked by asterisks. b Conventional immunoblot and phos-tag immunoblot analysis of HCT116 cell lysates transfected with Cdk7-HA T-loop mutants. Blots were probed with α-HA antibody to visualize transfected Cdk7-HA and in parallel with phospho-specific antibodies recognizing Cdk7 pT170 or Cdk7 pS164. c Theoretical Cdk7 T loop phospho-isoforms, indicating possible pathways of sequential phosphorylation of S164 and T170.

Article Snippet: The following antibodies were used in this study: α-pSer2 CTD, rat monoclonal, clone3E10, 1:100; α-pSer5 CTD, rat monoclonal, clone 3E8, 1:500; α-pSer7 CTD, rat monoclonal, clone 4E12, 1:100; α-pThr4 CTD, rat monoclonal, clone 1G7, 1:100 (CTD antibodies were a kind gift from Dirk Eick, Munich); rat monoclonal, clone 6D7, 1:1000 (ActiveMotif #61362); α-Cdk7 (total), mouse monoclonal, clone 31TF2-1F8, 1:1000, (Invitrogen #MA3-001); α-Cdk7 (total), mouse monoclonal, clone C4, 1:1000 (Santa Cruz Biotechnology #sc-7344); α-phospho Cdk7 (pT170), rabbit polyclonal, 1:1000 (Affinity Biosciences #CPA5749); α-phospho-Cdk7 (pT170), rabbit polyclonal, 1:1000 13 ; α-phospho-Cdk7 (pS164), rabbit polyclonal, 1:1000 (Invitrogen #PA5-105583); α-GST, mouse monoclonal, 1:1000 (Thermo Fisher Scientific # 740007M); α-HA tag, mouse monoclonal, clone F-7 (Santa Cruz Biotechnology #sc-7392); α-MBP, rabbit polyclonal, 1:10 for immobilization on SPR sensor ship (Novus Biologicals, NBP2-22462).

Techniques: SDS Page, Western Blot, Transfection, Phospho-proteomics

Journal: Nature Communications

Article Title: Structural basis of Cdk7 activation by dual T-loop phosphorylation

doi: 10.1038/s41467-024-50891-z

Figure Lengend Snippet: a Superimposition of the crystal structure of T170 phosphorylated monomeric Cdk7 (PDB 1ua2; yellow) with the doubly T-loop phosphorylated Cdk7 structure determined here (8pyr, blue). Despite T170 phosphorylation, Cdk7 adopts a completely different conformation of the T loop in the absence of Cyclin H and Mat1. b Overlay of the unphosphorylated CAK structure (8orm, sand) with the doubly phosphorylated complex structure (8pyr). Only subtle differences exist in the positioning of the side chains, while the conformation of the T loop is largely unchanged. c Display of the electrostatic surface potential for the doubly phosphorylated CAK structure (8pyr). The phosphate groups of pS164 and pT170, each carrying two negative charges, oppose the basic residues and neutralize the surface charge. ATP•Mg 2+ and the N-terminal residues 10-54 of Cdk7 were modelled into the CAK complex as described in Supplementary Fig. . d The same view on the unphosphorylated CAK structure bound to the inhibitor THZ1 (8orm) reveals an extended basic surface patch. The electrostatic surface charge is shown from −5 k B T (red) to +5 k B T (blue).

Article Snippet: The following antibodies were used in this study: α-pSer2 CTD, rat monoclonal, clone3E10, 1:100; α-pSer5 CTD, rat monoclonal, clone 3E8, 1:500; α-pSer7 CTD, rat monoclonal, clone 4E12, 1:100; α-pThr4 CTD, rat monoclonal, clone 1G7, 1:100 (CTD antibodies were a kind gift from Dirk Eick, Munich); rat monoclonal, clone 6D7, 1:1000 (ActiveMotif #61362); α-Cdk7 (total), mouse monoclonal, clone 31TF2-1F8, 1:1000, (Invitrogen #MA3-001); α-Cdk7 (total), mouse monoclonal, clone C4, 1:1000 (Santa Cruz Biotechnology #sc-7344); α-phospho Cdk7 (pT170), rabbit polyclonal, 1:1000 (Affinity Biosciences #CPA5749); α-phospho-Cdk7 (pT170), rabbit polyclonal, 1:1000 13 ; α-phospho-Cdk7 (pS164), rabbit polyclonal, 1:1000 (Invitrogen #PA5-105583); α-GST, mouse monoclonal, 1:1000 (Thermo Fisher Scientific # 740007M); α-HA tag, mouse monoclonal, clone F-7 (Santa Cruz Biotechnology #sc-7392); α-MBP, rabbit polyclonal, 1:10 for immobilization on SPR sensor ship (Novus Biologicals, NBP2-22462).

Techniques: Phospho-proteomics

Journal: bioRxiv

Article Title: Dual Modes of Gene Regulation by CDK12

doi: 10.1101/2025.09.22.677923

Figure Lengend Snippet: A. In theory, acute inhibition of transcriptional CDKs, particularly those involved in phosphorylating RPB1 CTD Ser2 phosphorylation, is expected to globally suppress gene expression, predominantly affecting mRNAs with short half-lives. B. Volcano plots of gene expression derived from 4-6 hours treatment of CDK7 inhibitor THZ1 (250 nM) in ovarian cancer cell line Kuramochi , CDK9 inhibitor HH1 (10 µM) in a cell line (YB5) derived from the SW48 colon cancer cell line , or CDK12 inhibitor SR-4835 (90 nM) in triple-negative breast cancer line MDA-MB-231 . RNA-seq data were downloaded from the Gene Expression Omnibus (GEO) and analyzed. C. A volcano plot of nascent RNA expression from neuroblastoma cells (IMR32) treated with 400 nM THZ531 for 2 hours . Note that the sequencing involved 4-thiouridine-pulse labeling and included RNA spike-in control. D. (Top) selection of TCGA ovarian serous adenocarcinoma samples with low or high expression of the indicated CDK genes (the top and bottom 5% in terms of CDK7, CDK9 or CDK12 mRNA expression in ovarian cancer samples with expression data; n = 17 each group). (Bottom) volcano plots of differential gene expression in tumors with low expressing CDKs compared to those with high expressing CDKs. For all volcano plots, genes significantly upregulated or downregulated (absolute log 2 fold change (FC) ≥ 1, p < 0.1) are colored in red and blue, respectively.

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: Inhibition, Phospho-proteomics, Gene Expression, Derivative Assay, RNA Sequencing, RNA Expression, Sequencing, Labeling, Control, Selection, Expressing

Journal: bioRxiv

Article Title: Dual Modes of Gene Regulation by CDK12

doi: 10.1101/2025.09.22.677923

Figure Lengend Snippet: A. HCC1954 cells were treated with THZ531 or NVP-2 for 4 hours, after which whole cell lysates were harvested for fluorescent immunoblotting. The molecular weights of the fluorescent protein markers and clone identities for monoclonal antibodies are indicated. Merged images show signals from two primary antibodies raised in different species. B. Volcano plots of gene expression. HCC1954 cells were treated for 4 h with THZ531 (200 nM), or NVP-2 (200 nM), or vehicle control. Total RNA was extracted and subjected to library construction and deep sequencing. Dotted lines in the volcano plots (left) indicate the thresholds used for log-transformed fold change (1, -1). The Tukey box plots (right) illustrate the comparison between the magnitudes of change in significantly up- or down-regulated genes. **** p < 0.0001; ns, not significant (Mann-Whitney test). C. Analysis of gene size among groups of genes impacted by CDK12 or 9 inhibition. **** p < 0.0001; ns, not significant (Mann-Whitney test). D. Traces of RNA-seq coverage over the gene BLM. Note that CDK12 inhibition decreases coverage over exons, but concomitantly produces reads at positions known to harbor intronic polyadenylation. The boxed regions are also displayed with re-scaled y-axis (bottom). E. Metagene profile plots of genes that are commonly downregulated in HCC1954 cells treated with THZ531, NVP-2, or THZ1 and that have been segmented by gene size. Note that CDK12 inhibition is uniquely associated with a size-dependent elongation defect. F. Overrepresentation analysis of significantly downregulated genes in HCC1954 cells that were treated as indicated in the x-axis. Gene sets in the KEGG and Reactome pathway databases were evaluated. Dot color denotes the statistical significance of gene set enrichment within the group of downregulated genes (-log q) and dot size denotes the fraction of downregulated genes corresponding to that gene set. G. Summary plot of normalized enrichment scores (NES) for gene sets that are significantly downregulated in HCC1954 cells treated with CDK12 inhibitor (nominal p-value < 0.05). Blue dots denote gene sets implicated in the DNA damage response. H. Venn diagram for gene sets that are downregulated by CDK12, CDK9, and CDK7 inhibition. Note that gene sets involved in the DNA damage response are specific to CDK12 inhibition. The right bottom illustrates metagene profile plots of genes in the gene set Reactome_ Homologous DNA Pairing and Strand Exchange in HCC1954 cells treated with THZ531, NVP-2, or THZ1 and that have been segmented by gene size.

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: Western Blot, Bioprocessing, Gene Expression, Control, Sequencing, Transformation Assay, Comparison, MANN-WHITNEY, Inhibition, RNA Sequencing

Journal: bioRxiv

Article Title: Dual Modes of Gene Regulation by CDK12

doi: 10.1101/2025.09.22.677923

Figure Lengend Snippet: A. A volcano plot of gene expression in SKBR3 cells treated with THZ531 (200 nM, 4 h) compared with vehicle control (0.04% DMSO, v/v). Blue and red dots denote individual genes with significant change in expression (fold of change > 1, q < 0.1). B. (Left) A Tukey box plot indicates that, for genes significantly altered in expression by THZ531 treatment in SKBR3 cells, the magnitude of upregulation is even greater than gene downregulation (**p < 0.01, Mann-Whitney test). (Right) Comparison of gene size among groups of genes that differentially respond to CDK12 inhibition (****p < 0.0001, Mann-Whitney test). C. HCC1954 cells were treated with vehicle control, 200 nM THZ531 or THZ1 for 4 h, followed by lysis with 1x SDS sample buffer and fluorescent immunoblotting. Note that THZ1 treatment caused a complete electrophoretic mobility shift of total RBP1, indicating a substantial loss of CTD phosphorylation. Note that THZ1 also targets CDK12 and CDK13, and thus lacks a desired selectivity. THZ1 was chosen for the current study, instead of a more selective version of CDK7 inhibitor YKL-5-124, primarily because YKL-5-125 does not have any effect on CTD phosphorylation (Olson et al., 2019). D. A volcano plot of gene expression in HCC1954 cells treated with THZ1 (200 nM, 4 h) compared with vehicle control. Blue and red dots are genes with significant change in expression (fold of change > 1, q < 0.1). E. (Left) A Tukey box plot indicating that, for genes with expression significantly altered by THZ1 treatment in HCC1954 cells, the magnitude of downregulation is greater than gene upregulation (****p < 0.01). (Right) A comparison of gene size among groups of genes that differentially respond to CDK12 inhibition (n.s., not significant; Mann-Whitney test). F. Fluorescent immunoblotting of total cell lysates from HCC1954 cells treated with indicated doses of THZ531 for 4 or 24 hours. Note that cells treated with THZ531 for 24 hours demonstrate a reduced protein abundance of ATM, BRCA1, and EGFR, all of which are encoded by large genes (>100 kb). G. (Top) A schematic depicting mRNA with proximal or distal polyA. (Bottom) The percentage of mRNA in each treatment group (HCC1954) showing positive (proximal polyA) or negative (distal polyA) change of proximal polyadenylation site usage (△PPAU). H. Traces of RNA Seq coverage over the MAP3K9 gene. Note that CDK12 inhibition in HCC1954 cells abolishes reads corresponding to a distal polyA site (boxed) while exerting no effect on coverage over the exons.

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: Gene Expression, Control, Expressing, MANN-WHITNEY, Comparison, Inhibition, Lysis, Western Blot, Electrophoretic Mobility Shift Assay, Phospho-proteomics, Quantitative Proteomics, RNA Sequencing

Journal: bioRxiv

Article Title: Dual Modes of Gene Regulation by CDK12

doi: 10.1101/2025.09.22.677923

Figure Lengend Snippet: A. (Left) Summary plot of normalized enrichment scores (NES) for gene sets that are significantly upregulated in HCC1954 cells treated with CDK12 inhibitor (nominal p-value < 0.05). Blue dots denote MYC signature. (Right) GSEA plot of Hallmark_MYC Targets V1 for genes altered by THZ531 (200 nM, 4h) in HCC1954 (top) and SKBR3 (bottom) cells. Normalized enrichment score (NES) and p values are indicated. B. Traces of RNA-seq coverage over MYC in cells that were treated as indicated. Enlarged views for traces of CDK9 and CDK7 inhibition are shown as inserts with the y-axis scaled down. C. HCC1954 cells were treated as indicated for 4 h followed by total RNA extraction and reverse transcription. Quantitative PCR was then performed for the indicated genes. Note that MYC expression is increased, while other select DDR genes demonstrated significant downregulation. *p<0.05, **p<0.01, ***p<0.001 (Student’s t tests). D. HER2+ breast cancer cells were treated with THZ531 (200 nM) for the indicated time points. Cell lysates were prepared in SDS sample buffer and subjected to fluorescent immunoblotting. E. HCC1954 cells were treated with increasing concentrations of THZ531 (0, 12.5, 25, 50,100, 200, 400, and 800 nM) for 4 hours. Cell lysates were prepared and analyzed as in (G). F. HCC1954 cells were treated with Actinomycin D at the indicated doses in the presence of vehicle control or THZ531 (400 nM). Four hours post-treatment, cells were lysed with 1x SDS sample buffer, and lysates were prepared for fluorescent immunoblotting. Merged images are shown for blots using antibodies raised in different species. The very left lane was loaded with protein markers that emit near-infrared fluorescence. G. THZ531-induced MYC expression relies on de novo protein synthesis. HCC1954 cells were treated with cycloheximide (6.25 μg/ml) and THZ531 (200 nM), either individually or in combination. Cells were harvested in 3 hours for immunoblotting. H. Tumors with low expression of CDK12 exhibit activation of a MYC signature. (Right) The consolidated MYC signature was developed by combining all 12 gene sets from MSigDB ( http://www.gsea-msigdb.org/ ) and identifying genes whose expression positively correlates with MYC across the 1305 cancer cell lines in the Cancer Cell Line Encyclopedia (CCLE, Pearson correlation r > 0.2). (Left) Expression of the 235 genes was then evaluated in CDK12, CDK7, CDK9-low ovarian tumors in comparison to their high expression counterparts. The top dot plot displays the differential expression of all 235 genes, while the bottom plot shows genes with significant differential expression (q < 0.1; n = 106, 60, 6 for the CDK12, CDK7, CDK9 groups, respectively). *p<0.05, ****p<0.0001 (Mann-Whitney test).

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, Inhibition, RNA Extraction, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Control, Fluorescence, Activation Assay, Comparison, Quantitative Proteomics, MANN-WHITNEY

Journal: Nature cell biology

Article Title: Functional transcription promoters at DNA double-strand breaks mediate RNA-driven phase separation of damage-response factors.

doi: 10.1038/s41556-019-0392-4

Figure Lengend Snippet: Fig. 2 | RNAP II and PIC components localize to DSB as detected by super-resolution imaging and DI–PLA. a–c, Analysis of U2OS cells treated with NCS for 30 min and labelled with EU (15 min pulse) using super-resolution imaging. Representative images of γH2AX and EU stainings with POLR2A-pS5 (a) and PIC components TBP (b) and CDK7 (c). Scale bars, 5,000 nm (left) and 200 nm (right). The experiments were repeated independently 4, 3 and 3 times for a–c, respectively. d, Representative image of γH2AX and MCM6 in a U2OS nucleus treated with NCS for 30 min. Scale bar, 5,000 nm. The experiment was repeated independently twice. e,f, Statistical analysis of the cross-pair correlation between POLR2A-pS5, TBP or CDK7, and γH2AX, compared with their self-randomized correlation signal (RND-control; e), or compared with the correlation between MCM6 and γH2AX (f). Data are mean ± s.d.; n = 184, n = 109, n = 102 and n = 40 nuclei were collected from 4, 3, 3 and 2 biologically independent experiments for cross-pair correlation analyses between POLR2A-pS5, TBP, CDK7 or MCM and γH2AX, respectively. P values were calculated using two-sample unpaired t-tests. g, The fraction of γH2AX colocalizing with either MCM (black; negative control) or EU (red). Data are mean ± s.d.; n = 40 and 162 nuclei collected from 2 and 4 biologically independent experiments for MCM and EU, respectively. P = 2 × 10−6; the statistical analysis was performed using a two-sample unpaired t-test. h, DI–PLA between biotin and TFIIB, CDK7, TBP or γH2AX in U2OS cells irradiated (2 Gy) and analysed 30 min later or untreated (−IR). Each plot represents at least 80 nuclei analysed. Data are mean ± s.e.m. of n = 3 biologically independent experiments. P values were calculated using unpaired t-tests; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Representative images are shown; scale bars, 10 μm. Statistics source data are provided in Supplementary Table 2.

Article Snippet: 3 nature research | reporting sum m ary O ctober 2018 Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology Animals and other organisms Human research participants Clinical data Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used TFIIA – TFIIA1 (rabbit Bethyl A302-777A 1:1000) TFIIB – (mouse Abcam ab12094 1:1000) TFIID – TBP (mouse Abcam ab51841 1:1000) TFIIE – TFIIE1 (rabbit Abcam Ab28177 1:1000) TFIIF – TFIIF1 (rabbit Abcam Ab28179 1:1000) TFIIH –CDK7 (ChIP and WB rabbit Bethyl A300-405A 1:1000, mouse, Santa Cruz Biotechnology sc-7344 1:200) MED – MED1 (mouse Millipore 17-10530 1:1000) CDK9 – (rabbit Abcam Ab6544 1:1000) RNAPI – POLR1A (mouse Santa Cruz sc-48385 1:1000) RNAPII – POLR2A pSer5 (mouse, Abcam ab5408, 1:1000) – ChIP analysis total POLR2A (mouse, Abcam, ab817 1:1000) – immunoblot total POLR2A (mouse, Santa Cruz 8WG16 1:100) RNAPIII POLR3A (rabbit Novus Biologicals NBP1-53051 1:1000) ATM – (mouse Sigma-Aldrich A1106 1:1000) pATM – ATM-pS1981 (mouse Millipore 05-740 clone 10H11.E12 1:1000) MDC1 – (mouse Sigma-Aldrich M2444 1:1000) 53BP1 - (rabbit, Novus Biologicals NB100-304, 1:1000) MRE11 - (rabbit polyclonal raised against recombinant MRE11, kind gift from S. P. Jackson 1:1000) RAD50 - (Millipore (13B3/2C6) 05-525 1:1000) NBS1 - (rabbit, Novus Biologicals NB100-143 1:1000) H2AX - pS139 (mouse, Millipore 05-636, 1:1000) - pS139 (rabbit, Abcam ab2893 1:1000) H4 – Histone H4 (rabbit Abcam ab10158 1:1000) MCM6 - (rabbit Abcam ab211916 1:1000) if not stated otherwise the same antibody and dilution was used for different techniques. for STORM analysis γH2AX , ms to γH2AX AF647 conjugated , 05-636-AF647, Millipore 1/10000 RNAPII-pS5, ms to RNAPII-pS5, Ab5408, Abcam 1/1000 TBP, ms to TBP Ab51841, Abcam 1/1000 CDK7, ms to Cdk7, Sc-7344, scbt 1/200 Validation All antibodies were validated by the manufacturer and were previously used in peer reviewed works.

Techniques: Imaging, Control, Negative Control, Irradiation

Journal: Nature cell biology

Article Title: Functional transcription promoters at DNA double-strand breaks mediate RNA-driven phase separation of damage-response factors.

doi: 10.1038/s41556-019-0392-4

Figure Lengend Snippet: Fig. 3 | MRN complex controls PIC recruitment at endogenous DSB. a, Bar plot shows the percentage of enrichment relative to input of POLR2A, POLR2A-pS5, TBP, CDK7 and MRE11 as detected at the DAB1 locus by ChIP at 100 bp from the DSB induced by I-PpoI in HeLa cells knocked down for MRN (siMRN), TBP (siTBP) or Luciferase (siLuc) as a negative control; n = 3 independent experiments. b, Induction of dilncRNAs by I-PpoI cut measured by strand-specific qPCR with reverse transcription (RT–qPCR) in HeLa cells knocked down for MRN (siMRN), TBP (siTBP) or Luciferase (siLuc) as a negative control. Bar plots show mean of enrichment of the indicated RNA sets after DSB, relative to uncut; n = 3 independent experiments. c, Co-immunoprecipitation of TBP and RAD50 following IR exposure. HEK293T cells were irradiated (+IR) or not (−IR) and samples were collected 10 min after IR, followed by immunoprecipitation of individual components of the MRN complex (RAD50) and PIC (TBP). Whole-cell extract (input) and immunoprecipitated samples were analysed by immunoblotting. Mouse immunoglobulin G (IgG) was used as a control. This experiment was repeated twice with similar results. Statistics source data are provided in Supplementary Table 2.

Article Snippet: 3 nature research | reporting sum m ary O ctober 2018 Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology Animals and other organisms Human research participants Clinical data Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used TFIIA – TFIIA1 (rabbit Bethyl A302-777A 1:1000) TFIIB – (mouse Abcam ab12094 1:1000) TFIID – TBP (mouse Abcam ab51841 1:1000) TFIIE – TFIIE1 (rabbit Abcam Ab28177 1:1000) TFIIF – TFIIF1 (rabbit Abcam Ab28179 1:1000) TFIIH –CDK7 (ChIP and WB rabbit Bethyl A300-405A 1:1000, mouse, Santa Cruz Biotechnology sc-7344 1:200) MED – MED1 (mouse Millipore 17-10530 1:1000) CDK9 – (rabbit Abcam Ab6544 1:1000) RNAPI – POLR1A (mouse Santa Cruz sc-48385 1:1000) RNAPII – POLR2A pSer5 (mouse, Abcam ab5408, 1:1000) – ChIP analysis total POLR2A (mouse, Abcam, ab817 1:1000) – immunoblot total POLR2A (mouse, Santa Cruz 8WG16 1:100) RNAPIII POLR3A (rabbit Novus Biologicals NBP1-53051 1:1000) ATM – (mouse Sigma-Aldrich A1106 1:1000) pATM – ATM-pS1981 (mouse Millipore 05-740 clone 10H11.E12 1:1000) MDC1 – (mouse Sigma-Aldrich M2444 1:1000) 53BP1 - (rabbit, Novus Biologicals NB100-304, 1:1000) MRE11 - (rabbit polyclonal raised against recombinant MRE11, kind gift from S. P. Jackson 1:1000) RAD50 - (Millipore (13B3/2C6) 05-525 1:1000) NBS1 - (rabbit, Novus Biologicals NB100-143 1:1000) H2AX - pS139 (mouse, Millipore 05-636, 1:1000) - pS139 (rabbit, Abcam ab2893 1:1000) H4 – Histone H4 (rabbit Abcam ab10158 1:1000) MCM6 - (rabbit Abcam ab211916 1:1000) if not stated otherwise the same antibody and dilution was used for different techniques. for STORM analysis γH2AX , ms to γH2AX AF647 conjugated , 05-636-AF647, Millipore 1/10000 RNAPII-pS5, ms to RNAPII-pS5, Ab5408, Abcam 1/1000 TBP, ms to TBP Ab51841, Abcam 1/1000 CDK7, ms to Cdk7, Sc-7344, scbt 1/200 Validation All antibodies were validated by the manufacturer and were previously used in peer reviewed works.

Techniques: Luciferase, Negative Control, Reverse Transcription, Quantitative RT-PCR, Immunoprecipitation, Irradiation, Western Blot, Control