phospho cdk7  (Cell Signaling Technology Inc)

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: Gene expression and protein analysis of human FLC samples (A) mRNA expression of various known super-enhancer-driven genes in FLC were assessed in human tumor samples ( n = 35) and paired normal liver samples ( n = 10) ( t test, ∗∗ p < 0.0001). (B) Individual patient tumor samples with matched normal liver (FCF 82, 83, and 106) were evaluated for phosphorylated RPB-1 (Ser 2, 5, and 7) and CDK7. Fibrolamellar cancer was confirmed by demonstrating presence (tumor) and absence (normal) of the DNAJ-PKAc oncoprotein. (C) A phosphorylation index was calculated for each sample and phosphorylation level is presented as Phosphorylation index (Tumor)/Phosphorylation index (Adjacent) for RPB-1 (Ser 2, 5, and 7) and CDK7. Seven to nine sets of samples are shown from FLC tumor and adjacent normal tissue samples (including samples from ). (D) The data suggest that DNAJ-PKAc is correlated with heightened CDK7 activity (dotted green line). CDK7 forms a trimeric complex with cyclin H and MAT1 to phosphorylate serine-5 (preferentially) and serine-7 residues of a 52 heptad repeat in RNA polymerase II. Similarly, CDK9 dimerizes with cyclin T to preferentially phosphorylate serine-2 residues. SY-5609 and YKL-5-124 inhibit CDK7 activity; similarly VIP-152 and NVP-2 inhibit CDK9 activity. [Image created with Biorender.com ].

Article Snippet: Membranes were incubated in 10% w/v BSA blocking buffer (Thermo Fisher, Waltham, MA) at room temperature for 1 hour and hybridized with primary antibody (PKA(c): 610981 (BD Biosciences, Franklin Lakes, NJ), CDK2: #2546 (Cell Signaling Technology, Danvers, MA), phospho-CDK2 (Thr160): #2561 (Cell Signaling Technology, Danvers, MA), CDK7: #2916 (Cell Signaling Technology, Danvers, MA), phospho-CDK7 (T170): ab155976 (Abcam, Cambrige, MA), RNA polymerase II RPB-1: RPB-1 NTD; #14958, Phospho-CTD (Ser2); #13499, Phospho-CTD (Ser5); #13523, Phospho-CTD (Ser7); #13780 (Cell Signaling Technology, Danvers, MA), SCG2; PA5-115018 (Thermo Fisher, Waltham, MA), PARP; #9542 (Cell Signaling Technology, Danvers, MA), Cleaved PARP (Asp214); #5625 (Cell Signaling Technology, Danvers, MA), β-Actin: #4967 (Cell Signaling Technology, Danvers, MA), MAB8929 (R&D Systems, Minneapolis, MN) at 4°C overnight.

Techniques: Gene Expression, Expressing, Phospho-proteomics, Activity Assay

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: CDK7 regulation of RNA Polymerase II phosphorylation and super-enhancer gene expression (A) Protein analysis for phosphorylated RNA polymerase II (serine-2, serine-5, and serine-7), CDK2 and CDK7 was performed and phosphorylation levels were quantified using ImageJ. Shown are two separate biological replicates for each line. Statistically significant differences between HepG2 and H33 are shown ( t test, ∗ p < 0.05). (B) RNA sequencing of HepG2 cells and H33 cells treated at varying doses of SY-5609 for 24 h ( n = 6 biological replicates per group) were evaluated for prominent super-enhancer-associated genes, including SLC16A14 and LINC00473 . Findings from the H33 clone were confirmed in a separate DNAJB1-PRKACA -expressing clone (H12) ( t test, ∗∗ p < 0.01). (C) DNAJB1-PRKACA -expressing H33 cells were treated with a selective CDK7 inhibitor, SY5609 (100 nM, 1 μM, and 10 μM), or DMSO control (0) for 24 h ( n = 3 biological replicates per group, two representative images shown per group for western blot). Known substrate targets of CDK7 were assessed including RNA Pol II CTD (Ser 2, 5, and 7), Thr160 phosphorylated CDK2 (pCDK2) and Thr170 phosphorylated CDK7 (pCDK7) ( t test, ∗ p < 0.05). (D and E) To assess for CDK7-dependent expression of FLC-specific genes, H33 cells were treated with SY-5609 (1–300 nM) for 24 h and levels of mRNA expression (RT-qPCR) versus DMSO control were evaluated, including SLC16A14 and LINC00473 . This was repeated with a separate covalent-binding selective and specific CDK7 inhibitor (YKL-5-124). Shown are three biological replicates per drug dose per mRNA with statistical significance denoted as compared to DMSO control ( t test, ∗ p < 0.05, ∗∗ p < 0.01).

Article Snippet: Membranes were incubated in 10% w/v BSA blocking buffer (Thermo Fisher, Waltham, MA) at room temperature for 1 hour and hybridized with primary antibody (PKA(c): 610981 (BD Biosciences, Franklin Lakes, NJ), CDK2: #2546 (Cell Signaling Technology, Danvers, MA), phospho-CDK2 (Thr160): #2561 (Cell Signaling Technology, Danvers, MA), CDK7: #2916 (Cell Signaling Technology, Danvers, MA), phospho-CDK7 (T170): ab155976 (Abcam, Cambrige, MA), RNA polymerase II RPB-1: RPB-1 NTD; #14958, Phospho-CTD (Ser2); #13499, Phospho-CTD (Ser5); #13523, Phospho-CTD (Ser7); #13780 (Cell Signaling Technology, Danvers, MA), SCG2; PA5-115018 (Thermo Fisher, Waltham, MA), PARP; #9542 (Cell Signaling Technology, Danvers, MA), Cleaved PARP (Asp214); #5625 (Cell Signaling Technology, Danvers, MA), β-Actin: #4967 (Cell Signaling Technology, Danvers, MA), MAB8929 (R&D Systems, Minneapolis, MN) at 4°C overnight.

Techniques: Phospho-proteomics, Gene Expression, RNA Sequencing, Expressing, Control, Western Blot, Quantitative RT-PCR, Binding Assay

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: CDK7 is a novel therapeutic target in DNAJB1-PRKACA-expressing cells (A) To assess for CDK7 effect on cell viability, HepG2 cells and H33 cells underwent 48-h drug treatment with either SY5609 (1 pM–5 μM) or YKL-5-124 (100 pM–10 μM). Percent viability was determined by normalizing to control (DMSO treated). To confirm the findings in the DNAJB1-PRKACA -expressing H33 cells, a separate clone (H12) was tested with the same drugs over the same dose range. In each figure, the LC 50 (IC 50 ) is represented by the straight line. (B) HepG2 cells and H33 cells were synchronized and treated with either DMSO or SY5609 (1 μm) for 24 h. Percent of cells in G0/G1, S, and G2/M were determined by flow cytometry. Shown are four biological replicates per cell line per treatment ( t test, ∗ p = 0.001, ∗∗ p < 0.0001). (C) HepG2 cells and H33 cells were treated with DMSO (control) or increasing doses of SY5609 for 24 h and caspase 3/7 activity measured. To confirm the increased apoptotic activity in the H33 cells, a separate clone (H12) was utilized. ( t test ∗ p < 0.0001, for H33 vs. HepG2 and H12 vs. HepG2). (D) To validate the results, PARP and cleaved-PARP (marker for apoptosis) protein were evaluated in HepG2 and H33 cells, using two separate antibodies that either recognize both PARP and cleaved-PARP (top bands) or only cleaved-PARP alone (bottom band). (E) Primary human hepatocytes (PHHs) isolated fresh from human donor liver transplant specimens and H33 cells were treated with DMSO, SY5609 100 nM, or SY5609 1 μM for 24 and 48 h. The percent of viable cells was determined by normalizing to DMSO control. There were four biological replicates per group per time/treatment dose ( t test ∗ p = 0.02, ∗∗ p = 0.01, ∗∗∗ p < 0.0001). (F) PHHs and H33 cells were treated with SY5609 (100 pM–5 μM) for 48, 72, or 120 h and percent viability assessed, normalized to DMSO control. The LC 50 (IC 50 ) is demarcated by the solid line. There were four biological replicates for each drug dose at each time point for each line (PHH and H33).

Article Snippet: Membranes were incubated in 10% w/v BSA blocking buffer (Thermo Fisher, Waltham, MA) at room temperature for 1 hour and hybridized with primary antibody (PKA(c): 610981 (BD Biosciences, Franklin Lakes, NJ), CDK2: #2546 (Cell Signaling Technology, Danvers, MA), phospho-CDK2 (Thr160): #2561 (Cell Signaling Technology, Danvers, MA), CDK7: #2916 (Cell Signaling Technology, Danvers, MA), phospho-CDK7 (T170): ab155976 (Abcam, Cambrige, MA), RNA polymerase II RPB-1: RPB-1 NTD; #14958, Phospho-CTD (Ser2); #13499, Phospho-CTD (Ser5); #13523, Phospho-CTD (Ser7); #13780 (Cell Signaling Technology, Danvers, MA), SCG2; PA5-115018 (Thermo Fisher, Waltham, MA), PARP; #9542 (Cell Signaling Technology, Danvers, MA), Cleaved PARP (Asp214); #5625 (Cell Signaling Technology, Danvers, MA), β-Actin: #4967 (Cell Signaling Technology, Danvers, MA), MAB8929 (R&D Systems, Minneapolis, MN) at 4°C overnight.

Techniques: Expressing, Control, Flow Cytometry, Activity Assay, Marker, Isolation

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: CDK7 inhibition is lethal in human FLC (A) An FLC cell line derived from human FLC (FLC-H) was grown for six days (control) or treated with DMSO (Control DMSO) versus SY5609 at two separate doses (500 nM and 1 μM). Viable cells were quantified at day zero and day six. Additionally, the day 6 percent survival compared to controls was calculated. Shown are two separate experiments with three biological replicates per experiment ( t test ∗ p < 0.01, ∗∗ p < 0.001 versus control and control DMSO). (B) In a separate experiment, an FLC cell line was derived from a patient FLC liver tumor (FLC1025), and another discrete cell line was derived from patient metastatic FLC tumor implants (FLCMet). Each line was treated with SY5609 (10 nM–10 μM). Percent survival was determined normalized to DMSO control, with three biological replicates per dose. Shown is the dose response with curve (and 95% CI) and LC 50 (IC 50 ) demonstrated by the straight line (LC 50 FLC1025 ∼300 nM, LC 50 FLCMet ∼20 nM). (C) Human tissue slices derived from a patient with FLC (FLC217) were treated with DMSO or SY5609 (500 nM), and percent viability determined compared to DMSO control (∗∗ p = 0.003). (D) In a separate experiment, tissue slices derived from human FLC grown in a patient derived xenograft (PDX) model were treated with DMSO, SY5609 (100 nM, 500 nM, and 1 μM) and staurosporine (STS) 500 nM (positive control). There were 3–4 biological replicates per group ( t test ∗ p < 0.01, ∗∗ p < 0.001).

Article Snippet: Membranes were incubated in 10% w/v BSA blocking buffer (Thermo Fisher, Waltham, MA) at room temperature for 1 hour and hybridized with primary antibody (PKA(c): 610981 (BD Biosciences, Franklin Lakes, NJ), CDK2: #2546 (Cell Signaling Technology, Danvers, MA), phospho-CDK2 (Thr160): #2561 (Cell Signaling Technology, Danvers, MA), CDK7: #2916 (Cell Signaling Technology, Danvers, MA), phospho-CDK7 (T170): ab155976 (Abcam, Cambrige, MA), RNA polymerase II RPB-1: RPB-1 NTD; #14958, Phospho-CTD (Ser2); #13499, Phospho-CTD (Ser5); #13523, Phospho-CTD (Ser7); #13780 (Cell Signaling Technology, Danvers, MA), SCG2; PA5-115018 (Thermo Fisher, Waltham, MA), PARP; #9542 (Cell Signaling Technology, Danvers, MA), Cleaved PARP (Asp214); #5625 (Cell Signaling Technology, Danvers, MA), β-Actin: #4967 (Cell Signaling Technology, Danvers, MA), MAB8929 (R&D Systems, Minneapolis, MN) at 4°C overnight.

Techniques: Inhibition, Derivative Assay, Control, Positive Control

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: Synergistic combination between CDK7 and CDK9 inhibition in vitro (A) DNAJB1-PRKACA expressing H33 cells were treated with SY-5609 alone, VIP-152 alone, or in combination for 24 h. Protein was isolated and western blot performed for measurement and quantification of phosphorylated RPB-1 (Ser 2, 5, and 7) ( n = 3 biological replicates per group, two representative western blots are shown, t test, ∗ p < 0.05). (B) Expression of SLC16A14 and LINC00473 was determined in H33 cells treated with SY-5609 alone (blue), VIP-152 alone (purple), or in combination (green). Statistical significance is denoted as compared to control ( t test, ∗∗ p < 0.01, ∗ p < 0.05). (C and D) Synergistic response to combination therapy of SY-5609 and VIP-152 in H33 cells was determined with potent reduction in percent survival (normalized to DMSO control). Depicted is a dose-response curve which demonstrates shift of the curve to the left for SY-5609 with increasing concentration of VIP-152 up to a dose of 30 nM VIP-152. The drug combination showed strong synergy using all metrics including HSA (mean 18.73, p = 6.31e-5), Bliss (mean 14.55, p = 3.46e-4), Loewe (mean 15.48, p = 1.98e-4), and ZIP (mean 13.54, p = 6.04e-4). The strongest synergistic doses occurred at 30 nM SY-5609 + 30 nM VIP152 (synergy score ∼38) and 10 nM SY-5609 + 30 nM VIP152 (synergy score ∼37).

Article Snippet: Membranes were incubated in 10% w/v BSA blocking buffer (Thermo Fisher, Waltham, MA) at room temperature for 1 hour and hybridized with primary antibody (PKA(c): 610981 (BD Biosciences, Franklin Lakes, NJ), CDK2: #2546 (Cell Signaling Technology, Danvers, MA), phospho-CDK2 (Thr160): #2561 (Cell Signaling Technology, Danvers, MA), CDK7: #2916 (Cell Signaling Technology, Danvers, MA), phospho-CDK7 (T170): ab155976 (Abcam, Cambrige, MA), RNA polymerase II RPB-1: RPB-1 NTD; #14958, Phospho-CTD (Ser2); #13499, Phospho-CTD (Ser5); #13523, Phospho-CTD (Ser7); #13780 (Cell Signaling Technology, Danvers, MA), SCG2; PA5-115018 (Thermo Fisher, Waltham, MA), PARP; #9542 (Cell Signaling Technology, Danvers, MA), Cleaved PARP (Asp214); #5625 (Cell Signaling Technology, Danvers, MA), β-Actin: #4967 (Cell Signaling Technology, Danvers, MA), MAB8929 (R&D Systems, Minneapolis, MN) at 4°C overnight.

Techniques: Inhibition, In Vitro, Expressing, Isolation, Western Blot, Control, Concentration Assay

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: CDK7 and CDK9 inhibition in an organoid model (A) Tissue from a patient with FLC (FLC4-PDX) was propagated and developed into a patient-derived cancer organoid model, FLC4 (organoid). Western blot shows the presence of DNAJ-PKAc oncoprotein and phosphorylated RPB-1 (Ser 2, 5, and 7) in the organoid compared to immortalized human hepatocytes (IHH, n = 4) which displays only native PKAc and low levels of phosphorylated RPB-1. Phosphorylated RPB-1 (Ser 2, 5, and 7) was measured and quantified. Statistical significance was calculated ( t test, ∗∗∗ p < 0.001, ∗∗ p < 0.01). (B) RNA was isolated from organoids treated with SY-5609 (1 nM, 3 nM, 10 nM, and 30 nM) and VIP-152 (30 nM, 100 nM, 300 nM, and 500 nM) and SLC16A14 and LINC00473 expression was determined by qPCR relative to DMSO control. Due to the limited availability of organoid tissue available only one replicate is represented per dose.

Article Snippet: Membranes were incubated in 10% w/v BSA blocking buffer (Thermo Fisher, Waltham, MA) at room temperature for 1 hour and hybridized with primary antibody (PKA(c): 610981 (BD Biosciences, Franklin Lakes, NJ), CDK2: #2546 (Cell Signaling Technology, Danvers, MA), phospho-CDK2 (Thr160): #2561 (Cell Signaling Technology, Danvers, MA), CDK7: #2916 (Cell Signaling Technology, Danvers, MA), phospho-CDK7 (T170): ab155976 (Abcam, Cambrige, MA), RNA polymerase II RPB-1: RPB-1 NTD; #14958, Phospho-CTD (Ser2); #13499, Phospho-CTD (Ser5); #13523, Phospho-CTD (Ser7); #13780 (Cell Signaling Technology, Danvers, MA), SCG2; PA5-115018 (Thermo Fisher, Waltham, MA), PARP; #9542 (Cell Signaling Technology, Danvers, MA), Cleaved PARP (Asp214); #5625 (Cell Signaling Technology, Danvers, MA), β-Actin: #4967 (Cell Signaling Technology, Danvers, MA), MAB8929 (R&D Systems, Minneapolis, MN) at 4°C overnight.

Techniques: Inhibition, Derivative Assay, Western Blot, Isolation, Expressing, Control

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: Synergistic combination of CDK7 and CDK9 inhibition in an organoid model (A) FLC organoids were treated with SY-5609 alone, VIP-152 alone or in combination and western blot was performed showing phosphorylated RPB-1 (Ser 2, 5, and 7). Protein levels were measured and quantified. (B) FLC organoids were treated with SY-509 alone and in combination with VIP-152 at various doses which showed a dose-dependent decrease in cell survival as compared to DMSO. (C) RNA was isolated from FLC organoids treated with combination SY-5609 and VIP-152 for 96 h (1nM/10 nM, 10nM/10 nM, and 100nM/100 nM) and expression of SLC16A14 and LINC00473 was determined. Due to the limited availability of organoid tissue available only one replicate is represented per dose.

Article Snippet: Membranes were incubated in 10% w/v BSA blocking buffer (Thermo Fisher, Waltham, MA) at room temperature for 1 hour and hybridized with primary antibody (PKA(c): 610981 (BD Biosciences, Franklin Lakes, NJ), CDK2: #2546 (Cell Signaling Technology, Danvers, MA), phospho-CDK2 (Thr160): #2561 (Cell Signaling Technology, Danvers, MA), CDK7: #2916 (Cell Signaling Technology, Danvers, MA), phospho-CDK7 (T170): ab155976 (Abcam, Cambrige, MA), RNA polymerase II RPB-1: RPB-1 NTD; #14958, Phospho-CTD (Ser2); #13499, Phospho-CTD (Ser5); #13523, Phospho-CTD (Ser7); #13780 (Cell Signaling Technology, Danvers, MA), SCG2; PA5-115018 (Thermo Fisher, Waltham, MA), PARP; #9542 (Cell Signaling Technology, Danvers, MA), Cleaved PARP (Asp214); #5625 (Cell Signaling Technology, Danvers, MA), β-Actin: #4967 (Cell Signaling Technology, Danvers, MA), MAB8929 (R&D Systems, Minneapolis, MN) at 4°C overnight.

Techniques: Inhibition, Western Blot, Isolation, Expressing

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, 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:

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, 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, 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

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:

Journal: bioRxiv

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

doi: 10.1101/2024.02.14.580246

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.

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); α-Cdk7 (total), mouse monoclonal, clone 31TF2-1F8, 1:1000, (Invitrogen #MA3-001); α-Cdk7 (total), mouse monoclonal, clone C4, 1:1000 (Santa Cruz #sc-7344); α-phospho Cdk7 (pT170), rabbit polyclonal, 1:1000 (Affinity Biosciences #CPA5749); α-phospho-Cdk7 (pT170), rabbit polyclonal, 1:1000 ; α-phospho-Cdk7 (pS164), rabbit polyclonal, 1:1000 (Invitrogen #PA5-105583); anti-GST, mouse monoclonal, 1:1000 (Thermo Fisher Scientific # 740007M).

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

Journal: bioRxiv

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

doi: 10.1101/2024.02.14.580246

Figure Lengend Snippet: a , Additional SDS-PAGE analysis of the samples shown in . Running parameters were changed to separate phosphorylated Cdk7 from Cyclin H. 10 µl at a concentration of 2.6 µM were separated on a 15% SDS-PAGE until the 35 kDa marker band of the prestained marker had migrated ∼60% through the gel. b , Cartoon of the running behavior of Cdk7 phospho-isoforms in SDS-PAGE. c , Merged images of the western blots shown in . d-f , Molecular masses of intact proteins determined by ESI–(LC)–MS indicating the phosphorylation status. d , Protein masses from the co-expression of the full length Cdk7/Cyclin H/Mat1 1-309 complex (corresponding to lane 2 in panel a ). e , Protein masses from the co-expression of truncated Cdk7/Cyclin H/Mat1 230-309 (lane 3 in panel a ). f , Protein masses upon addition of Mat1 230-309 to co-expressed Cdk7/Cyclin H (lane 4 in panel a ).

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); α-Cdk7 (total), mouse monoclonal, clone 31TF2-1F8, 1:1000, (Invitrogen #MA3-001); α-Cdk7 (total), mouse monoclonal, clone C4, 1:1000 (Santa Cruz #sc-7344); α-phospho Cdk7 (pT170), rabbit polyclonal, 1:1000 (Affinity Biosciences #CPA5749); α-phospho-Cdk7 (pT170), rabbit polyclonal, 1:1000 ; α-phospho-Cdk7 (pS164), rabbit polyclonal, 1:1000 (Invitrogen #PA5-105583); anti-GST, mouse monoclonal, 1:1000 (Thermo Fisher Scientific # 740007M).

Techniques: SDS Page, Concentration Assay, Marker, Western Blot, Liquid Chromatography with Mass Spectroscopy, Expressing

Journal: bioRxiv

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

doi: 10.1101/2024.02.14.580246

Figure Lengend Snippet: a , SPR binding analyses of Cdk7/Cyclin H/Mat1 directed VHHs. b , Epitope binning of VHHs highlights two different epitopes. c , Impact of VHHs on Cdk7 activity. 0.1 µM Cdk7/Cyclin H/Mat1 230-309 was pre-incubated with 1 µM VHH and 10 µM GST-CTD [52] . Reaction was started with 1 mM ATP and stopped after 15 min. Phosphorylation of the CTD was determined by SDS-PAGE analysis. d , Radiometric kinase assay to analyze the inhibitory potential of VHH RD7-01 . Assay was performed as in c but with varying concentrations of VHH RD7-01 and with 1 mM ATP containing 0.35 µCi [P]γ-ATP. Cdk9/CycT1 1-272 was used as a control for nanobody specificity. Data are depicted as dot plots with mean of duplicate measurements. e , f , Interaction networks of the VHH RD7-01 CDRs with Cdk7/Cyclin H/Mat1 230-309

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); α-Cdk7 (total), mouse monoclonal, clone 31TF2-1F8, 1:1000, (Invitrogen #MA3-001); α-Cdk7 (total), mouse monoclonal, clone C4, 1:1000 (Santa Cruz #sc-7344); α-phospho Cdk7 (pT170), rabbit polyclonal, 1:1000 (Affinity Biosciences #CPA5749); α-phospho-Cdk7 (pT170), rabbit polyclonal, 1:1000 ; α-phospho-Cdk7 (pS164), rabbit polyclonal, 1:1000 (Invitrogen #PA5-105583); anti-GST, mouse monoclonal, 1:1000 (Thermo Fisher Scientific # 740007M).

Techniques: Binding Assay, Activity Assay, Incubation, SDS Page, Kinase Assay

Journal: bioRxiv

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

doi: 10.1101/2024.02.14.580246

Figure Lengend Snippet: a , Electron density of pT170 and surrounding residues. The structural model is drawn in stick representation. The blue mesh is a 2mF o -DF c electron density map contoured at 1.0 σ. b , Representative electron density of pS164 and surrounding residues. The structural model and the electron density map are displayed as in panel a . c , Stacking interactions between R165 of Cyclin H, R295 of Mat1, and W132 of Cdk7, with pS164 of Cdk7 making salt-bridge interactions with R165 of Cyclin H. d , Electrostatic surface display of the Cdk7/Cyclin H/Mat1 complex structure determined here (8pyr) fused with residues 10-54, ATPγS and Mg 2+ of Cdk7 from structure 6xbz and superimposed with the substrate peptide P 3 T 4 S 5 P 6 S 7 Y 1 S 2 that was modeled on the peptide PKTPKKA from the Cdk2/CycA/substrate complex structure 3qhr . The register of the PxxP binding motif in the substrate sequence aligns to S5 phosphorylation within the CTD and an P 3 xSP 6 xY 1 interaction motif.

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); α-Cdk7 (total), mouse monoclonal, clone 31TF2-1F8, 1:1000, (Invitrogen #MA3-001); α-Cdk7 (total), mouse monoclonal, clone C4, 1:1000 (Santa Cruz #sc-7344); α-phospho Cdk7 (pT170), rabbit polyclonal, 1:1000 (Affinity Biosciences #CPA5749); α-phospho-Cdk7 (pT170), rabbit polyclonal, 1:1000 ; α-phospho-Cdk7 (pS164), rabbit polyclonal, 1:1000 (Invitrogen #PA5-105583); anti-GST, mouse monoclonal, 1:1000 (Thermo Fisher Scientific # 740007M).

Techniques: Binding Assay, Sequencing

Journal: bioRxiv

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

doi: 10.1101/2024.02.14.580246

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. Residues belonging to Cdk7 are written in letters without outlines; those belonging to Cyclin H are outlined in white and Mat1 residues are outlined in magenta. 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); α-Cdk7 (total), mouse monoclonal, clone 31TF2-1F8, 1:1000, (Invitrogen #MA3-001); α-Cdk7 (total), mouse monoclonal, clone C4, 1:1000 (Santa Cruz #sc-7344); α-phospho Cdk7 (pT170), rabbit polyclonal, 1:1000 (Affinity Biosciences #CPA5749); α-phospho-Cdk7 (pT170), rabbit polyclonal, 1:1000 ; α-phospho-Cdk7 (pS164), rabbit polyclonal, 1:1000 (Invitrogen #PA5-105583); anti-GST, mouse monoclonal, 1:1000 (Thermo Fisher Scientific # 740007M).

Techniques:

Journal: bioRxiv

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

doi: 10.1101/2024.02.14.580246

Figure Lengend Snippet: a , SDS-PAGE and immunoblot analysis of GST-Cdk7/Cyclin H/MBP-Mat1 complexes. Protein (3 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 GST-Cdk2. Each Cdk7 complex at 0.1 µM was incubated with 10 µM GST-CTD [52] or 15 µM GST-Cdk2 in the presence of 1 mM ATP containing 0.35 µCi [ 32 P]-γ-ATP for 15 min at 30°C. Bars represent mean ± SD. c , SDS-PAGE analyses of GST-Cdk7/Cyclin H and MBP-Mat1; 2 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] of Cdk7/Cyclin H without Mat1 and upon pre-incubation with MBP-Mat1 230-309 or MBP-Mat1 1-309 for 10 min (left panel). Cdk7/Cyclin H (0.1 µM) was pre-incubated with buffer or 0.4 µM Mat1, and 10 µM GST-CTD [52] . Reaction was started by addition of 1 mM ATP containing 0.35 µCi [ 32 P]-γ-ATP and incubated for 15 min at 30°C. 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). Assay conditions were as described above. Each data point represents an individual measurement monitored at least in duplicate. Bars represent mean ± SEM. e , Molecular interaction network of R295 of Mat1 with neighboring amino acids. f , Radiometric kinase activity assay was performed as in d . 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 separate experiments, each performed in triplicate.

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); α-Cdk7 (total), mouse monoclonal, clone 31TF2-1F8, 1:1000, (Invitrogen #MA3-001); α-Cdk7 (total), mouse monoclonal, clone C4, 1:1000 (Santa Cruz #sc-7344); α-phospho Cdk7 (pT170), rabbit polyclonal, 1:1000 (Affinity Biosciences #CPA5749); α-phospho-Cdk7 (pT170), rabbit polyclonal, 1:1000 ; α-phospho-Cdk7 (pS164), rabbit polyclonal, 1:1000 (Invitrogen #PA5-105583); anti-GST, mouse monoclonal, 1:1000 (Thermo Fisher Scientific # 740007M).

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

Journal: bioRxiv

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

doi: 10.1101/2024.02.14.580246

Figure Lengend Snippet: a , SDS-PAGE analysis of Cdk7/Cyclin H complex and MBP-Mat1 230-309 , 3 µg each. b , Radiometric kinase activity assay. 0.1 µM Cdk7/Cyclin H complex was incubated with 0.4 µM MBP or MBP-Mat1 230-309 and 10 µM GST-CTD [52] for 10 min prior to starting the kinase reaction by addition of 1 mM ATP containing 0.35 µCi [ 32 P]-γ-ATP. Samples were incubated for 15 min at 30°C. c , SDS-PAGE analysis of a kinase assay titrating MBP-Mat1 230-309 . Cdk7/Cyclin H complex (0.1 µM)—wild-type or S164 mutant variants, as indicated—was incubated with increasing concentrations of MBP-Mat1 230-309 . Samples were incubated in the presence of 10 µM GST-CTD [52] for 10 min prior to starting the assay with 1 mM ATP. After 15 min at 30°C, assay was stopped with 2xSDS sample buffer. d , Radiometric kinase assay. As in panel c , but with addition of 0.35 µCi [ 32 P]-γ-ATP. Phosphorylation was measured by liquid scintillation counting. Data represent mean of duplicate measurements. e , SDS-PAGE analysis of the GST-Cdk7/Cyclin H complexes used in , 2 µg each. f , Absolute counts of one experiment of the data shown in , highlighting similar activity of the binary preparations. Data represent mean ± SD.

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); α-Cdk7 (total), mouse monoclonal, clone 31TF2-1F8, 1:1000, (Invitrogen #MA3-001); α-Cdk7 (total), mouse monoclonal, clone C4, 1:1000 (Santa Cruz #sc-7344); α-phospho Cdk7 (pT170), rabbit polyclonal, 1:1000 (Affinity Biosciences #CPA5749); α-phospho-Cdk7 (pT170), rabbit polyclonal, 1:1000 ; α-phospho-Cdk7 (pS164), rabbit polyclonal, 1:1000 (Invitrogen #PA5-105583); anti-GST, mouse monoclonal, 1:1000 (Thermo Fisher Scientific # 740007M).

Techniques: SDS Page, Kinase Assay, Incubation, Mutagenesis, Activity Assay

Journal: bioRxiv

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

doi: 10.1101/2024.02.14.580246

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. 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.

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); α-Cdk7 (total), mouse monoclonal, clone 31TF2-1F8, 1:1000, (Invitrogen #MA3-001); α-Cdk7 (total), mouse monoclonal, clone C4, 1:1000 (Santa Cruz #sc-7344); α-phospho Cdk7 (pT170), rabbit polyclonal, 1:1000 (Affinity Biosciences #CPA5749); α-phospho-Cdk7 (pT170), rabbit polyclonal, 1:1000 ; α-phospho-Cdk7 (pS164), rabbit polyclonal, 1:1000 (Invitrogen #PA5-105583); anti-GST, mouse monoclonal, 1:1000 (Thermo Fisher Scientific # 740007M).

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

Journal: bioRxiv

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

doi: 10.1101/2024.02.14.580246

Figure Lengend Snippet: a , Immunoblot analysis of site-specific CTD kinase activity of binary Cdk7/Cyclin H complex and Cdk7/Cyclin H incubated with MBP-Mat1 230-309 prior to activity assay. b , Immunoblot analysis of Cdk7 substrate site specificity compared to P-TEFb. c , Radiometric activity assay. Peptides composed of three heptad repeats containing different pre-phosphorylations are shown in schematic diagram at right. Pre-phosphorylation was either continuous (pS5/pS7) or within a single repeat (pS5/7-C-terminal or middle or N-terminal); cons.: (consensus sequence with no pre-phosphorylation). Data represent mean ± SD of a single experiment measured in triplicate.

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); α-Cdk7 (total), mouse monoclonal, clone 31TF2-1F8, 1:1000, (Invitrogen #MA3-001); α-Cdk7 (total), mouse monoclonal, clone C4, 1:1000 (Santa Cruz #sc-7344); α-phospho Cdk7 (pT170), rabbit polyclonal, 1:1000 (Affinity Biosciences #CPA5749); α-phospho-Cdk7 (pT170), rabbit polyclonal, 1:1000 ; α-phospho-Cdk7 (pS164), rabbit polyclonal, 1:1000 (Invitrogen #PA5-105583); anti-GST, mouse monoclonal, 1:1000 (Thermo Fisher Scientific # 740007M).

Techniques: Western Blot, Activity Assay, Incubation, Sequencing

Journal: bioRxiv

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

doi: 10.1101/2024.02.14.580246

Figure Lengend Snippet: a , Experimental setup for SPR measurements. b , SPR analysis of Mat1 binding to Cdk7(S164E)/Cyclin H. c , Stabilization matrix, highlighting the melting-temperature changes between different Cdk7 preparations upon phosphorylation or Mat1 addition. The least stable Cdk7/Cyclin H complex tested (binary A_pT) was used as reference.

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); α-Cdk7 (total), mouse monoclonal, clone 31TF2-1F8, 1:1000, (Invitrogen #MA3-001); α-Cdk7 (total), mouse monoclonal, clone C4, 1:1000 (Santa Cruz #sc-7344); α-phospho Cdk7 (pT170), rabbit polyclonal, 1:1000 (Affinity Biosciences #CPA5749); α-phospho-Cdk7 (pT170), rabbit polyclonal, 1:1000 ; α-phospho-Cdk7 (pS164), rabbit polyclonal, 1:1000 (Invitrogen #PA5-105583); anti-GST, mouse monoclonal, 1:1000 (Thermo Fisher Scientific # 740007M).

Techniques: Binding Assay

Journal: bioRxiv

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

doi: 10.1101/2024.02.14.580246

Figure Lengend Snippet: a , b , Multi-cycle kinetics SPR measurement. 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.

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); α-Cdk7 (total), mouse monoclonal, clone 31TF2-1F8, 1:1000, (Invitrogen #MA3-001); α-Cdk7 (total), mouse monoclonal, clone C4, 1:1000 (Santa Cruz #sc-7344); α-phospho Cdk7 (pT170), rabbit polyclonal, 1:1000 (Affinity Biosciences #CPA5749); α-phospho-Cdk7 (pT170), rabbit polyclonal, 1:1000 ; α-phospho-Cdk7 (pS164), rabbit polyclonal, 1:1000 (Invitrogen #PA5-105583); anti-GST, mouse monoclonal, 1:1000 (Thermo Fisher Scientific # 740007M).

Techniques: Protein Concentration, Fluorescence, Nano Differential Scanning Fluorimetry

Journal: bioRxiv

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

doi: 10.1101/2024.02.14.580246

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. b , c , Phos-tag-SDS-PAGE and conventional SDS-PAGE 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 in parallel with phospho-specific antibodies recognizing Cdk7 pT170 ( b ) or Cdk7 pS164 ( c ). d , 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); α-Cdk7 (total), mouse monoclonal, clone 31TF2-1F8, 1:1000, (Invitrogen #MA3-001); α-Cdk7 (total), mouse monoclonal, clone C4, 1:1000 (Santa Cruz #sc-7344); α-phospho Cdk7 (pT170), rabbit polyclonal, 1:1000 (Affinity Biosciences #CPA5749); α-phospho-Cdk7 (pT170), rabbit polyclonal, 1:1000 ; α-phospho-Cdk7 (pS164), rabbit polyclonal, 1:1000 (Invitrogen #PA5-105583); anti-GST, mouse monoclonal, 1:1000 (Thermo Fisher Scientific # 740007M).

Techniques: SDS Page, Western Blot, Transfection

Journal: bioRxiv

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

doi: 10.1101/2024.02.14.580246

Figure Lengend Snippet: Electrostatic surface display of the Cdk7/Cyclin H/Mat1 complex structure determined here (8pyr) fused with residues 10-54, ATPγS and Mg 2+ of Cdk7 from structure 6xbz and superimposed with a substrate peptide at the catalytic site aligned to S5 phosphorylation that was modeled on the peptide PKTPKKAKKL from the Cdk2/CycA/substrate complex structure 3qhr . The initial substrate model was extended at the N-terminus by residues Y 1 S 2 and at the C-terminus by three heptad repeats, each repeat being alternately colored yellow and white. In this model, the fifth repeat was directed towards a basic patch on the second cyclin box of Cyclin H, which is formed by residues R197, R223, K253, R256 and K260.

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); α-Cdk7 (total), mouse monoclonal, clone 31TF2-1F8, 1:1000, (Invitrogen #MA3-001); α-Cdk7 (total), mouse monoclonal, clone C4, 1:1000 (Santa Cruz #sc-7344); α-phospho Cdk7 (pT170), rabbit polyclonal, 1:1000 (Affinity Biosciences #CPA5749); α-phospho-Cdk7 (pT170), rabbit polyclonal, 1:1000 ; α-phospho-Cdk7 (pS164), rabbit polyclonal, 1:1000 (Invitrogen #PA5-105583); anti-GST, mouse monoclonal, 1:1000 (Thermo Fisher Scientific # 740007M).

Techniques:

Journal: bioRxiv

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

doi: 10.1101/2024.02.14.580246

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.

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); α-Cdk7 (total), mouse monoclonal, clone 31TF2-1F8, 1:1000, (Invitrogen #MA3-001); α-Cdk7 (total), mouse monoclonal, clone C4, 1:1000 (Santa Cruz #sc-7344); α-phospho Cdk7 (pT170), rabbit polyclonal, 1:1000 (Affinity Biosciences #CPA5749); α-phospho-Cdk7 (pT170), rabbit polyclonal, 1:1000 ; α-phospho-Cdk7 (pS164), rabbit polyclonal, 1:1000 (Invitrogen #PA5-105583); anti-GST, mouse monoclonal, 1:1000 (Thermo Fisher Scientific # 740007M).

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

Journal: bioRxiv

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

doi: 10.1101/2024.02.14.580246

Figure Lengend Snippet: a , Additional SDS-PAGE analysis of the samples shown in . Running parameters were changed to separate phosphorylated Cdk7 from Cyclin H. 10 µl at a concentration of 2.6 µM were separated on a 15% SDS-PAGE until the 35 kDa marker band of the prestained marker had migrated ∼60% through the gel. b , Cartoon of the running behavior of Cdk7 phospho-isoforms in SDS-PAGE. c , Merged images of the western blots shown in . d-f , Molecular masses of intact proteins determined by ESI–(LC)–MS indicating the phosphorylation status. d , Protein masses from the co-expression of the full length Cdk7/Cyclin H/Mat1 1-309 complex (corresponding to lane 2 in panel a ). e , Protein masses from the co-expression of truncated Cdk7/Cyclin H/Mat1 230-309 (lane 3 in panel a ). f , Protein masses upon addition of Mat1 230-309 to co-expressed Cdk7/Cyclin H (lane 4 in panel a ).

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); α-Cdk7 (total), mouse monoclonal, clone 31TF2-1F8, 1:1000, (Invitrogen #MA3-001); α-Cdk7 (total), mouse monoclonal, clone C4, 1:1000 (Santa Cruz #sc-7344); α-phospho Cdk7 (pT170), rabbit polyclonal, 1:1000 (Affinity Biosciences #CPA5749); α-phospho-Cdk7 (pT170), rabbit polyclonal, 1:1000 ; α-phospho-Cdk7 (pS164), rabbit polyclonal, 1:1000 (Invitrogen #PA5-105583); anti-GST, mouse monoclonal, 1:1000 (Thermo Fisher Scientific # 740007M).

Techniques: SDS Page, Concentration Assay, Marker, Western Blot, Liquid Chromatography with Mass Spectroscopy, Expressing

Journal: bioRxiv

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

doi: 10.1101/2024.02.14.580246

Figure Lengend Snippet: a , SPR binding analyses of Cdk7/Cyclin H/Mat1 directed VHHs. b , Epitope binning of VHHs highlights two different epitopes. c , Impact of VHHs on Cdk7 activity. 0.1 µM Cdk7/Cyclin H/Mat1 230-309 was pre-incubated with 1 µM VHH and 10 µM GST-CTD [52] . Reaction was started with 1 mM ATP and stopped after 15 min. Phosphorylation of the CTD was determined by SDS-PAGE analysis. d , Radiometric kinase assay to analyze the inhibitory potential of VHH RD7-01 . Assay was performed as in c but with varying concentrations of VHH RD7-01 and with 1 mM ATP containing 0.35 µCi [P]γ-ATP. Cdk9/CycT1 1-272 was used as a control for nanobody specificity. Data are depicted as dot plots with mean of duplicate measurements. e , f , Interaction networks of the VHH RD7-01 CDRs with Cdk7/Cyclin H/Mat1 230-309

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); α-Cdk7 (total), mouse monoclonal, clone 31TF2-1F8, 1:1000, (Invitrogen #MA3-001); α-Cdk7 (total), mouse monoclonal, clone C4, 1:1000 (Santa Cruz #sc-7344); α-phospho Cdk7 (pT170), rabbit polyclonal, 1:1000 (Affinity Biosciences #CPA5749); α-phospho-Cdk7 (pT170), rabbit polyclonal, 1:1000 ; α-phospho-Cdk7 (pS164), rabbit polyclonal, 1:1000 (Invitrogen #PA5-105583); anti-GST, mouse monoclonal, 1:1000 (Thermo Fisher Scientific # 740007M).

Techniques: Binding Assay, Activity Assay, Incubation, SDS Page, Kinase Assay

Journal: bioRxiv

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

doi: 10.1101/2024.02.14.580246

Figure Lengend Snippet: a , Electron density of pT170 and surrounding residues. The structural model is drawn in stick representation. The blue mesh is a 2mF o -DF c electron density map contoured at 1.0 σ. b , Representative electron density of pS164 and surrounding residues. The structural model and the electron density map are displayed as in panel a . c , Stacking interactions between R165 of Cyclin H, R295 of Mat1, and W132 of Cdk7, with pS164 of Cdk7 making salt-bridge interactions with R165 of Cyclin H. d , Electrostatic surface display of the Cdk7/Cyclin H/Mat1 complex structure determined here (8pyr) fused with residues 10-54, ATPγS and Mg 2+ of Cdk7 from structure 6xbz and superimposed with the substrate peptide P 3 T 4 S 5 P 6 S 7 Y 1 S 2 that was modeled on the peptide PKTPKKA from the Cdk2/CycA/substrate complex structure 3qhr . The register of the PxxP binding motif in the substrate sequence aligns to S5 phosphorylation within the CTD and an P 3 xSP 6 xY 1 interaction motif.

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); α-Cdk7 (total), mouse monoclonal, clone 31TF2-1F8, 1:1000, (Invitrogen #MA3-001); α-Cdk7 (total), mouse monoclonal, clone C4, 1:1000 (Santa Cruz #sc-7344); α-phospho Cdk7 (pT170), rabbit polyclonal, 1:1000 (Affinity Biosciences #CPA5749); α-phospho-Cdk7 (pT170), rabbit polyclonal, 1:1000 ; α-phospho-Cdk7 (pS164), rabbit polyclonal, 1:1000 (Invitrogen #PA5-105583); anti-GST, mouse monoclonal, 1:1000 (Thermo Fisher Scientific # 740007M).

Techniques: Binding Assay, Sequencing

Journal: bioRxiv

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

doi: 10.1101/2024.02.14.580246

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. Residues belonging to Cdk7 are written in letters without outlines; those belonging to Cyclin H are outlined in white and Mat1 residues are outlined in magenta. 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); α-Cdk7 (total), mouse monoclonal, clone 31TF2-1F8, 1:1000, (Invitrogen #MA3-001); α-Cdk7 (total), mouse monoclonal, clone C4, 1:1000 (Santa Cruz #sc-7344); α-phospho Cdk7 (pT170), rabbit polyclonal, 1:1000 (Affinity Biosciences #CPA5749); α-phospho-Cdk7 (pT170), rabbit polyclonal, 1:1000 ; α-phospho-Cdk7 (pS164), rabbit polyclonal, 1:1000 (Invitrogen #PA5-105583); anti-GST, mouse monoclonal, 1:1000 (Thermo Fisher Scientific # 740007M).

Techniques:

Journal: bioRxiv

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

doi: 10.1101/2024.02.14.580246

Figure Lengend Snippet: a , SDS-PAGE and immunoblot analysis of GST-Cdk7/Cyclin H/MBP-Mat1 complexes. Protein (3 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 GST-Cdk2. Each Cdk7 complex at 0.1 µM was incubated with 10 µM GST-CTD [52] or 15 µM GST-Cdk2 in the presence of 1 mM ATP containing 0.35 µCi [ 32 P]-γ-ATP for 15 min at 30°C. Bars represent mean ± SD. c , SDS-PAGE analyses of GST-Cdk7/Cyclin H and MBP-Mat1; 2 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] of Cdk7/Cyclin H without Mat1 and upon pre-incubation with MBP-Mat1 230-309 or MBP-Mat1 1-309 for 10 min (left panel). Cdk7/Cyclin H (0.1 µM) was pre-incubated with buffer or 0.4 µM Mat1, and 10 µM GST-CTD [52] . Reaction was started by addition of 1 mM ATP containing 0.35 µCi [ 32 P]-γ-ATP and incubated for 15 min at 30°C. 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). Assay conditions were as described above. Each data point represents an individual measurement monitored at least in duplicate. Bars represent mean ± SEM. e , Molecular interaction network of R295 of Mat1 with neighboring amino acids. f , Radiometric kinase activity assay was performed as in d . 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 separate experiments, each performed in triplicate.

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); α-Cdk7 (total), mouse monoclonal, clone 31TF2-1F8, 1:1000, (Invitrogen #MA3-001); α-Cdk7 (total), mouse monoclonal, clone C4, 1:1000 (Santa Cruz #sc-7344); α-phospho Cdk7 (pT170), rabbit polyclonal, 1:1000 (Affinity Biosciences #CPA5749); α-phospho-Cdk7 (pT170), rabbit polyclonal, 1:1000 ; α-phospho-Cdk7 (pS164), rabbit polyclonal, 1:1000 (Invitrogen #PA5-105583); anti-GST, mouse monoclonal, 1:1000 (Thermo Fisher Scientific # 740007M).

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

Journal: bioRxiv

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

doi: 10.1101/2024.02.14.580246

Figure Lengend Snippet: a , SDS-PAGE analysis of Cdk7/Cyclin H complex and MBP-Mat1 230-309 , 3 µg each. b , Radiometric kinase activity assay. 0.1 µM Cdk7/Cyclin H complex was incubated with 0.4 µM MBP or MBP-Mat1 230-309 and 10 µM GST-CTD [52] for 10 min prior to starting the kinase reaction by addition of 1 mM ATP containing 0.35 µCi [ 32 P]-γ-ATP. Samples were incubated for 15 min at 30°C. c , SDS-PAGE analysis of a kinase assay titrating MBP-Mat1 230-309 . Cdk7/Cyclin H complex (0.1 µM)—wild-type or S164 mutant variants, as indicated—was incubated with increasing concentrations of MBP-Mat1 230-309 . Samples were incubated in the presence of 10 µM GST-CTD [52] for 10 min prior to starting the assay with 1 mM ATP. After 15 min at 30°C, assay was stopped with 2xSDS sample buffer. d , Radiometric kinase assay. As in panel c , but with addition of 0.35 µCi [ 32 P]-γ-ATP. Phosphorylation was measured by liquid scintillation counting. Data represent mean of duplicate measurements. e , SDS-PAGE analysis of the GST-Cdk7/Cyclin H complexes used in , 2 µg each. f , Absolute counts of one experiment of the data shown in , highlighting similar activity of the binary preparations. Data represent mean ± SD.

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); α-Cdk7 (total), mouse monoclonal, clone 31TF2-1F8, 1:1000, (Invitrogen #MA3-001); α-Cdk7 (total), mouse monoclonal, clone C4, 1:1000 (Santa Cruz #sc-7344); α-phospho Cdk7 (pT170), rabbit polyclonal, 1:1000 (Affinity Biosciences #CPA5749); α-phospho-Cdk7 (pT170), rabbit polyclonal, 1:1000 ; α-phospho-Cdk7 (pS164), rabbit polyclonal, 1:1000 (Invitrogen #PA5-105583); anti-GST, mouse monoclonal, 1:1000 (Thermo Fisher Scientific # 740007M).

Techniques: SDS Page, Kinase Assay, Incubation, Mutagenesis, Activity Assay

Journal: bioRxiv

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

doi: 10.1101/2024.02.14.580246

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. 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.

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); α-Cdk7 (total), mouse monoclonal, clone 31TF2-1F8, 1:1000, (Invitrogen #MA3-001); α-Cdk7 (total), mouse monoclonal, clone C4, 1:1000 (Santa Cruz #sc-7344); α-phospho Cdk7 (pT170), rabbit polyclonal, 1:1000 (Affinity Biosciences #CPA5749); α-phospho-Cdk7 (pT170), rabbit polyclonal, 1:1000 ; α-phospho-Cdk7 (pS164), rabbit polyclonal, 1:1000 (Invitrogen #PA5-105583); anti-GST, mouse monoclonal, 1:1000 (Thermo Fisher Scientific # 740007M).

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

Journal: bioRxiv

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

doi: 10.1101/2024.02.14.580246

Figure Lengend Snippet: a , Immunoblot analysis of site-specific CTD kinase activity of binary Cdk7/Cyclin H complex and Cdk7/Cyclin H incubated with MBP-Mat1 230-309 prior to activity assay. b , Immunoblot analysis of Cdk7 substrate site specificity compared to P-TEFb. c , Radiometric activity assay. Peptides composed of three heptad repeats containing different pre-phosphorylations are shown in schematic diagram at right. Pre-phosphorylation was either continuous (pS5/pS7) or within a single repeat (pS5/7-C-terminal or middle or N-terminal); cons.: (consensus sequence with no pre-phosphorylation). Data represent mean ± SD of a single experiment measured in triplicate.

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); α-Cdk7 (total), mouse monoclonal, clone 31TF2-1F8, 1:1000, (Invitrogen #MA3-001); α-Cdk7 (total), mouse monoclonal, clone C4, 1:1000 (Santa Cruz #sc-7344); α-phospho Cdk7 (pT170), rabbit polyclonal, 1:1000 (Affinity Biosciences #CPA5749); α-phospho-Cdk7 (pT170), rabbit polyclonal, 1:1000 ; α-phospho-Cdk7 (pS164), rabbit polyclonal, 1:1000 (Invitrogen #PA5-105583); anti-GST, mouse monoclonal, 1:1000 (Thermo Fisher Scientific # 740007M).

Techniques: Western Blot, Activity Assay, Incubation, Sequencing

Journal: bioRxiv

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

doi: 10.1101/2024.02.14.580246

Figure Lengend Snippet: a , Experimental setup for SPR measurements. b , SPR analysis of Mat1 binding to Cdk7(S164E)/Cyclin H. c , Stabilization matrix, highlighting the melting-temperature changes between different Cdk7 preparations upon phosphorylation or Mat1 addition. The least stable Cdk7/Cyclin H complex tested (binary A_pT) was used as reference.

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); α-Cdk7 (total), mouse monoclonal, clone 31TF2-1F8, 1:1000, (Invitrogen #MA3-001); α-Cdk7 (total), mouse monoclonal, clone C4, 1:1000 (Santa Cruz #sc-7344); α-phospho Cdk7 (pT170), rabbit polyclonal, 1:1000 (Affinity Biosciences #CPA5749); α-phospho-Cdk7 (pT170), rabbit polyclonal, 1:1000 ; α-phospho-Cdk7 (pS164), rabbit polyclonal, 1:1000 (Invitrogen #PA5-105583); anti-GST, mouse monoclonal, 1:1000 (Thermo Fisher Scientific # 740007M).

Techniques: Binding Assay

Journal: bioRxiv

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

doi: 10.1101/2024.02.14.580246

Figure Lengend Snippet: a , b , Multi-cycle kinetics SPR measurement. 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.

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); α-Cdk7 (total), mouse monoclonal, clone 31TF2-1F8, 1:1000, (Invitrogen #MA3-001); α-Cdk7 (total), mouse monoclonal, clone C4, 1:1000 (Santa Cruz #sc-7344); α-phospho Cdk7 (pT170), rabbit polyclonal, 1:1000 (Affinity Biosciences #CPA5749); α-phospho-Cdk7 (pT170), rabbit polyclonal, 1:1000 ; α-phospho-Cdk7 (pS164), rabbit polyclonal, 1:1000 (Invitrogen #PA5-105583); anti-GST, mouse monoclonal, 1:1000 (Thermo Fisher Scientific # 740007M).

Techniques: Protein Concentration, Fluorescence, Nano Differential Scanning Fluorimetry

Journal: bioRxiv

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

doi: 10.1101/2024.02.14.580246

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. b , c , Phos-tag-SDS-PAGE and conventional SDS-PAGE 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 in parallel with phospho-specific antibodies recognizing Cdk7 pT170 ( b ) or Cdk7 pS164 ( c ). d , 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); α-Cdk7 (total), mouse monoclonal, clone 31TF2-1F8, 1:1000, (Invitrogen #MA3-001); α-Cdk7 (total), mouse monoclonal, clone C4, 1:1000 (Santa Cruz #sc-7344); α-phospho Cdk7 (pT170), rabbit polyclonal, 1:1000 (Affinity Biosciences #CPA5749); α-phospho-Cdk7 (pT170), rabbit polyclonal, 1:1000 ; α-phospho-Cdk7 (pS164), rabbit polyclonal, 1:1000 (Invitrogen #PA5-105583); anti-GST, mouse monoclonal, 1:1000 (Thermo Fisher Scientific # 740007M).

Techniques: SDS Page, Western Blot, Transfection

Journal: bioRxiv

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

doi: 10.1101/2024.02.14.580246

Figure Lengend Snippet: Electrostatic surface display of the Cdk7/Cyclin H/Mat1 complex structure determined here (8pyr) fused with residues 10-54, ATPγS and Mg 2+ of Cdk7 from structure 6xbz and superimposed with a substrate peptide at the catalytic site aligned to S5 phosphorylation that was modeled on the peptide PKTPKKAKKL from the Cdk2/CycA/substrate complex structure 3qhr . The initial substrate model was extended at the N-terminus by residues Y 1 S 2 and at the C-terminus by three heptad repeats, each repeat being alternately colored yellow and white. In this model, the fifth repeat was directed towards a basic patch on the second cyclin box of Cyclin H, which is formed by residues R197, R223, K253, R256 and K260.

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); α-Cdk7 (total), mouse monoclonal, clone 31TF2-1F8, 1:1000, (Invitrogen #MA3-001); α-Cdk7 (total), mouse monoclonal, clone C4, 1:1000 (Santa Cruz #sc-7344); α-phospho Cdk7 (pT170), rabbit polyclonal, 1:1000 (Affinity Biosciences #CPA5749); α-phospho-Cdk7 (pT170), rabbit polyclonal, 1:1000 ; α-phospho-Cdk7 (pS164), rabbit polyclonal, 1:1000 (Invitrogen #PA5-105583); anti-GST, mouse monoclonal, 1:1000 (Thermo Fisher Scientific # 740007M).

Techniques:

Journal: Frontiers in Oncology

Article Title: PKM2 Interacts With the Cdk1-CyclinB Complex to Facilitate Cell Cycle Progression in Gliomas

doi: 10.3389/fonc.2022.844861

Figure Lengend Snippet: PKM2 physically interacts with the Cdk1-cyclin B complex. (A) Levels (top) and activity (bottom) of Cdk7 in control and PKM2 knock-down U87, T98G and LN319 cells 10 hrs following release from serum deprivation-induced arrest. (B) Levels of cyclin B1, PKM2, and Cdk1 in total cell lysates in control or PKM2 shRNA U87 cells measured at the times indicated following release from serum deprivation-induced arrest. (C) Levels of Cdk1, cyclin B, or PKM2 from Cdk1, cyclin B or PKM2 immunoprecipitates measured at the times indicated following release of control or PKM2 shRNA U87 cells from serum deprivation-induced arrest. Data in M lanes were derived from cells isolated by mitotic shake-off. I:B = Immuno Blot, IP = immunoprecipitate. Bottom panel: Densitometric analysis of Cdk1, cyclin B, or PKM2 levels in U87 cells from Cdk1, cyclin B or PKM2 immunoprecipitates. (D) Western blot analysis of cyclin B1 in control and PKM2 knock-down U87, T98G and LN319 cells. Densitometric analysis (bottom right panel) of western blot demonstrating levels of cyclin B1 in different experimental groups. *p<.05, n=3.

Article Snippet: For Cdk1-cyclin B activity analysis, cell lysates were diluted and pipetted into recombinant Cdc7 pre-coated wells (MBL International Corp), after which Mg2+ and ATP were added, and the amount of phosphorylated substrate was measured by binding to an anti-phospho-Cdk7 (T 376) antibody and a horseradish peroxidase-conjugated anti-mouse IgG.

Techniques: Activity Assay, Control, Knockdown, shRNA, Derivative Assay, Isolation, Western Blot

Journal: Cell reports

Article Title: Transcriptional control of DNA repair networks by CDK7 regulates sensitivity to radiation in MYC-driven medulloblastoma.

doi: 10.1016/j.celrep.2021.109013

Figure Lengend Snippet: Figure 1. CDK7 expression in MB enhanced in group 3 tumors (A) Graphic of the Crispr-Cas9 druggable kinase screen performed on three group 3 MB cell lines. (B) Principal-component analysis (PCA) plot of before and after puromycin selection. (C) S curve plot. Previously identified genes are in black, and new genes are in red. (D) The fold change expression of CDK7 sgRNA. (E) CDK7 expression in MB by subtype. (F) Patient overall survival in group 3 MB in relation to CDK7 expression. n = 113.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit monoclonal Phospho-Rpb1 CTD (Ser5) Cell Signaling Cat#13523;RRID:AB_2798246 Rabbit monoclonal Phospho-Rpb1 CTD (Ser2) Cell Signaling Cat13499;RRID:AB_2798238 Mouse monoclonal CDK7 Cell Signaling Cat#2916;RRID:AB_2077142 Rabbit monoclonal CDK9 Cell Signaling Cat#2316;RRID:AB_2291505 Rabbit polyclonal PARP/cleaved PARP Cell Signaling Cat#9542;RRID:AB_2160739 Rabbit monoclonal cMYC Cell Signaling Cat#5605;RRID:AB_1903938 Rabbit polyclonal RNA Pol II Bethyl Laboratories Cat#A300-653A; RRID:AB_519334 Anti-Mouse IgG-HRP linked Cell Signaling Cat#7076;RRID:AB_330924 Anti-Rabbit IgG-HRP linked Cell Signaling Cat#7074;RRID:AB_2099233 Mouse monoclonal b-Actin-HRP linked Cell Signaling Cat#12262;RRID:AB_2566811 Rabbit monoclonal Phospho-Histone H2A.X (Ser139) Cell Signaling Cat#9718;RRID:AB_2118009 Rabbit polyclonal RPA70/RPA1 Cell Signaling Cat#2267;RRID:AB_2180506 Rabbit monoclonal Ki67 Thermo Fisher Scientific Cat#RM-9106; RRID:AB_2335745 Mouse monoclonal RAD51 Novus Biologicals Cat#NB100-148; RRID:AB_10002131 Rabbit polyclonal Cleaved Caspase 3 Cell Signaling Cat#9661:RRID:AB_2341188 Rabbit monoclonal RNA Polymerase II CTD abCAM Cat#ab817; RRID:AB_306327 Rabbit monoclonal cMYC Cell signaling Cat#13987;RRID:AB_2631168 Goat Anti-Rabbit IgG (H+L) Polyclonal Antibody, Alexa Fluor 488 Conjugated Thermo Fisher Scientific Cat#A-11008; RRID:AB_143165 Goat Anti-Rabbit IgG (H+L) Polyclonal Antibody, Alexa Fluor 555 Conjugated Thermo Fisher Scientific Cat# A-31572; RRID:AB_162543 Biological samples Normal Cerebellum paraffin blocks Dr. Nicholas Foreman lab N/A Patient embedded tumor paraffin blocks Dr. Nicholas Foreman lab N/A Chemicals, peptides, and recombinant proteins THZ1 Cayman Chemical Cat#9002215 THZ2 MedChem Express Cat#HY-12280/CS-3245 YKL-5-124 Selleckchem Cat#S8863 CT7001 MedChem Express Cat#HY-103712A Nitrotetrazolium blue chloride Sigma Aldrich Cat# N6876-50MG Critical commercial assays Guava Nexin Assay Millipore Cat#4500-0450 CellTiter 96 Aqueous One Solution Cell Proliferation Promega Cat#G3580 RNeasy Mini Kit QIAGEN Cat#74104 DNeasy Blood and Tissue Kit QIAGEN Cat#69504 High Capacity Reverse Transcription Kit Thermo Fisher Scientific Cat#4368814 TaqMan Universal Master Mix II, no UNG Thermo Fisher Scientific Cat#4400040 Venor GeM Mycoplasma Detection Kit Sigma Aldrich Cat#MP0025-1kt Qubit dsDNA High Sensitivity Assay Thermo Fisher Scientific Cat#Q32851 Lipofectamine 3000 Transfection Reagent Thermo Fisher Scientific Cat#L3000008 (Continued on next page) e1 Cell Reports 35, 109013, April 27, 2021

Techniques: Expressing, CRISPR, Selection

Journal: Cell reports

Article Title: Transcriptional control of DNA repair networks by CDK7 regulates sensitivity to radiation in MYC-driven medulloblastoma.

doi: 10.1016/j.celrep.2021.109013

Figure Lengend Snippet: Figure 2. Genetic depletion of CDK7 decreases proliferation and tumor growth (A) Immunoblot for indicated proteins of lysates transduced with three shRNAs against CDK7 or shNull. (B) Quantification of (A). n = 3, mean ± SD. Statistical analysis, two-way ANOVA, ****p < 0.0001; ***p < 0.001; *p < 0.05. (C) Neurosphere assay growth in CDK7-depleted D458 or D425 MB cell lines. Representative images at 10 days are shown. Total average growth of neurospheres was determined by NucRed fluorescence. n = 3, mean ± SD. Scale bar, 400 mm. (D) Methylcellulose assay colony count of CDK7 KD cell lines (D458,D425). n = 3. Scatterplot, mean ± SD. Statistical analysis, two-way ANOVA. ****p < 0.0001; ***p < 0.001. (E) Annexin V (+) staining assay. D458 and D425 KD cells were stained for Annexin V and measured by guava flow cytometry. Box and whisker ± min and max plot of the percentage of Annexin V (+) stained cells (y axis). n = 3. Statistical analysis, two-way ANOVA. ***p < 0.001; **p < 0.005; *p < 0.05.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit monoclonal Phospho-Rpb1 CTD (Ser5) Cell Signaling Cat#13523;RRID:AB_2798246 Rabbit monoclonal Phospho-Rpb1 CTD (Ser2) Cell Signaling Cat13499;RRID:AB_2798238 Mouse monoclonal CDK7 Cell Signaling Cat#2916;RRID:AB_2077142 Rabbit monoclonal CDK9 Cell Signaling Cat#2316;RRID:AB_2291505 Rabbit polyclonal PARP/cleaved PARP Cell Signaling Cat#9542;RRID:AB_2160739 Rabbit monoclonal cMYC Cell Signaling Cat#5605;RRID:AB_1903938 Rabbit polyclonal RNA Pol II Bethyl Laboratories Cat#A300-653A; RRID:AB_519334 Anti-Mouse IgG-HRP linked Cell Signaling Cat#7076;RRID:AB_330924 Anti-Rabbit IgG-HRP linked Cell Signaling Cat#7074;RRID:AB_2099233 Mouse monoclonal b-Actin-HRP linked Cell Signaling Cat#12262;RRID:AB_2566811 Rabbit monoclonal Phospho-Histone H2A.X (Ser139) Cell Signaling Cat#9718;RRID:AB_2118009 Rabbit polyclonal RPA70/RPA1 Cell Signaling Cat#2267;RRID:AB_2180506 Rabbit monoclonal Ki67 Thermo Fisher Scientific Cat#RM-9106; RRID:AB_2335745 Mouse monoclonal RAD51 Novus Biologicals Cat#NB100-148; RRID:AB_10002131 Rabbit polyclonal Cleaved Caspase 3 Cell Signaling Cat#9661:RRID:AB_2341188 Rabbit monoclonal RNA Polymerase II CTD abCAM Cat#ab817; RRID:AB_306327 Rabbit monoclonal cMYC Cell signaling Cat#13987;RRID:AB_2631168 Goat Anti-Rabbit IgG (H+L) Polyclonal Antibody, Alexa Fluor 488 Conjugated Thermo Fisher Scientific Cat#A-11008; RRID:AB_143165 Goat Anti-Rabbit IgG (H+L) Polyclonal Antibody, Alexa Fluor 555 Conjugated Thermo Fisher Scientific Cat# A-31572; RRID:AB_162543 Biological samples Normal Cerebellum paraffin blocks Dr. Nicholas Foreman lab N/A Patient embedded tumor paraffin blocks Dr. Nicholas Foreman lab N/A Chemicals, peptides, and recombinant proteins THZ1 Cayman Chemical Cat#9002215 THZ2 MedChem Express Cat#HY-12280/CS-3245 YKL-5-124 Selleckchem Cat#S8863 CT7001 MedChem Express Cat#HY-103712A Nitrotetrazolium blue chloride Sigma Aldrich Cat# N6876-50MG Critical commercial assays Guava Nexin Assay Millipore Cat#4500-0450 CellTiter 96 Aqueous One Solution Cell Proliferation Promega Cat#G3580 RNeasy Mini Kit QIAGEN Cat#74104 DNeasy Blood and Tissue Kit QIAGEN Cat#69504 High Capacity Reverse Transcription Kit Thermo Fisher Scientific Cat#4368814 TaqMan Universal Master Mix II, no UNG Thermo Fisher Scientific Cat#4400040 Venor GeM Mycoplasma Detection Kit Sigma Aldrich Cat#MP0025-1kt Qubit dsDNA High Sensitivity Assay Thermo Fisher Scientific Cat#Q32851 Lipofectamine 3000 Transfection Reagent Thermo Fisher Scientific Cat#L3000008 (Continued on next page) e1 Cell Reports 35, 109013, April 27, 2021

Techniques: Western Blot, Transduction, Neurosphere Assay, Methylcellulose Assay, Annexin V Staining Assay, Staining, Cytometry, Whisker Assay

Journal: Cell reports

Article Title: Transcriptional control of DNA repair networks by CDK7 regulates sensitivity to radiation in MYC-driven medulloblastoma.

doi: 10.1016/j.celrep.2021.109013

Figure Lengend Snippet: Figure 5. CDK7 inhibition abrogates RNA Pol II and MYC promoter association (A) ChIP sequencing with RNA Pol II and MYC antibody performed on D458 cells treated with DMSO versus 10 nM THZ1. Heatmap of normalized RNA Pol II or MYC at the TSS. (Bottom) average read density of RNA Pol II or MYC ChIP sequencing at the TSS. DMSO (blue) or 10 nM THZ1 (yellow). (B) GO functional categories for RNA Pol II ChIP cluster 1 genes affected by THZ1 treatment. Enrichment scores are shown as -log10(Pval). (C) GO functional categories for MYC ChIP cluster 1 genes affected by THZ1 treatment. Enrichment scores are shown as -log10(Pval). (D) Box-and-whisker plots ± min and max of the total number of reads within 200 bp of the TSS for the top genes within the DNA Repair ontology of cluster 1 for RNA Pol II ChIP and MYC ChIP. MYC ChIP read numbers for top DNA repair genes (right). Statistical analysis, two-tailed, unpaired t test. ****p < 0.0001; **p < 0.01. Color scale indicates high (red) to low (white) reads. (E) Top genes within the mRNA processing ontology of cluster 2 for RNA Pol II and MYC ChIPs. Statistical analysis, two-tailed, unpaired t test. ns, not significant. See also Figure S5.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit monoclonal Phospho-Rpb1 CTD (Ser5) Cell Signaling Cat#13523;RRID:AB_2798246 Rabbit monoclonal Phospho-Rpb1 CTD (Ser2) Cell Signaling Cat13499;RRID:AB_2798238 Mouse monoclonal CDK7 Cell Signaling Cat#2916;RRID:AB_2077142 Rabbit monoclonal CDK9 Cell Signaling Cat#2316;RRID:AB_2291505 Rabbit polyclonal PARP/cleaved PARP Cell Signaling Cat#9542;RRID:AB_2160739 Rabbit monoclonal cMYC Cell Signaling Cat#5605;RRID:AB_1903938 Rabbit polyclonal RNA Pol II Bethyl Laboratories Cat#A300-653A; RRID:AB_519334 Anti-Mouse IgG-HRP linked Cell Signaling Cat#7076;RRID:AB_330924 Anti-Rabbit IgG-HRP linked Cell Signaling Cat#7074;RRID:AB_2099233 Mouse monoclonal b-Actin-HRP linked Cell Signaling Cat#12262;RRID:AB_2566811 Rabbit monoclonal Phospho-Histone H2A.X (Ser139) Cell Signaling Cat#9718;RRID:AB_2118009 Rabbit polyclonal RPA70/RPA1 Cell Signaling Cat#2267;RRID:AB_2180506 Rabbit monoclonal Ki67 Thermo Fisher Scientific Cat#RM-9106; RRID:AB_2335745 Mouse monoclonal RAD51 Novus Biologicals Cat#NB100-148; RRID:AB_10002131 Rabbit polyclonal Cleaved Caspase 3 Cell Signaling Cat#9661:RRID:AB_2341188 Rabbit monoclonal RNA Polymerase II CTD abCAM Cat#ab817; RRID:AB_306327 Rabbit monoclonal cMYC Cell signaling Cat#13987;RRID:AB_2631168 Goat Anti-Rabbit IgG (H+L) Polyclonal Antibody, Alexa Fluor 488 Conjugated Thermo Fisher Scientific Cat#A-11008; RRID:AB_143165 Goat Anti-Rabbit IgG (H+L) Polyclonal Antibody, Alexa Fluor 555 Conjugated Thermo Fisher Scientific Cat# A-31572; RRID:AB_162543 Biological samples Normal Cerebellum paraffin blocks Dr. Nicholas Foreman lab N/A Patient embedded tumor paraffin blocks Dr. Nicholas Foreman lab N/A Chemicals, peptides, and recombinant proteins THZ1 Cayman Chemical Cat#9002215 THZ2 MedChem Express Cat#HY-12280/CS-3245 YKL-5-124 Selleckchem Cat#S8863 CT7001 MedChem Express Cat#HY-103712A Nitrotetrazolium blue chloride Sigma Aldrich Cat# N6876-50MG Critical commercial assays Guava Nexin Assay Millipore Cat#4500-0450 CellTiter 96 Aqueous One Solution Cell Proliferation Promega Cat#G3580 RNeasy Mini Kit QIAGEN Cat#74104 DNeasy Blood and Tissue Kit QIAGEN Cat#69504 High Capacity Reverse Transcription Kit Thermo Fisher Scientific Cat#4368814 TaqMan Universal Master Mix II, no UNG Thermo Fisher Scientific Cat#4400040 Venor GeM Mycoplasma Detection Kit Sigma Aldrich Cat#MP0025-1kt Qubit dsDNA High Sensitivity Assay Thermo Fisher Scientific Cat#Q32851 Lipofectamine 3000 Transfection Reagent Thermo Fisher Scientific Cat#L3000008 (Continued on next page) e1 Cell Reports 35, 109013, April 27, 2021

Techniques: Inhibition, ChIP-sequencing, Functional Assay, Whisker Assay, Two Tailed Test

Journal: Cell reports

Article Title: Transcriptional control of DNA repair networks by CDK7 regulates sensitivity to radiation in MYC-driven medulloblastoma.

doi: 10.1016/j.celrep.2021.109013

Figure Lengend Snippet: Figure 6. CDK7 inhibition minimizes DNA damage response enhancing susceptibility to IR (A) GSEA from THZ1 D458 treatment RNA-seq. Kauffman DNA repair, KEGG homologous recombination. FDR q value = 0.0. (B) Volcano plot of Kauffman DNA repair (green), KEGG homologous recombination (yellow), and MYC targets (blue) from THZ1-treated D458 cells. (C) Individual ChIP gene tracks of RNA Pol II, MYC signals, and RNA transcript profile for DMSO and THZ1 10-nM D458 treatments. Shown are the y axis signal density (rpm/bp) of BRCA2 promoter and RAD51C promoter.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit monoclonal Phospho-Rpb1 CTD (Ser5) Cell Signaling Cat#13523;RRID:AB_2798246 Rabbit monoclonal Phospho-Rpb1 CTD (Ser2) Cell Signaling Cat13499;RRID:AB_2798238 Mouse monoclonal CDK7 Cell Signaling Cat#2916;RRID:AB_2077142 Rabbit monoclonal CDK9 Cell Signaling Cat#2316;RRID:AB_2291505 Rabbit polyclonal PARP/cleaved PARP Cell Signaling Cat#9542;RRID:AB_2160739 Rabbit monoclonal cMYC Cell Signaling Cat#5605;RRID:AB_1903938 Rabbit polyclonal RNA Pol II Bethyl Laboratories Cat#A300-653A; RRID:AB_519334 Anti-Mouse IgG-HRP linked Cell Signaling Cat#7076;RRID:AB_330924 Anti-Rabbit IgG-HRP linked Cell Signaling Cat#7074;RRID:AB_2099233 Mouse monoclonal b-Actin-HRP linked Cell Signaling Cat#12262;RRID:AB_2566811 Rabbit monoclonal Phospho-Histone H2A.X (Ser139) Cell Signaling Cat#9718;RRID:AB_2118009 Rabbit polyclonal RPA70/RPA1 Cell Signaling Cat#2267;RRID:AB_2180506 Rabbit monoclonal Ki67 Thermo Fisher Scientific Cat#RM-9106; RRID:AB_2335745 Mouse monoclonal RAD51 Novus Biologicals Cat#NB100-148; RRID:AB_10002131 Rabbit polyclonal Cleaved Caspase 3 Cell Signaling Cat#9661:RRID:AB_2341188 Rabbit monoclonal RNA Polymerase II CTD abCAM Cat#ab817; RRID:AB_306327 Rabbit monoclonal cMYC Cell signaling Cat#13987;RRID:AB_2631168 Goat Anti-Rabbit IgG (H+L) Polyclonal Antibody, Alexa Fluor 488 Conjugated Thermo Fisher Scientific Cat#A-11008; RRID:AB_143165 Goat Anti-Rabbit IgG (H+L) Polyclonal Antibody, Alexa Fluor 555 Conjugated Thermo Fisher Scientific Cat# A-31572; RRID:AB_162543 Biological samples Normal Cerebellum paraffin blocks Dr. Nicholas Foreman lab N/A Patient embedded tumor paraffin blocks Dr. Nicholas Foreman lab N/A Chemicals, peptides, and recombinant proteins THZ1 Cayman Chemical Cat#9002215 THZ2 MedChem Express Cat#HY-12280/CS-3245 YKL-5-124 Selleckchem Cat#S8863 CT7001 MedChem Express Cat#HY-103712A Nitrotetrazolium blue chloride Sigma Aldrich Cat# N6876-50MG Critical commercial assays Guava Nexin Assay Millipore Cat#4500-0450 CellTiter 96 Aqueous One Solution Cell Proliferation Promega Cat#G3580 RNeasy Mini Kit QIAGEN Cat#74104 DNeasy Blood and Tissue Kit QIAGEN Cat#69504 High Capacity Reverse Transcription Kit Thermo Fisher Scientific Cat#4368814 TaqMan Universal Master Mix II, no UNG Thermo Fisher Scientific Cat#4400040 Venor GeM Mycoplasma Detection Kit Sigma Aldrich Cat#MP0025-1kt Qubit dsDNA High Sensitivity Assay Thermo Fisher Scientific Cat#Q32851 Lipofectamine 3000 Transfection Reagent Thermo Fisher Scientific Cat#L3000008 (Continued on next page) e1 Cell Reports 35, 109013, April 27, 2021

Techniques: Inhibition, RNA Sequencing, Homologous Recombination

Journal: Cell reports

Article Title: Transcriptional control of DNA repair networks by CDK7 regulates sensitivity to radiation in MYC-driven medulloblastoma.

doi: 10.1016/j.celrep.2021.109013

Figure Lengend Snippet: Figure 7. In vivo IR with CDK7 inhibition (A) Representative bioluminescence images of xenograft D458 vehicle or THZ2 15 mg/kg treated with 1.5 Gy over 5 days starting at day 15 post-injection. Color scales indicate bioluminescence radiance in photons/sec/cm2/steradian. (B) Representative MRI of vehicle + IR and THZ2 + IR treated D458 xenograft mice at 23 and 49 days. White arrows indicate tumor. (C) Box-and-whisker plot ± min and max of mean total radiance in photons/sec/cm2/steradian. Statistical analysis, two-way ANOVA, ****p < 0.0001. (D) Kaplan-Meier survival curve of D458 xenograft mice treated with vehicle (n = 9) or THZ2 (n = 10) and 1.5 Gy for 5 days (violet box). THZ2 treatment period for 25 days, shaded box. Statistical analysis, log-rank (Mantel-Cox) test, *p < 0.05. (E) MYC gene amplification in group 3 MB drives highly active transcription of DNA repair, proliferation, and self-renewal genes shielding cells against IR and sustaining growth. Treatment with THZ1/THZ2 blocks RNA Pol II association at the MYC promoter, limiting MYC transcription, and blocks RNA Pol II and MYC association with select gene promoters, removing MB tumor cells’ defense mechanisms and leading to cell death. Image created with BioRender.com.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit monoclonal Phospho-Rpb1 CTD (Ser5) Cell Signaling Cat#13523;RRID:AB_2798246 Rabbit monoclonal Phospho-Rpb1 CTD (Ser2) Cell Signaling Cat13499;RRID:AB_2798238 Mouse monoclonal CDK7 Cell Signaling Cat#2916;RRID:AB_2077142 Rabbit monoclonal CDK9 Cell Signaling Cat#2316;RRID:AB_2291505 Rabbit polyclonal PARP/cleaved PARP Cell Signaling Cat#9542;RRID:AB_2160739 Rabbit monoclonal cMYC Cell Signaling Cat#5605;RRID:AB_1903938 Rabbit polyclonal RNA Pol II Bethyl Laboratories Cat#A300-653A; RRID:AB_519334 Anti-Mouse IgG-HRP linked Cell Signaling Cat#7076;RRID:AB_330924 Anti-Rabbit IgG-HRP linked Cell Signaling Cat#7074;RRID:AB_2099233 Mouse monoclonal b-Actin-HRP linked Cell Signaling Cat#12262;RRID:AB_2566811 Rabbit monoclonal Phospho-Histone H2A.X (Ser139) Cell Signaling Cat#9718;RRID:AB_2118009 Rabbit polyclonal RPA70/RPA1 Cell Signaling Cat#2267;RRID:AB_2180506 Rabbit monoclonal Ki67 Thermo Fisher Scientific Cat#RM-9106; RRID:AB_2335745 Mouse monoclonal RAD51 Novus Biologicals Cat#NB100-148; RRID:AB_10002131 Rabbit polyclonal Cleaved Caspase 3 Cell Signaling Cat#9661:RRID:AB_2341188 Rabbit monoclonal RNA Polymerase II CTD abCAM Cat#ab817; RRID:AB_306327 Rabbit monoclonal cMYC Cell signaling Cat#13987;RRID:AB_2631168 Goat Anti-Rabbit IgG (H+L) Polyclonal Antibody, Alexa Fluor 488 Conjugated Thermo Fisher Scientific Cat#A-11008; RRID:AB_143165 Goat Anti-Rabbit IgG (H+L) Polyclonal Antibody, Alexa Fluor 555 Conjugated Thermo Fisher Scientific Cat# A-31572; RRID:AB_162543 Biological samples Normal Cerebellum paraffin blocks Dr. Nicholas Foreman lab N/A Patient embedded tumor paraffin blocks Dr. Nicholas Foreman lab N/A Chemicals, peptides, and recombinant proteins THZ1 Cayman Chemical Cat#9002215 THZ2 MedChem Express Cat#HY-12280/CS-3245 YKL-5-124 Selleckchem Cat#S8863 CT7001 MedChem Express Cat#HY-103712A Nitrotetrazolium blue chloride Sigma Aldrich Cat# N6876-50MG Critical commercial assays Guava Nexin Assay Millipore Cat#4500-0450 CellTiter 96 Aqueous One Solution Cell Proliferation Promega Cat#G3580 RNeasy Mini Kit QIAGEN Cat#74104 DNeasy Blood and Tissue Kit QIAGEN Cat#69504 High Capacity Reverse Transcription Kit Thermo Fisher Scientific Cat#4368814 TaqMan Universal Master Mix II, no UNG Thermo Fisher Scientific Cat#4400040 Venor GeM Mycoplasma Detection Kit Sigma Aldrich Cat#MP0025-1kt Qubit dsDNA High Sensitivity Assay Thermo Fisher Scientific Cat#Q32851 Lipofectamine 3000 Transfection Reagent Thermo Fisher Scientific Cat#L3000008 (Continued on next page) e1 Cell Reports 35, 109013, April 27, 2021

Techniques: In Vivo, Inhibition, Injection, Whisker Assay