Engineering of the viral genome
DH10B™ cells (ThermoFisher scientific) containing the Kanamycin and Chloramphenicol resistant AcMNPV bacmid BAC10:KO16297 were transformed with the plasmid pSC101-BAD-gbaA28. This Tetracycline resistant plasmid harbors mCherry encoding gene under the control of the pBAD promoter which is tightly regulated by l-arabinose and the temperature-sensitive pSC101 replicon. A single colony was used to inoculate LB media complemented with Chloramphenicol and Tetracycline. After overnight growth at 30 °C, 1:100 dilution of the culture was transferred to 100 mL of medium and once OD600 reached 0.3, l-arabinose (A3256, Sigma) was added to 0.5% w/v. After 60 min, the culture was pelleted at 4 °C, washed three times with ice-cold 10% glycerol, re-suspended in the minimal volume of ice-cold 10% glycerol, frozen in liquid nitrogen and stored at − 80 °C.
For the recombineering experiments (Suppl. Fig. 1a), a donor PCR product containing the ampicillin resistance gene and an expression cassette for a fluorescent reporter gene flanked by chiA and v-cath homology regions was amplified from the pKI shuttle vector (Suppl. Fig. 1b), by PCR using primers chia-FW and v-cath-RV. Amplification was performed with Phusion™ High-Fidelity DNA polymerase (F503, ThermoFisher scientific) with an initial denaturation step at 95 °C for 5 min, followed by 30 cycles of 95 °C for 45 s, 60 °C for 60 s and 72 °C for 2 min and a final extension step at 72 °C for 10 min. The PCR product was treated with DpnI (10 u for a 50 µL reaction), purified using a PCR-Clean Up kit and its concentration adjusted to 100 ng/µL.
For each DNA transformation, 200 ng of PCR products were added to a 50-µL aliquot of competent cells, gently mixed and then transferred to a 0.1-cm cuvette for electroporation using a BTX ECM630 electroporator (1350 V, 600 Ohms, 25 µF). After pulsing, 500 µL of LB without antibiotic was added to the cells and the suspension was incubated 90 min at 30 °C, of which 100 µL were plated onto an LB agar plate containing Chloramphenicol and Ampicillin and incubated at 37 °C. Colonies were further analyzed by PCR using the primer pairs Chia_control_FW/Amp_FW and v-cath_control_RV/mCherry_RV (Suppl. Table 3).
AcMNPV bacmids were purified using a Nucleobond PC500 DNA purification kit (740574 Macherey–Nagel) as described earlier29. Eluted DNA was precipitated with 0.7 volume of isopropanol, re-suspended at a concentration of 0.1 µg/µL and digested with Bsu36I enzyme (NEB) (Suppl. Fig. 1c). After enzyme was inactivated by heating at 80 °C during 20 min, aliquots were stored at − 80 °C.
Design of modified transfer vectors
Plasmids were propagated in DH5α cells with the exception of pSPL/pUCDM derivatives, which were propagated in PIR1 bacteria. All modifications of vectors were validated by sequencing. Plasmids pAC8_GWs and pAC8_MF (Fig. 2b) were obtained from the corresponding pAC8 vectors15:
pAC8_GWs
A LoxP sequence was inserted in the unique EcoRV site of pAC8 plasmids using the set of complementary oligonucleotides LoxP_FW and LoxP_RV by restriction/ligation. Resulting plasmids were adapted to Gateway cloning by inserting the RfA conversion cassette (11828029, ThermoFisher scientific) into the PmeI site of the NdeI-BamHI linker. cDNAs inserted into Gateway Entry vectors can be transferred into the pAC8_GWs destination vectors using LR reactions. Note that the AAA-AAA triplet in the AttL1 site of the entry vector should be in phase with the reading frame of the target gene.
pMF-dual
A DNA fragment containing a LoxP site and dual expression cassette was amplified from pSPL with the primer pair pMF-LoxP-F/pMF-Rev and inserted between the restriction sites EcoRV and PacI of the pBacPak8 plasmid (Clontech) by restriction-ligation. The different pAC8_MF derivatives (Fig. 2b) harboring N- or C-terminal tags (10His, 6His, Flag, HA, c-myc, Twin Strep, see Suppl. Table 2) were obtained by annealing of complementary oligonucleotides or by PCR and inserted into the XhoI, NheI (p10 Nter, p10 Cter respectively) or BamHI, XbaI (PH Nter, pH Cter respectively) restriction sites, by leaving the structure of the pAC8_MF Multiple Cloning Site unchanged. cDNAs encoding the GOIs are inserted into the dual expression cassettes of pAC8_MF using the XhoI/NheI restriction sites for the gene under the control of p10 promoter and the BamHI/XbaI restriction sites for the gene under the control of pH promoter. Cloning is performed as a single step reaction based on the Sequence and Ligation Independent Cloning (SLIC) or other assembly techniques (In-Fusion, Gibson Assembly, NEBuilder). Amplified cDNAs are assembled with the promoters and the backbone regions of the plasmid isolated by restriction (XhoI/BamHI for the 5′ and XbaI/NheI for the 3′ ends of the cDNA) or amplified with primers Prom-Bam-F, Prom-Xho-R, Backbone-F, Backbone-R25.
pKI_Ø, pKI_mCherry and pKI_EGFP
The pKI shuttle vectors contain the integration cassette used to replace the chiA and v-cath genes in the baculovirus genome (Suppl. Fig. 1b). The cassette is composed of the homology arms HomA and HomB from the chiaA and v-cath genes which flank an ampicilin resistance gene and, in the case of pKI_mCherry and pKI_EGFP, the mCherry and EGFP expression units. The plasmid pKI_φ was obtained by introducing an AvrII site into the plasmid pKIloxP13 using PCR with the oligonucleotides pKI-FW and pKI-RV. The unique PmeI and AvrII restriction sites from pKI_ φ were used to insert the fluorescent reporter cassette obtained by digestion of pSPL_p10/mCherry and pSPL_p10/EGFP with the SpeI and PmeI restriction enzymes.
Generation of recombinant baculoviruses and protein production
Preparation of recombinant baculoviruses and protein production was performed using established protocols15,20,29. In brief, 750 ng of pAC8 plasmid preparation and 250 ng linearized AcMNPV bacmid were co-transfected using 3.0 µL of Lipofectamine™ 2000 (ThermoFisher Scientific) in 35 mm dishes or individual wells of 6-well plates (9.6 cm2) seeded with 0.75 × 106 Sf9 cells grown in Grace Insect medium supplemented with 10% FCS. As Lipofectamine transfection is inhibited by serum, we carefully adhered to the manufacturer’s instructions and performed transfection in serum free medium. After 4 h incubation, the transfection mixture was replaced by serum-supplemented medium and cells were incubated for 5–7 days at 27 °C. The supernatant constitutes the initial virus stock (V0). Viruses were amplified by adding 50 µL of V0 to 50 mL suspension cultures of Sf9 cells (1 × 106 cells/mL) that were incubated for 5–7 days. Protein expression was evaluated and optimised in 6 well plates or 10 mL suspension culture prior to scale-up. For production at larger scale, suspension cultures of Sf9 or Sf21 cells (1 × 106 cells/mL) cultivated in Sf900-II were infected with the appropriate volume of virus (V1) and collected 2–3 days post-infection. Cells were washed in PBS containing 10% glycerol and stored at − 80 °C.
Baculovirus isolation and PCR analysis
Plaque purification
Serial dilutions of the V0 virus stock obtained from the co-transfection of the pAC8_MF_CAK transfer plasmid and the BAC10:KO1629, ∆v-cath/chiA, mCherry viral DNA (101 to 106) were seeded on plates containing 2.7 × 106 Sf9 cells and incubated 1 h at 27 °C. The inoculum was removed, and infected cells were covered by medium containing 10% FCS, 40% LB medium and 50% agarose. After 5–6 days incubation at 27 °C, 24 virus-forming plaques were identified using mCherry fluorescence microscope and plugs of the overlay from the selected plaques were transferred in 1 mL of medium. Following overnight incubation at 27 °C, 60 µL of each isolate were amplified in 6-well plate seeded with 1 × 106 Sf9 cells, leading to clonal virus stocks.
PCR analysis
Baculoviral DNA were extracted by using the Nucleospin® Virus kit (740983, Marcherey-Nagel). For PCR amplification, 250 ng of viral DNA were added to a 20 µL mix containing 0.02 U/µL Phusion™ High-Fidelity DNA Polymerase (F503, ThermoFisher Scientific), 200 µM dNTP (R0192, ThermoFisher Scientific), 1X GC buffer, 3% DMSO and 0.5 µM of the primer pairs: orf 1629 F-CDK7 R (PCR1), CCNH F-CCNH-R (PCR2) or MAT1 F-Lef2, orf603 R (PCR3) (Fig. 4a and Suppl. Table 2). PCR products obtained from 4 of the tested cloned were sequenced to verify that the expected fragment was amplified.
Production and purification of P-TEFb and XPG.
Expression of human P-TEFb
CDK9 fused to an N-terminal TwinStrep tag was cloned under the control of the PH promoter while N-terminal Flag-cyclin T1 was inserted under the control of p10. The CDK9/ cyclin T1 expression cassette assembled in the pKL vector from the Multibac suite19 was transferred into the pAC8_MF backbone using the unique PmeI and AvrII restriction sites yielding pAC8_MF-CDK9/ cyclin T1. Viruses for P-TEFb expression were generated by co-transfection of the pAC8_MF-CDK9/ cyclin T1 transfer vector with the AcMNPV BAC10:KO1629, Δv-cath/chiA, mCherry bacmid. The virus for P-TEFb expression obtained by Tn7-mediated transposition was previously described21.
Expression of XPG
The cDNA encoding the Komagataella phaffii (Pichia pastoris) XPG homologue (C4R890_KOMPG) was cloned under the control of the PH promoter of a pAC8_MF-pH-TwinStrep-Nter transfer vector already containing the mCherry cDNA yielding the pAC8_MF TwinStrep-XPG/mCherry plasmid. Viruses for XPG expression were generated by co-transfection of the transfer vector with the AcMNPV BAC10:KO1629, Δv-cath/chiA bacmid.
Purification of pTEFb and XPG
Cell pellets from infected suspension cultures (1 mL for 25 × 106 cells) were disrupted by sonication in buffer A (20 mM Tris/HCl pH8, 250 mM NaCl, 0.1% NP40, 1 mM DTT and EDTA free protease inhibitor cocktail (Roche)) and the clarified lysate subjected to Strep-Tactin®XT affinity chromatography (IBA-Lifesciences). Proteins were eluted in the same buffer supplemented with 10 mM desthiobiotin.
Production and purification of nuclear hormone receptor complexes
Expression of PPAR/RXR
The cDNA was transferred from the gateway entry vector pDONR207 into pAC8-HGWs using an LR reaction performed with the Gateway™ LR Clonase™ II enzyme mix (11791020, Invitrogen) yielding pAC8-HGWs-PPAR. The latter was fused to pSPL-RXR, a pSPL derivative14 comprising the gene encoding for RXR under the control of the PH promoter. For in vitro Cre/LoxP reactions 0.3 µg of each vector are mixed with 1 µL of Cre recombinase buffer 10× (500 mM Tris–HCl, 330 mM NaCl, 100 mM MgCl2, pH 7.5 @ 25 °C) and 1 µL of Cre recombinase enzyme prepared as described in. Reaction is performed at 37 °C during 15 min in 10 µL final volume. Reaction is stopped by heating the reaction mixture during 15 min at 70 °C, which is transformed in DH5α. For expression of PPAR and of the PPAR/RXR heterodimer, viruses were generated by co-transfection of the transfer vectors with the AcMNPV BAC10:KO1629, Δv-cath/chiA bacmid.
Purification of PPAR/RXR
Cells re-suspended in buffer B (50 mM Hepes pH7.5, 500 mM NaCl, 2 mM Chaps, 5% glycerol, 5 mM imidazole, and EDTA free protease inhibitor cocktail (Roche™)), were lysed by sonication (Vibracell sonicator, 3 mm probe at 20% intensity for 30 s (2 s ON/2 s OFF) and the clarified extract (25,000g for 30 min) was subjected to Ni-affinity chromatography using an HisTrap™ FF column (Cytiva). In the case of the His-tagged PPAR/RXR heterodimer, after addition of 2 molar equivalent of rosiglitazone and 9-cis-retionic acid (PPAR and RXR ligands required to stabilize the heterodimer), complex containing fractions were injected on Superdex™ 200 Increase 10/300 size exclusion column (Cytiva) equilibrated in buffer C (50 mM Hepes pH 7.5, 75 mM NaCl, 75 mM KCl, 2 mM Chaps, 5% glycerol and 2 mM TCEP).
Expression and purification RAR/RXR
The RAR and RXR cDNAs were inserted under the control of the PH and p10 promoters of pAC8_MF-pH-TwinStrep-Nter and the corresponding virus was generated by co-transfection with the AcMNPV BAC10:KO1629, Δv-cath/chiA bacmid. The complex was produced in Sf9 cells and, after addition of 2 molar equivalent of 9-cis-retionic acid (RAR and RXR ligand required to stabilize the heterodimer) the corresponding clarified extract was subjected to Strep-Tactin®XT affinity chromatography (IBA-Lifesciences). Proteins were eluted in the same buffer supplemented with 10 mM desthiobiotin.
Production and purification of CAK and CAK/XPD
Expression of CAK and CAK/XPD
The genes coding for the CDK7 kinase, cyclin H, and MAT1 were cloned in pAC8_MF. The organization of the promoter cassette in pAC8_MF is identical to that of the pAcAB3 triple expression vector30 with ORF coding for CDK7 cloned under the control of p10 promoter, and ORFs coding for cyclin H and MAT1 proteins cloned under the control of PH promoters but in opposite orientation. The expression levels of cyclin H and MAT1 being higher than that of CDK7 (Suppl. Fig. 5b), the kinase was cloned with a C-terminal strep tag to ensure stochiometric amounts of each subunit after purification. The cDNA encoding full length XPD fused to an N-terminal Flag sequence was inserted under the control of the PH promoter in the pSPL_p10/DsRed donor vector and the resulting vector fused to the pCA8_MF-CAK acceptor plasmid using in vitro Cre-mediated recombination as described above. Viruses were generated by co-transfection of the transfer vectors with the AcMNPV BAC10:KO1629, Δv-cath/chiA bacmids.
Purification of CAK and CAK/XPD
For purification of CAK, a cell pellet from an infected suspension culture is disrupted by sonication in buffer A (20 mM Tris/HCl pH8, 250 mM NaCl, 0.1% NP40, 1 mM DTT and EDTA free protease inhibitor cocktail (Roche)) and the clarified lysate subjected to Strep-Tactin®XT affinity chromatography (IBA-Lifesciences). Proteins were eluted in the same buffer supplemented with 10 mM desthiobiotin. For purification of the CAK/XPD complex cells were disrupted by sonication in buffer A, incubated with Anti Flag®M2 affinity gel (Sigma-Aldrich) using 1 mL of resin for 1 L of culture for 2 h at 4 °C with gentle shaking. After extensive washes in buffer D (20 mM Tris–HCl pH 7.5, 250 mM NaCl), bound proteins were eluted with the same buffer containing 1 mg/mL Flag peptide (sequence: DYKDDDDK) and further subjected to Strep-Tactin®XT affinity chromatography (IBA-Lifesciences).
