D biotin

Restriction enzymes and Phusion HF DNA polymerase (used for standard PCR reactions according to the manual) were ordered from Thermo Fisher Scientific (Vienna, Austria) and single stranded DNA oligonucleotides were purchased from Integrated DNA Technologies (Leuven Belgium). Zeocin™ was obtained from InvivoGen (Toulouse, France), D(+)-biotin from Sigma-Aldrich (Vienna, Austria), Difco yeast nitrogen base w/o amino acids from Becton Dickinson (Schwechat, Austria) and Bacto yeast extract was obtained from (Thermo Fisher Scientific, Vienna, Austria). All other chemicals were purchased from Carl Roth (Karlsruhe, Germany).

Enzymes were obtained from Thermo Fisher Scientific, Vienna, Austria. D(+)‐biotin was obtained from Sigma‐Aldrich, Vienna, Austria. Difco yeast nitrogen base w/o amino acids, Bacto tryptone and Bacto yeast extract were obtained from Becton Dickinson, Schwechat, Austria. Zeocin was obtained from Thermo Fisher Scientific. Other chemicals were purchased from Carl Roth, Karlsruhe, Germany. Oligonucleotides were ordered from Integrated DNA Technologies, Leuven Belgium, see supplementary Table S1 for the sequences.

All cell lines were obtained from ATCC. PC3 and DU145 cells were cultured in F-12K medium and Eagle’s minimum essential medium supplemented with fetal bovine serum (FBS) to a final concentration of 10%, respectively. C4-2B cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (4:1) supplemented with heat-inactivated FBS (to 10%) and T-medium supplement (to 1×). To prepare 10×T-medium supplement in 100 ml of 0.1% BSA (Sigma cat# A8022) in PBS, the following components were added: 50 mg insulin (Gibco cat# 12585-014), 136 ng triiodo-l-thyronine (Sigma cat# T2877), 50 mg transferrin (Sigma cat# T4382), 2.5 mg d-Biotin (Sigma cat# 47868), and 250 mg adenine (Sigma cat# A3159). RWPE-1 and PZ-HPV-7 cells were maintained in keratinocyte serum free medium (K-SFM, Gibco 17005-042), supplemented with 0.05 mg/ml of bovine pituitary extract and 5 ng/ml of human recombinant epidermal growth factor. HPrEC cells were maintained in prostate epithelial cell basal medium (ATCC, PCS-440-030) supplemented with prostate epithelial cell growth kit (ATCC, PCS-440-040). HeLa cells were cultured in DMEM supplemented with 5% FBS.

All Trim28 constructs were expressed in LOBSTR (DE3) cells³⁸ grown in LB media supplemented with 100 μM ZnCl. Expression was induced by the addition of 1 mM isopropyl β‐_D‐1‐thiogalactopyranoside (IPTG) to log phase cultures prior to subsequent growth overnight at 18°C. Cells were lysed by cell disruption in 20 mM Tris–HCl (pH 8.0), 200 mM NaCl, 0.5 mM Tris(2‐carboxyethyl)phosphine (TCEP), 10% (v/v) glycerol, 0.1% (v/v) Triton X‐100. The MBP‐His‐tagged Trim28 constructs were purified using amylose affinity chromatography. The N‐terminal purification tags were removed by cleavage with 3C‐protease prior to further purification by size exclusion chromatography (SEC). The AviTagged ZFP932 KRAB domain was co‐expressed with pBirA in E. coli BL21 (DE3) LOBSTR grown in LB at 37°C to approximately 0.5 OD₆₀₀. Cells were then induced with 1 mM IPTG and supplemented with 20 μM D‐biotin (Sigma) and grown overnight at 18°C. The MBP‐His‐tagged ZFP932_KRAB (5–76) was then purified as described above.

HeLa cell lines carrying tetracycline-inducible HA-Strep-tagged NUP58 and HA-Strep-tagged GFP were generated using a HeLa Flp-In T-REx cell line (Häfner et al., 2014 (link)). Expression of HASt-NUP58 and HASt-GFP were induced by addition of 0.1 μg/ml tetracycline (Sigma). Correct expression and localization of affinity-tagged Nup58 was validated by immunofluorescence and Western blotting (see below). For affinity purifications (AP), bait expression was induced for 48 hrs before synchronizing the cells in interphase or prometaphase as described above. After washing and pelleting, cells were snap-frozen in liquid nitrogen. Thawed cells were lysed by resuspension in lysis buffer containing 25 mM Tris-HCl pH 7.6, 125 mM NaCl, 2 mM MgCl2, 1 mM DTT, 0.5% NP-40, protease and phosphatase inhibitors and sonicated. After lysate clearance by centrifugation (15,000 rpm, 30 min, 4°C), HA-Strep-Nup58 and HA-Strep-GFP were purified by affinity chromatography with StrepTactin Sepharose (IBA) for 45 min at 4°C. Beads were washed three times with lysis buffer and one time with lysis buffer without NP40 and protease inhibitors. Bound protein was eluted with elution buffer containing 25 mM Tris-HCl pH 7.6, 125 mM NaCl, 2 mM MgCl2, 1 mM DTT and 2.5 mM D-biotin (Sigma). Elutions and input samples were further analyzed using SDS-PAGE followed by Western blotting.

Cloning of MICAL-L1 into pEGFP-C1 and mCherry vectors were previously described (Abou-Zeid et al., 2011 (link)). All the RUSH plasmids used in this study, use streptavidin-KDEL as a hook. Briefly, the hook (streptavidin-tagged protein) allows retention of the SBP-tagged cargo in the ER in the absence of biotin thanks to streptavidin–SBP interaction (Boncompain et al., 2012 (link)). The release of the RUSH cargoes was induced by addition of 40 µM of D-biotin (Sigma-Aldrich).