Ruler elements in chromatin remodelers set nucleosome array spacing and phasing

Elisa Oberbeckmann; Vanessa Niebauer; Shinya Watanabe; Lucas Farnung; Manuela Moldt; Andrea Schmid; Patrick Cramer; Craig L. Peterson; Sebastian Eustermann; Karl-Peter Hopfner; Philipp Korber

The copyright holder for this preprint (which was not peer-reviewed) is the . https: //doi.org/10.1101 //doi.org/10. /2020 terminal deletion mutant (Ino80 ΔN , deletion of the first 461 amino acids of the N terminus of Ino80) and two INO80 (2xFlag) Nhp10 module mutants (ΔNhp10 (INO80 complex without Ies1, Ies3, Ies5 and Nhp10 but with Ino80 N-terminus) and HMGII ( Figure 4G ) were generated and integrated into baculoviruses using MultiBac Technology. Expression and purification of mutant INO80 complexes was carried out as described above. The INO80 core complex from Chaetomium thermophilum (equivalent to the S. cerevisiae N-terminal deletion mutant) was essentially purified as described in Expression and purification of full-length Chd1 and FACT. Hi5 cells (600 mL) were grown in ESF-921 media (Expression Systems) and infected with V1 virus for full-length Chd1 (tagged with a N-terminal 6×His tag, followed by a MBP tag, and a tobacco etch virus protease cleavage site) or FACT (Spt16 carries an N-terminal 6×His tag, followed by an MBP tag, and a tobacco etch virus protease cleavage site) for protein expression. Cells were grown for 72 hours at 72 °C and subsequently harvested by centrifugation (238 g, 4 °C, 30 min). Supernatant was discarded and cell pellets resuspended in lysis buffer (300 mM NaCl, 20 mM Na·HEPES pH 7.4, 10 % (v/v) glycerol, 1 mM DTT, 30 mM imidazole pH 8.0, 0.284 μg/mL leupeptin, 1.37 μg/mL pepstatin A, 0.17 mg/mL PMSF, 0.33 mg/mL benzamidine). Resuspended cells were snap frozen and stored at -80 °C. All protein purifications were performed at 4 °C. Frozen cell pellets were thawed and lysed by sonication. Lysates were cleared using centrifugation (18,000 g, 4 °C, 30 min and 235,000 g, 4 °C, 60 min). The supernatant containing Chd1 was filtered with 0.8-μm syringe filters (Millipore) and applied onto a GE HisTrap HP 5 mL (GE Healthcare). The column was washed with 10 column volumes (CV) lysis buffer, 5 CV high salt buffer (1 M NaCl, 20 mM Na·HEPES pH 7.4, 10 % (v/v) glycerol, 1 mM DTT, 30 mM imidazole pH 8.0, 0.284 μg/mL leupeptin, 1.37 μg/mL pepstatin A, 0.17 mg/mL PMSF, 0.33 mg/mL benzamidine), and 5 CV lysis buffer. Chd1 was eluted using a 40-minutes gradient of 0-100 % elution buffer (300 mM NaCl, 20 mM Na·HEPES pH 7.4, 10 % (v/v) glycerol, 1 mM DTT, 500 mM imidazole pH 8.0, 0.284 μg/mL leupeptin, 1.37 μg/mL pepstatin A, 0.17 mg/mL PMSF, 0.33 mg/mL benzamidine). Fractions containing Chd1 were pooled and subjected to dialysis/TEV protease digestion for 16 hours (300 mM NaCl, 20 mM Na·HEPES pH 7.4, 10 % (v/v) glycerol, 1 mM DTT, 30 mM imidazole with 2 mg His6-TEV protease). The dialyzed sample was again applied to a GE HisTrap HP 5 mL. The flow-through, which contained cleaved tag-less Chd1, was concentrated using an Amicon Millipore 15 mL 50,000 MWCO centrifugal concentrator. The concentrate was applied to a GE S200 16/600 pg size exclusion column in 300 mM NaCl, 20 mM Na·HEPES pH 7.4, 10 % (v/v) glycerol, 1 mM DTT. Fractions containing Chd1 were concentrated to ~100 μM. The sample was aliquoted, snap frozen and stored at -80 °C. FACT was purified as above with minor modifications. After dialysis, the sample was subjected to a tandem GE HisTrap HP 5 mL and GE HiTrap Q 5 mL columns combination. After sample application, the columns were washed with lysis buffer and the HisTrap removed. FACT was eluted by applying a high salt buffer gradient from 0-100 % high salt buffer (1 M NaCl, 20 mM Na·HEPES pH 7.4, 10 % (v/v) glyc