Pet 28a vector

The DNA sequence encoding full-length IniA (residues 1–602) from M. smegmatis mc²155 was PCR-amplified using Phanta Max DNA polymerase (Vazyme) and inserted into the NcoI-HindIII sites of pET-28a vector (GE Healthcare) using ClonExpress^®II (One Step Cloning Kit, Vazyme). This construct includes a C-terminal 6 × His-tag. For expression in M. smegmatis, the encoding sequence of IniA-GFP fusion protein was inserted into the SspI site of the engineered plasmid pMV261 and the mutant construct (residues L480-K492 was mutated into GGGGSGGGGS liker) was generated by recombinant circle PCR. Site-directed mutagenesis was performed using the TaKaRa MutanBEST Kit. The mutants were introduced by the PCR method using the IniA expression plasmid as a template, with pairs of primers encoding the mutations at the sites of substitution. DNA sequencing of the constructs was performed to validate that the mutagenesis experiments were successful. All primer sequences used in this study are shown in the Supplementary Table 1.
E. coli BL21 (DE3) ATCC strain (Supplementary Table 2) was used to express IniA for purification. The M. smegmatis mc²155 strain was used to observe cellular localization of IniA.

DNA fragment encoding AF9 YEATS domain (residues 1–138) was amplified and cloned into the pET28a vector (GE Healthcare, Chicago, IL, USA). The construct was then transformed into Escherichia coli BL21 (DE3) by heat shock. The protein expression was induced at A600 = 0.8–1.0 using 0.3 mM Isopropyl β-d-1-thiogalactoside. After incubating at 25 °C for 20 h, the cell pellets were harvested by centrifugation and resuspended in a binding buffer of 20 mM HEPES, 0.5 M NaCl at pH 7.4. The cells were then lysed by a high-pressure cracker and centrifuged at 13,000× g for 30 min. The supernatant was purified on a HisTrap nickel column (GE Healthcare) and a gel-filtration column (Superdex 75 30/100 GL) in 20 mM Tris, 150 mM NaCl, 2 mM DTT, 1 mM EDTA at pH 7.4. Pure fractions as analyzed by SDS-PAGE were concentrated. Uniformly 15N-labeled protein was prepared from cells grown in LR medium supplemented with 15NH4Cl. The purified protein was dialyzed into PBS buffer for following NMR experiments.