Binding of the human pioneer transcription factor OCT4 to nucleosomes containing endogenous DNA sequences causes changes to the nucleosome structure and facilitates the cooperative assembly of multiple pioneer transcription factors, a property that can be affected by histone modifications.
), suggesting that not all TFs might be affected by epigenetic marks. In conclusion, our findings suggest that the pre-existing epigenetic landscape could tune pioneer TF activity.histones for nucleosome assembly were overexpressed in theThe cells were grown in LB medium at 37 °C and induced with 1 mM IPTG when ODreached 0.6. After 3 h of expression, the cells were pelleted down, resuspended in lysis buffer , 150 mM NaCl, 1 mM EDTA, 1 mM DTT and 0.1 mM PMSF) and frozen.
Each histone protein was extracted from the purified inclusion body pellet in a buffer containing 50 mM Tris , 2 M NaCl, 6 M guanidine hydrochloride and 1 mM DTT for overnight at room temperature. Any insoluble components were removed by centrifugation. Proteins making histone pairs were combined in equimolar ratios and dialysed two times in 1 l of refolding buffer , 2 M NaCl and 1 mM DTT) at 4 °C. Any precipitate was removed by centrifugation for 20 min at 13,000 rpm at 4 °C.
For cryo-EM grid freezing of ‘assembly 1’ , commercially available OCT4 from Abcam was used. The protein was fused with the herpes simplex virus VP16 transactivation domain at the N terminus and a 11R tag at the C terminus. For the ‘assembly 2’ for cryo-EM and all the other assays, His-tagged OCT4 was expressed in a pET28 vector and purified under denaturing conditions from inclusion body using Talon affinity resins.
All the OCT4 variants were generated using the inverse PCR strategy. Oligo primers used for mutagenesis were purchased from Integrated DNA Technology and are listed in the Supplementary Table. The inverse PCRs were set up in a total volume of 25 μl. After amplification, 10 μl of purified PCR product was incubated with 5 U of T4 PNK in 20 μl of 1× T4 DNA ligase buffer for 1 h at 37 °C. Of T4 DNA ligase, 200 U was added to the reaction and incubated for 1 h at room temperature.
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