Fig 2: SPF activity is not sufficient, in isolation, to generate chromatin accessibility.a, b Upper panels. Results of ATAC-qPCR experiments in FR1_OCT4SOX2 ESCs (a) and FR1_CTCF ESCs (b) containing WT and Sc motifs in -SssI and +SssI conditions. We measured very low levels of chromatin accessibility at the FR1 locus in all four conditions when compared to known accessible (Zfp345 large ATAC-peak; Kif3b medium ATAC-peak) and inaccessible (Intergenic small ATAC peak) regions of the chromatin in mESCs97. Results are shown as mean+SD of n = 3 biologically independent replicates. Source data are provided as a Source data file. Lower panels. Representative genome browser tracks of the chromatin accessibility landscape around the FR1 locus. ATAC-Seq experiments were performed in FR1-OCT4SOX2 ESCs containing WT/±SssI and Sc/±SssI motifs (a) and in FR1-CTCF ESCs containing WT/-SssI and Sc/-SssI motifs (b). The ATAC-Seq signal is low over the FR1 locus compared to neighbouring accessibility peaks, highlighting the low levels of chromatin accessibility, which remain unchanged despite SPF binding in the WT conditions. c OCT4 and SOX2 ChIP-Seq in FR1 OCT4SOX2 ESCs reveals binding of both TFs at the FR1 locus. Displayed are representative genome browser tracks of the ChIP-Seq data across the FR1 locus.

Fig 3: Histone modifications modulate OCT4 binding to the nMATN1 nucleosome.a, Structural model of human OCT4 (orange) bound to a nucleosome (grey) assembled with a 186-bp DNA fragment from the nMATN1 regulatory element. b, Surface model showing the electrostatic potential of the OCT4-POUS domain and the positively charged histone H3 tail. c, Representative native gel electrophoresis showing OCT4 binding to unmodified or nMATN1-H3K27ac nucleosomes (left). The coloured asterisks mark molecules bound to the nucleosome: 1-OCT4 is in red, 2-OCT4 is in blue, 3-OCT4 is in green and 4-OCT4 is in purple. Quantification of OCT4 binding to nMATN1-H3K27ac relative to unmodified nucleosomes (right), using bands marked with asterisks. Data shown are mean ± s.e.m. of four independent experiments; *P = 0.02 and **P = 0.008, one-sided Student’s t-test. d, Representative native gel electrophoresis showing OCT4 binding to unmodified or nMATN1-H3K27me3 nucleosomes (left). The coloured asterisks are as in panel c. Quantification of OCT4 binding to nMATN1-H3K27me3 relative to unmodified (right), using bands marked with asterisks. Data shown are mean ± s.e.m. of four independent experiments; ***P = 0.0007 (2-OCT4) and ***P = 0.0004 (3-OCT4), one-sided Student’s t-test.

Fig 4: N-terminal disordered region of OCT4 is required for chromatin de-compaction.a) Native gels showing Mg2+ induced compaction of nucleosomes and nucleosomes bound to OCT4 (n = 4), OCT4ΔNtail (n = 4) and OCT4ΔCtail (n = 3). OCT4 binding reduces nucleosome compaction. Deletion of OCT4 N-terminal disordered region eliminates OCT4 effect on nucleosome compaction. b) Native agarose gel showing assembly of nucleosome and nucleosome array (n > 3). c) Negative stain micrographs showing Mg2+ induced compaction of the LIN28B nucleosome array (n = 26 micrographs), the OCT4 bound LIN28B nucleosome array (n=32 micrographs) and the OCT4ΔNtail bound LIN28B nucleosome array (n = 23 micrographs). Most nucleosomes are compacted (red circle) in sample containing LIN28B arrays and OCT4ΔNtail bound LIN28B arrays. Many more open arrays (green circle) are detectable when OCT4 is bound to LIN28B arrays. d) Native gels showing binding of OCT4ΔNtail (n = 4) and OCT4ΔCtail (n = 2) to the LIN28B nucleosome. OCT4ΔNtail and OCT4ΔCtail binds nucleosome comparably to wild type OCT4. e) Quantification of data from d). Deletion of OCT4 C-terminal disordered tail does not reduce Oct4 effect on nucleosome compaction. Data are mean and s.e.m., n = 4. f) A native gel stained for DNA showing OCT4 binding to nucleosome and DNA. Binding to DNA and nucleosome generates distinct bands. g) A native gel stained for DNA showing OCT4 binding to nucleosome and western blot with anti-H3 showing presence of histones in these complexes (left). A native gel stained for DNA showing OCT4 binding to nucleosome and western blot with anti-OCT4 showing presence of OCT4 in these complexes (right). Each experiment has been performed > 3 times. h) Native gel showing OCT4 binding to the LIN28B nucleosome and LIN28B nucleosome with mutated binding site 2 (LIN28B-2M). Mutation of the binding site 2 did not affect binding of 1st OCT4. i) Native gel showing OCT4 binding to the LIN28B nucleosome and LIN28B nucleosome with mutated binding site 3 (LIN28B-3M). Mutation of the binding site 3 did not affect binding of 1st OCT4. j) Cryo-EM maps from a subset of data showing OCT4 interaction with H3 and H2A tails. The maps are colored by local resolution. Histone tails are marked with red dots. Model showing interaction of OCT4 with histone tails is shown below. Model of OCT4-nucleosome complex was rigid body fitted into cryo-EM maps.

Fig 5: Histone modifications modulate OCT4 and SOX2 cooperativity on various human DNA.a) Cryo-EM map of OCT4 region from the OCT4_nucleosome complex from Fig. 8g). Focused classification and refinements improved the resolution of this 20 kDa fragment to 8.1 Å. b) Fourier shell correlation (FSC) curve showing the resolution of the map in a). c) Directional FSC plot showing uniform resolution in all directions. d) Representative regions showing map quality and fit of the model are shown for the nucleosome with bound OCT4. Right: bases in the DNA are well resolved. e) Quantification of sequencing of Mnase I digested OCT4-bound nMATN1 nucleosomes. The y-axis shows fraction of nucleosome size reads starting at defined position, the x-axis shows position of the first base pair relative to the most abundant position (0 as observed in the structure). Data are mean and spread of 2 independent experiments. f) The model of the OCT4 bound to DNA (Extended Data Fig. 2g) was refined into the cryo-EM map. The representative region showing map quality and fit of the model is shown. g) Cryo-EM models of OCT4 bound to the nMATN1 nucleosome containing 186bp of DNA at 2.2-5.6 Å resolution for two most dominant conformations. h) DNA sequence and schematic representation showing nMATN1 DNA positioning on the OCT4_nucleosome complex. Potential OCT4 binding sites are labeled in green. OCT4 binding site occupied in the structure is labeled in orange. i) Close-up views of the nucleosome entry/exit site showing interaction of the OCT4_POUS domain with the H3 N-terminal tail. Ribbon representation shows OCT4_POUS helix 4 and helix 5 interacting with histone H3 N-terminal tail. j) In LIN28B nucleosome OCT4 (light green) and SOX2 (light blue) binding sites are wrapped around the histone octamer. LIN28B nucleosomes are mobile, and nucleosome sliding transiently exposes the OCT4 binding site 1 (green), which leads to binding of OCT4 (green box). OCT4 binding (green box) traps DNA in a more defined position, which exposes internal OCT4 and SOX2 binding sites (blue). OCT4 bound to the OBS1 interacts with the histone H3 tail. H3K27ac modifies this interaction leading to DNA movement towards the histone octamer, which exposes internal OCT4 and SOX2 sites even more, leading to increased binding. k) The canonical H4 tail conformation (yellow, facing outward) favors inter-nucleosome interactions by interacting with the acidic patch of neighboring nucleosomes. These interactions are essential for chromatin compaction. OCT4 DNA binding domain binds linker DNA whereas disordered activation domain binds H4 near the H4 tail. This repositions the H4 tail to an inward conformation that reduces inter-nucleosome interactions in chromatin.