Journal: Nature Communications

Article Title: Cell-type-specific functionality encoded within the intrinsically disordered regions of OCT4

doi: 10.1038/s41467-025-63806-3

Figure Lengend Snippet: a Graph of intrinsic disorder of OCT4 by PONDR-VSL2 (blue) and MoRF prediction by MoRFpred (red). Heatmap of OCT4 reprogramming activity and OCT4 domains are indicated above. SLiPERs and nonSLiPERs are highlighted in grey and magenta, respectively. b Immunofluorescence showing punctate staining of OCT4-WT and mutants in OSKM-48h (insets of zoomed-in views in yellow). Scalebar, 20 µm. c The transactivation of Luciferase reporter constructs (schematics) by OCT4-WT and mutants normalized against Renilla luciferase and expressed as a ratio over empty vector (pGL3-tk). d Cartoon representation of OCT4 structure by AlphaFold2 colour-coded by pLDDT scores. Examples of SLiPERs and nonSLiPERs are indicated. e Representative EMSA of increasing amounts (0–8 nM) of recombinant OCT4-WT and mutants binding to LIN28B -DNA (top-left panel) and LIN28B -nucleosome (top-right panel) at 1 nM concentration. The affinity of recombinant OCT4 WT and mutants (3 nM) for LIN28B -DNA (bottom-left panel) and LIN28B -nucleosome (bottom-right panel) in the presence of 30-fold molar excess (60 nM) of specific competitor (“s” lanes) or non-specific competitor (“n” lanes) or absence of competitor (“-” lanes). f ChIP-SICAP experimental workflow for identifying OCT4 partners on chromatin. Dual crosslinking with formaldehyde and glutaraldehyde to preserve protein-DNA and protein-protein interactions, respectively. g ChIP-seq read density heatmaps (blue) of OCT4 WT and mutants in OSKM-48h relative to chromatin accessibility (red) spanning ±1 kb from the centre of OCT4 peaks. The sequences were rank-ordered according to ChIP-seq read density. The number of OCT4 sites ( n ) and the colour scales are indicated. Motifs enriched in OCT4 sites are shown. h Profile plots showing the average ATAC-seq signal around OCT4 sites shown in ( g ) . i Bar plots showing the caspase activity to indicate the level of apoptosis induced by OCT4 wild type and mutants in OSKM-48h. Data are presented as mean values +/- SEM of n = 4 ( c ) and n = 3 ( i ) biological replicates. Statistical significance assessed by unpaired t -test as indicated by P values (*≤ 0.05, ***≤ 0.001, ****≤ 0.0001) ( c and i ). Images are representative of n = 6 ( b ) and n = 3 ( e ) biological replicates. Source data are provided as a Source Data file.

Article Snippet: The 119 OCT4 deletion mutants were synthesized as linear DNA fragments by Twist Bioscience (San Francisco, CA), each containing five amino acid deletion tiled with a two amino acid overlap (see Supplementary Data for DNA sequence) and flanked by two adaptors for sub-cloning.

Techniques: Activity Assay, Immunofluorescence, Staining, Luciferase, Construct, Plasmid Preparation, Recombinant, Binding Assay, Concentration Assay, Protein-Protein interactions, ChIP-sequencing

Journal: Nature Communications

Article Title: Cell-type-specific functionality encoded within the intrinsically disordered regions of OCT4

doi: 10.1038/s41467-025-63806-3

Figure Lengend Snippet: a Schematic representation of the knock-in strategy used to replace OCT4-SLiPER with a linker in the endogenous Pou5f1 gene in ES cells. A crRNA was designed to introduce a double-stranded break (DSB) downstream OCT4-SLiPER coding for a.a. 29-42 (green) by Cas9 RNP (blue) bound to PAM (yellow). A single-stranded donor DNA that harbours glycine-rich linker coding sequence (purple) flanked by 50-mer homology arms on both sides was used for homology-directed repair (HDR). b Western blot showing the OCT4 levels in three independent ESC clones with Pou5f1 lin/ Δ knock-in compared to the E14tg2a parental line ( Pou5f1 wt/wt ). Molecular weight standards (kDa) are indicated. c Immunofluorescence of LaminB1, Nanog, and GFP, showing the contribution of Pou5f1 lin/ Δ ES lines to the inner cell mass of blastocysts compared to Pou5f1 wt/wt . Scalebar, 20 μm. d Same as ( c ) for E6.5 embryos. e Table summarizing the contribution of Pou5f1 wt/wt and Pou5f1 lin/ Δ ESC lines to chimeric embryos at different developmental stages. f Same as ( c ) for E8.5 embryos. Inset showing the lateral view representation of embryos. g Pou5f1 lin/ Δ ESCs caused increased foetal resorption and failed to generate chimeric embryos at E13.5 compared to Pou5f1 wt/wt . h The contribution of Pou5f1 wt/wt ESCs to chimeric embryos at E13.5 were assessed by transgene GFP fluorescence. i Colony morphology of ESCs in the presence and absence of LIF, showing the robustness of Pou5f1 lin/ Δ ESC self-renewal even in the absence of LIF. Scalebar, 100 µm. j Quantitation of colony types formed by different Pou5f1 lin/ Δ ES lines under different conditions as shown in ( h ). Data are presented as mean values +/- SEM of n = 3 biological replicates. Statistical significance was assessed by multi-comparison one-way ANOVA as indicated by P value (***≤0.001). Images are representatives of n = 3 ( b , h and i ) and n = 59, 47, 68 ( c , d and f ) biological replicates. Source data are provided as a Source Data file.

Article Snippet: The 119 OCT4 deletion mutants were synthesized as linear DNA fragments by Twist Bioscience (San Francisco, CA), each containing five amino acid deletion tiled with a two amino acid overlap (see Supplementary Data for DNA sequence) and flanked by two adaptors for sub-cloning.

Techniques: Knock-In, Introduce, Sequencing, Western Blot, Clone Assay, Molecular Weight, Immunofluorescence, Fluorescence, Quantitation Assay, Comparison

Journal: Nature Communications

Article Title: Cell-type-specific functionality encoded within the intrinsically disordered regions of OCT4

doi: 10.1038/s41467-025-63806-3

Figure Lengend Snippet: a Principal component analysis of RNA-seq showing distinct gene expression of Pou5f1 lin/ Δ ESC lines compared to Pou5f1 wt/wt under LIF+2i and LIF conditions. b Volcano plots showing TF DEGs between Pou5f1 lin/ Δ and Pou5f1 wt/wt ESCs under LIF+2i and LIF conditions. The significance threshold is represented by dotted lines denoting log2 (fold change) of 1.5 and FDR adjusted P value of 0.05 by unpaired t -test. c Genome browser screenshot showing OCT4 ChIP-seq, ATAC-seq and RNA-seq around the Nanog locus in Pou5f1 lin/ Δ and Pou5f1 wt/wt ES cells. OCT4 binding is diminished in Nanog promoter is highlighted in yellow. d Volcano plot showing differential chromatin accessibility measured by ATAC-seq in Pou5f1 lin/ Δ and Pou5f1 wt/wt ES lines. The significance threshold is represented by green and blue circles denoting; log2 (fold change) of 2 and FDR adjusted P value of 0.001 by unpaired t -test. The number of peaks ( n ) of each category are indicated. e ChIP-seq read density heatmaps (blue) of OCT4 WT and mutants in ES cells relative to ATAC-seq (red) spanning ±1 kb from the centre of OCT4 peaks. The corresponding profile plots are shown above. The number of sites ( n ) of each group and the enrichment colour scale (RPGC) are indicated. f Read-density heatmaps of MNase-seq around nucleosome dyads (±500 bp) bound by OCT4 as in ( e ). The sites were rank ordered based on the distance between OCT4 peak summits and the closest nucleosome dyad. Density heatmaps of de novo motifs (logos on top) around nucleosome dyads. Motif density is scored on both DNA strands (red and blue) following the colour gradient scale shown at the bottom. The number ( n ) of nucleosomes are indicated. g Average profile plots of motif density scores on the two DNA strands (red and blue) around nucleosome dyads (±200 bp) targeted by OCT4 as in ( f ). Weighted frequency values were generated using kernel smoothing in 3 bp windows. DNA 10 bp twists around nucleosome are shown in grey-white stripes. h GO terms associated with genes targeted by OCT4 in the three binding site groups shown in ( e ).

Article Snippet: The 119 OCT4 deletion mutants were synthesized as linear DNA fragments by Twist Bioscience (San Francisco, CA), each containing five amino acid deletion tiled with a two amino acid overlap (see Supplementary Data for DNA sequence) and flanked by two adaptors for sub-cloning.

Techniques: RNA Sequencing, Gene Expression, ChIP-sequencing, Binding Assay, Generated