Journal: iScience

Article Title: Structurally-discovered KLF4 variants accelerate and stabilize reprogramming to pluripotency

doi: 10.1016/j.isci.2021.103525

Figure Lengend Snippet: The reprogramming efficiency of Nanog-GFP MEF using KLF4 ZnF alanine-scanned mutants (A) Schematic of the protein sequence of the KLF4 ZnF domain. The amino acid residues highlighted in red are targeted by alanine scanning site mutagenesis in this study. (B) Schematic of the generation of iPSCs using alanine-scanned KLF4 ZnF mutants made by site mutagenesis of KLF4 wild-type (WT) retroviral vector together with OCT4, SOX2, and MYCL1 retroviral vectors. (C) The typical morphologies of generated iPSC colonies in NC (negative control with mRFP1 vector), WT (KLF4 wild-type), R497A mutant, D503A mutant, and L507A mutant. The images were shown in GFP (green channel fluorescence), Phase (phase contrast), and merged images. Scale bars, 200 μm. (D) The number of Nanog-GFP-positive iPSC colonies were counted 15 days after infection of retroviral vectors to Nanog-GFP MEF from 10,000 cells. Results are mean and SE, n = 4. p value was calculated from Dunnett's test between WT and L507A conditions. (E) The number of Nanog-GFP-positive colonies were counted 25 days after infection of retroviral vectors to Nanog-GFP MEF from 10,000 cells. Results are mean and SE, n = 4. p value was calculated from Dunnett's test between WT and L507A conditions. (F) The ratio of Nanog-GFP-positive colonies was calculated 25 days after infection of retroviral vectors. The total number of Nanog-GFP-positives was counted from whole wells. Results are mean and SE, n = 4. p value was calculated from Dunnett's test between WT and L507A conditions.

Article Snippet: Anti-KLF4 polyclonal Rabbit Ig Antibodies , MBL , Cat#PM057, RRID:AB_10598180.

Techniques: Sequencing, Mutagenesis, Retroviral, Plasmid Preparation, Generated, Negative Control, Fluorescence, Infection

Journal: iScience

Article Title: Structurally-discovered KLF4 variants accelerate and stabilize reprogramming to pluripotency

doi: 10.1016/j.isci.2021.103525

Figure Lengend Snippet: Efficient generation system of Nanog-GFP-positive miPSCs using SeVdp in feeder-free conditions (A) Schematic of miPSC generation from Nanog-GFP MEF using SeVdp infection. On day 3 after infection, the medium was changed to serum replacement (SR)-based conditioned medium which was collected from the culture of the SL10 feeder cell line. In addition, Shield1 to stabilize KLF4 protein in the ProteoTuner Shield system and Blasticidin S to select the transduced cells were added on day 3 after infection until day 10 and day 7, respectively. (B) A typical example of flow cytometric analysis of reprogrammed iPSCs in the conditioned medium or MEF medium using SeVdp on day 10 after infection. (C) Flow cytometric analysis of the percentage of Nanog-GFP-positive cells after the infection of SeVdp carrying WT or L507A KLF4. Flow cytometric experiments were performed on days 5, 7, 10, 13, and 15 after the infection. Results are mean and SE, n = 5. p values calculated with t test were shown based on the comparison between WT and L507A KLF4 conditions. (D) The number of Nanog-GFP-positive iPSC colonies were counted 13 days after infection of SeVdp to Nanog-GFP MEF from 10,000 cells. Results are mean and SE, n = 4. The p value calculated with the t test was shown based on the comparison between WT and L507A KLF4 conditions.

Article Snippet: Anti-KLF4 polyclonal Rabbit Ig Antibodies , MBL , Cat#PM057, RRID:AB_10598180.

Techniques: Infection, Comparison

Journal: iScience

Article Title: Structurally-discovered KLF4 variants accelerate and stabilize reprogramming to pluripotency

doi: 10.1016/j.isci.2021.103525

Figure Lengend Snippet: The reprogramming efficiency and heterogeneity of human somatic cells using KLF4 ZnF alanine-scanned mutants (A) Schematic of the reprogramming of human fibroblasts using alanine-scanned KLF4 ZnF mutants made by site mutagenesis of KLF4 wild-type (WT) retroviral vector together with OCT4, SOX2, and MYCL1 retroviral vectors. (B and C) Typical data of flow cytometric analysis of the percentage of TRA1-60-positive cells in KLF4 alanine-scanned mutants 7 days after infection of reprogramming retrovirus vectors carrying KLF4 ZnF alanine-substituted mutants. (D) Flow cytometric analysis of the percentage of TRA1-60-positive cells in KLF4 alanine-scanned mutants 7 days after infection of reprogramming retrovirus vectors carrying KLF4 ZnF alanine-substituted mutants. Results are mean and SE, n = 6. The p value was calculated with Dunnett's test between WT and L507A conditions. (E) Schematic of the generation of human iPSCs using SeVdp carrying KLF4 or L507A mutant in feeder-free conditions. On day 3 after infection, the medium was changed to primate ESC medium-based conditioned medium which was collected from the culture of a feeder cell line. Because KLF4 is fused with a ligand-dependent destabilization domain (DD) at the N-terminus in this SeVdp, Shield1 to stabilize KLF4 protein in the ProteoTuner Shield system was added into one group until picking up iPSC colonies. Blasticidin S was added on day 3 after infection until day 7. (F) Numbers of total colonies in each condition were counted by colony morphology on day 20. Results are mean and SE, n = 3. (G) Numbers of NANOG-positive colonies in each condition were counted by immunocytochemistry. Results are mean and SE, n = 3. The p value calculated with the t test was shown based on the comparison between WT and L507A KLF4 conditions. (H) The ratio of NANOG-positive colonies in each condition was calculated from numbers of total colonies and NANOG-positive colonies. Results are mean and SE, n = 3. The p value calculated with the t test was shown based on the comparison between WT and L507A KLF4 conditions. (I and J) Variations of NANOG expression and residual SeVdp NP RNA amount (G) and HERV-H and lincRNA-RoR (H) in human iPSC clones generated with SeVdp carrying WT or L507A KLF4 were shown in scatter plots. Numbers (N) of the clones used in each condition are shown. Results are shown as Z-scores of all the samples.

Article Snippet: Anti-KLF4 polyclonal Rabbit Ig Antibodies , MBL , Cat#PM057, RRID:AB_10598180.

Techniques: Mutagenesis, Retroviral, Plasmid Preparation, Infection, Immunocytochemistry, Comparison, Expressing, Clone Assay, Generated

Journal: iScience

Article Title: Structurally-discovered KLF4 variants accelerate and stabilize reprogramming to pluripotency

doi: 10.1016/j.isci.2021.103525

Figure Lengend Snippet: The reprogramming efficiency of Nanog-GFP MEF using every KLF4 L507 amino acid residue variant (A) Typical morphology of reprogrammed colonies Nanog-GFP MEF using retroviral vectors carrying every KLF4 L507 amino acid residue variant on day 25. Scale bars, 200 μm. (B) The number of Nanog-GFP-positive iPSC colonies were counted on day 15 after infection of retroviral vectors to Nanog-GFP MEF from 10,000 cells. Results are mean and SE, n = 4. (C) The number of Nanog-GFP-positive colonies was counted on day 25 after infection of retroviral vectors to Nanog-GFP MEF from 10,000 cells. Results are mean and SE, n = 4. (D) The ratio of Nanog-GFP-positive colonies was calculated on day 25 after infection of retroviral vectors. The total number of Nanog-GFP-positives was counted from whole wells. Results are mean and SE, n = 4. (E) Scatterplot shows a possible linear correlation between van der Waals volume (Å 3 ) of the variants' amino acid residue and the mean numbers of Nanog-GFP-positive colonies on day 25. The red dot line and the value of R 2 show the best-fitted trendline and the coefficient of determination (the square of the correlation coefficient), respectively.

Article Snippet: Anti-KLF4 polyclonal Rabbit Ig Antibodies , MBL , Cat#PM057, RRID:AB_10598180.

Techniques: Residue, Variant Assay, Retroviral, Infection

Journal: iScience

Article Title: Structurally-discovered KLF4 variants accelerate and stabilize reprogramming to pluripotency

doi: 10.1016/j.isci.2021.103525

Figure Lengend Snippet: The reprogramming efficiency of Nanog-GFP MEF using retrovirus vector-based L507A mutants with pro-reprogramming factors (A) Typical morphology of Nanog-GFP colonies in the reprogramming Nanog-GFP MEF on day 25 of reprogramming in KLF4 wildtype (WT) or L507A mutant with pro-programming factors. Scale bars, 200 μm. (B) The number of Nanog-GFP colonies in the reprogramming Nanog-GFP MEF calculated on day 25 of reprogramming in KLF4 WT or L507A mutant with pro-programming factors. Results are mean and SE, n = 4. p values calculated with the t test were shown based on the comparison between WT and L507A KLF4 conditions. (C) The ratio of Nanog-GFP colonies to the total colonies in the reprogramming Nanog-GFP MEF was calculated on day 25 of reprogramming in KLF4 WT or L507A mutant with pro-programming factors. Results are mean and SE, n = 4. p values calculated with the t test were shown based on the comparison between WT and L507A KLF4 conditions.

Article Snippet: Anti-KLF4 polyclonal Rabbit Ig Antibodies , MBL , Cat#PM057, RRID:AB_10598180.

Techniques: Plasmid Preparation, Mutagenesis, Comparison

Journal: iScience

Article Title: Structurally-discovered KLF4 variants accelerate and stabilize reprogramming to pluripotency

doi: 10.1016/j.isci.2021.103525

Figure Lengend Snippet: KLF4 L507A mutant preferentially binds promoter/enhancer of pluripotency-related genes in reprogramming cells (A) Heat maps of peak distribution and the numbers of peaks from the ChIP-seq data of KLF4. (B) The distribution of ChIP-seq reads between -3kb and +3kb around TSS. (C) A Venn diagram of gene sets obtained from the comparison of ChIP-seq data of KLF4 among reprogramming MEF induced by KLF4 WT or L507A on day 2 or 10. Genes that have specific ChIP-seq peaks within 5kb from TSS are categorized in each set. The numbers of the genes in each set are shown. (D) Wikipathway enrichment analysis was performed on the gene set derived from the sample of L507A on day 10. The top 5 pathways that are enriched with statistically significant are illustrated with the adjusted p values. (E–H) Mapped ChIP-seq peaks on pluripotency-related genes, Alpl (E), Dppa5 (F), Dsg2 (G), and Klf5 (H). These genes are extracted in the combination with RNA-seq data. Red bars indicate ChIP-seq peaks in each genomic region. (I and J) Motif discovery analysis was performed on ChIP-seq data of KLF4 in reprogramming MEF induced by KLF4 WT or L507A. Discovered known motifs from WT (I) and L507A (J) were shown with the name of transcription factor and p value. (K) A typical blot image of capillary Western blot assays on samples co-immunoprecipitated with anti-KLF4 antibody. Reprogramming MEFs infected with SeVdp carrying KLF4 WT or L507A or no transduction for 3 days were used. (L) Protein abundance was calculated from the data of co-IP assays. Results are mean and SE, n = 3. The p value calculated with a t test was shown based on the comparison between WT and L507A KLF4 conditions.

Article Snippet: Anti-KLF4 polyclonal Rabbit Ig Antibodies , MBL , Cat#PM057, RRID:AB_10598180.

Techniques: Mutagenesis, ChIP-sequencing, Comparison, Derivative Assay, RNA Sequencing, Western Blot, Immunoprecipitation, Infection, Transduction, Quantitative Proteomics, Co-Immunoprecipitation Assay

Journal: iScience

Article Title: Structurally-discovered KLF4 variants accelerate and stabilize reprogramming to pluripotency

doi: 10.1016/j.isci.2021.103525

Figure Lengend Snippet: Global expression similarity and differences between reprogramming cells between KLF4 WT and L507A (A) PCA of global gene expression in MEF, miPSCs, and reprogramming MEF induced by KLF4 WT or L507A on day 2 or 10 after transduction of each SeVdp vector. (B) A volcano plot shows log2 fold change as X axis and -log10 (FDR) as Y axis. All the genes were plotted in red circles. Significant genes with FDR<0.05 were rectangularly surrounded with pink (downregulated) and orange (upregulated). (C-E) GO (Gene Ontology) enrichment analysis was performed on the gene sets, composed of the genes which differentially expressed between WT and L507A conditions on day 10 samples. GO Cellular Component enriched from upregulated genes in L507A samples were shown in (C); GO Biological Process enriched from upregulated genes in L507A samples were shown in (D); GO Biological Process enriched from down-regulated genes in L507A samples were shown in (E). The top 5 GO terms that are enriched with statistically significant are illustrated with adjusted p values. (F) A Venn diagram of gene sets obtained from specifically regulated by L507A as shown above in RNA-seq data and ChIP-seq data. 33 genes, including Klf5 , were extracted in common. (G–K) As examples of the genes which are commonly identified in RNA-seq and ChIP-seq experiments in this study, the expression levels (i.e., TPM (Transcripts Per Million)) of Alpl , Dppa5a , Dsg2 , Esrp1 , and Klf5 , are shown from RNA-seq data. Results are mean and SE, n = 3. p values calculated with FDR correction were shown based on the comparison between WT and L507A KLF4 conditions on day 10 samples.

Article Snippet: Anti-KLF4 polyclonal Rabbit Ig Antibodies , MBL , Cat#PM057, RRID:AB_10598180.

Techniques: Expressing, Gene Expression, Transduction, Plasmid Preparation, RNA Sequencing, ChIP-sequencing, Comparison

Journal: iScience

Article Title: Structurally-discovered KLF4 variants accelerate and stabilize reprogramming to pluripotency

doi: 10.1016/j.isci.2021.103525

Figure Lengend Snippet: MD simulation analysis of the effect of the mouse Klf4 L477A mutation (identical to human KLF4 L507A) on the structure of the Klf4 ZnF-DNA complex (A) Conformational distribution of the mouse Klf4 (WT)-DNA complex (magenta contours) and the Klf4(L477A)-DNA complex (green contours). The level of the n -th contour is 2 n −1 × 10 −4 . The positions of the initial structure and the representative structures obtained from the cluster analysis of the MD trajectories of the Klf4(WT)-DNA and the Klf4(L477A)-DNA complexes are indicated by a black rectangle, magenta triangles, green triangles, respectively. The black line separates conventional conformation distribution, Region 1, and unique conformation distribution of the Klf4(L477A)-DNA complex as Region 2. (B–D) Comparison of the representative structure from cluster 1 of the Klf4(WT)-DNA complex (B) and that from cluster 4 of the Klf4(L477A)-DNA complex (C). A superposed view is also shown (D). L477 or A477 are highlighted in yellow circles. (E–F) Schematic representation of protein-DNA interactions of WT cluster 1(E) and L477A cluster 4 (F). The interaction between an amino acid residue and a DNA base or DNA phosphate group is shown in a blue dotted line or a blue solid line with rhombus, respectively. Amino acid residues highlighted in tangerine and red indicate the ones that changed target DNA bases and that formed interactions to DNA in either WT cluster 1 or L477A cluster 4, respectively. Hydrogen-bond occupancy was calculated for each possible atom pair between the protein and the DNA of each cluster. The occupancies of the atom pairs within a pair of a residue and the base or the phosphate group of a nucleotide were summed. The pairs with the sum of the occupancies >0.7 are shown. (G–J) Conformational changes and additional hydrogen bonds to phosphate groups of DNA in the representative structure from cluster 4 of the Klf4(L477A)-DNA complex. Y430, R452, R467, and S472 are highlighted in yellow circles. All the molecular graphic images were produced using UCSF Chimera ( Pettersen et al., 2004 ).

Article Snippet: Anti-KLF4 polyclonal Rabbit Ig Antibodies , MBL , Cat#PM057, RRID:AB_10598180.

Techniques: Mutagenesis, Comparison, Residue, Produced

Journal: iScience

Article Title: Structurally-discovered KLF4 variants accelerate and stabilize reprogramming to pluripotency

doi: 10.1016/j.isci.2021.103525

Figure Lengend Snippet:

Article Snippet: Anti-KLF4 polyclonal Rabbit Ig Antibodies , MBL , Cat#PM057, RRID:AB_10598180.

Techniques: Affinity Purification, Produced, Ubiquitin Proteomics, Control, Recombinant, Magnetic Beads, Transfection, Mutagenesis, Chromatin Immunoprecipitation, DNA Purification, Real-time Polymerase Chain Reaction, Isolation, Transgenic Assay, Retroviral, Plasmid Preparation, Software