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Structurally-discovered KLF4 variants accelerate and stabilize reprogramming to pluripotency

Summary Non-genetically modified somatic cells can only be inefficiently and stochastically reprogrammed to pluripotency by exogenous expression of reprogramming factors. Low competence of natural reprogramming factors may prevent the majority of cells to successfully and synchronously reprogram. Here we screened DNA-interacting amino acid residues in the zinc-finger domain of KLF4 for enhanced reprogramming efficiency using alanine-substitution scanning methods. Identified KLF4 L507A mutant accelerated and stabilized reprogramming to pluripotency in both mouse and human somatic cells. By testing all the variants of L507 position, variants with smaller amino acid residues in the KLF4 L507 position showed higher reprogramming efficiency. L507A bound more to promoters or enhancers of pluripotency genes, such as KLF5, and drove gene expression of these genes during reprogramming. Molecular dynamics simulations predicted that L507A formed additional interactions with DNA. Our study demonstrates how modifications in amino acid residues of DNA-binding domains enable next-generation reprogramming technology with engineered reprogramming factors.


INTRODUCTION
Reprogramming technology provides unprecedented opportunities for life sciences, drug discovery, and regenerative medicine (Gurdon and Melton, 2008;Yamanaka and Blau, 2010). Advances of reprogramming technology enable one type of somatic cells to turn into other desired cell types by ectopic expression of defined factors, which are mostly transcription factors (Adachi and Scholer, 2012;Gurdon, 2016;Takahashi and Yamanaka, 2016). These transcription factors cooperatively bind silenced or closed parts of DNA and initiate active transcriptional events as ''pioneer factors'' to regulate the expression of downstream genes and the modification of epigenetic status toward reprogrammed cell states (Guo and Morris, 2017;Iwafuchi-Doi and Zaret, 2014;Ninkovic and Gotz, 2018). Once a reprogramming method to generate a cell type from another has been developed, many studies have focused on searching additional or substitutive factors, epigenetic modifiers, culture conditions, and delivery methods of the reprogramming factors to improve the reprogramming results (Gonzalez et al., 2011;Hanna et al., 2010;Xu et al., 2015). However, the current stagnation of reprogramming technology still accounts for such drawbacks as a poor and low yield of high-quality reprogrammed cells from primary somatic cells, which results in delays of medical applications. On the other hand, the functional alteration of reprogramming factors remains disregarded or unexploited overall. Only a handful of studies have demonstrated that modification or addition of transcriptional activity in reprogramming factors enhanced or altered reprogramming results (Jauch, 2018;Tsang et al., 2014;Velychko et al., 2019). Generally, natural transcription factors are still used as reprogramming factors.
Among these reprogramming factors, KLF4 (Krü ppel-like factor 4; as known as EZF or GKLF) is a ZnF (Zinc-Finger) protein with both transcriptional activation and repression domains and regulates downstream gene expression to control cellular states in a context-dependent manner (Garrett-Sinha et al., 1996;Rowland et al., 2005;Shields et al., 1996). Although KLF4 regulates the development and homeostasis of various tissues including cancers in natural biological contexts (Mahatan et al., 1999;Segre et al., 1999;Shie et al., 2000;Zhang et al., 2000), it plays a central role in the reprogramming of somatic cells into iPSCs (induced pluripotent stem cells) and other types of cells (Adachi and Scholer, 2012;Hayashi et al., 2014;Hiramatsu et al., 2011;Kitazawa et al., 2019;Takahashi and Yamanaka, 2006;Thier et al., 2012). In iPSC reprogramming, KLF4 regulates genome-wide epigenetic status and chromatin reorganization as a pioneer factor as well as its direct downstream transcription (Di Giammartino et al., 2019;Soufi et al., 2012Soufi et al., , 2015. Although the functionality of KLF4 in reprogramming has been understood well as above, the structural basis of KLF4 that enables its unique reprogramming activity remains elusive. Therefore in this study, we took a structure-function approach to answer if rational engineering of reprogramming factors might improve reprogramming. We focused on the ZnF DNA-binding domain of KLF4 to delineate its unique contribution to iPSC generation. We scanned alanine-substituted mutants of DNA-interacting amino acid residues in the KLF4 ZnF domain in iPSC generation. Although many of these mutants lost or decreased their reprogramming activity, one mutant, L507A, increased the speed and efficiency of iPSC generation. Human iPSC clones generated with this mutant showed relatively low heterogeneity in the expression of NANOG gene, low expression of exogenous vector RNA from SeVdp (Sendai Virus vector), and low expression of differentiation-defective iPSC markers (i.e., HERV-H and lincRNA-RoR (Ohnuki et al., 2014). By testing all the variants of L507 position, smaller amino acid residues of L507 position in KLF4 showed higher reprogramming efficiency. ChIP-Seq (chromatin immunoprecipitation -sequencing) and RNA-Seq experiments revealed that this L507A mutant bound more to promoters or enhancers of some pluripotency genes, such as KLF5, and drove the gene expression of them during reprogramming. Finally, MD (molecular dynamics) simulation analysis predicted that this mutant may additionally acquire a unique structural conformation in the protein-DNA complex by forming additional hydrogen bonds between several other amino acid residues and DNA. Our study demonstrates that only one amino acid modulation in a reprogramming factor can markedly improve natural transcription factor function to achieve faster and effective reprogramming, which leads to cost-effective advantages in future scientific and medical applications.

RESULTS
Design of alanine scanning KLF4 ZnF mutants based on in silico identification of putative KLF4 DNA-interacting amino acid residues First, we identified amino acid residues in the KLF4 ZnF domain directly interacting with target DNA by using PDB (protein data bank) ePISA program (Krissinel and Henrick, 2007) (Table S1). In this program, structural data of mouse Klf4 ZnF-DNA complex from two studies that solved its X-ray crystal structures were analyzed (Liu et al., 2014;Schuetz et al., 2011). The sequences of amino acid residues in the KLF4 ZnF domain are perfectly conserved between humans and mice. Next, we selected conserved amino acid residue among KLF2, KLF4, and KLF5, which were reported to show similar functions in pluripotency and reprogramming (Jeon et al., 2016;Jiang et al., 2008;Nakagawa et al., 2008;Yamane et al., 2018). As a result, 20 amino acid residues were identified as evolutionarily conserved and directly interacted with DNA (Table S1). Then, we generated 19 mutants of human KLF4, substituting wild-type residue with alanine (A) by site-directed mutagenesis in retroviral vectors except for an original alanine amino acid residue (Figure 1A). Retroviral expression and the transduced protein expression of these 19 KLF4 ZnF mutants were examined in human fibroblasts by Western blot analysis (Figures S1A-S1C). Although many of the mutant proteins were expressed comparable to WT (wild-type) KLF4, protein levels of K443A, H446A, H450A, F471A, R479A, F499A, R501A, and R504A were substantially lower than WT.
iPSC generation from Nanog-GFP MEF using KLF4 ZnF mutants To examine the role of these DNA-interacting residues in iPSC reprogramming, we generated iPSCs from MEF (mouse embryonic fibroblasts), which carry Nanog-GFP (Nanog-promoter-driven green fluorescent protein) transgene , using KLF4 ZnF alanine substitution mutants ( Figure 1B). Retroviral vectors carrying KLF4 ZnF alanine substitution mutants were transduced together with OCT4, SOX2, and MYCL1 to generate iPSCs from these MEF Takahashi and Yamanaka, 2006). Nanog-GFP-positive colonies were observed and counted by a fluorescent microscope ( Figure 1C). As . Continued (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.

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iScience 25, 103525, January 21, 2022 3 iScience Article early as day 15 after transduction, GFP-positive cells can be detected by fluorescent microscopy. On days 15 and 25 after transduction, more GFP-positive iPSC colonies were found in L507A mutant conditions than WT conditions ( Figures 1D and E). Fewer or comparable numbers of GFP-positive iPSC colonies were found in the conditions using T442A, K443A, F471A, K483A, R497A, S500A mutants, and no or few GFP-positive iPSC colonies were found in the conditions using K439A, Y441A, H446A, H450A, R473A, D475A, E476A, R479A, F499A, R501A, D503A, and H504A mutants. The ratio of Nanog-GFP-positive iPSC colonies to total colonies was also calculated on day 25 after transduction to evaluate the heterogeneity of iPSC generation. L507A mutant conditions resulted in Nanog-GFP-positive colonies at more than 60%, whereas WT or all the other mutant conditions resulted in Nanog-GFP-positive colonies at around 30% ( Figure 1F). These results indicated that L507A mutant improved both the efficiency and the speed of iPSC reprogramming from MEF and that targeted amino acid residues contained many critical sequences in reprogramming activity.
To test the effect of L507A mutant on iPSC generation in another vector system, we developed rapid and efficient iPSC generation from primary MEF using SeVdp (replication-defective and persistent Sendai virus) vectors in feeder-free conditions ( Figure 2A). iPSC generation system using SeVdp vectors, which remains persistently in the cytoplasm without integrating into the host genome, has been developed considering safety and efficacy . In this study, we constructed L507A KLF4 mutant harboring SeVdp vector from SeVdp vector expressing WT KLF4 (Nishimura et al., , 2017. Also in this system, because KLF4 is fused with a ligand-dependent destabilization domain (DD) at the N-terminus, we treated Shield-1 to stabilize exogenous KLF4 protein during reprogramming. In this culture condition, we analyzed 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 Nterminus 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.

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iScience 25, 103525, January 21, 2022 5 iScience Article the ratio of Nanog-GFP-positive cells using flow cytometry on day 5 -15 after the infection of these SeVdp vectors and counted the Nanog-GFP-positive colony numbers on day 15. From day 7, Nanog-GFP-positive cells appeared, and the ratio of them was significantly higher in the L507A condition throughout these days ( Figures 2B and 2C). The number of Nanog-GFP-positive colonies was also significantly higher in the L507A condition ( Figure 2D). Compared with the retroviral vector system, these results suggested that SeVdp vector system carrying KLF4 L507A produced mouse iPSCs (miPSCs) from somatic cells more efficiently and faster.

L507A mutant accelerated and stabilized human iPSC generation
To determine whether KLF4 ZnF mutants affect the efficiency of iPSC reprogramming in human cells, we transduced KLF4 ZnF mutants together with OCT4, SOX2, and MYCL1 to human neonatal fibroblasts (Figure 3A). TRA (Tumor-Related Antigen)-1-60 was used to detect faithfully-reprogramming cells by flow cytometry (Chan et al., 2009). On day 7 after transduction, a higher ratio of TRA-1-60-positive cells was produced by L507A compared to WT ( Figures 3B-3D). These results indicated that L507A enhanced the reprogramming process in human somatic cells. Consistent with earlier induction of Nanog-GFP-positive colonies in MEF on day 15 after transduction ( Figure 1D), the effect of L507A on iPSC reprogramming may be common in mammals.
Next, we generated human iPSCs with these vectors from human neonatal fibroblasts using SeVdp vector system in feeder-free conditions ( Figure 2E). Comparable numbers of reprogrammed iPS-like colonies were observed based only on their morphologies ( Figure 3F); however, the number and the ratio of NANOG-positive colonies were significantly higher in L507A condition ( Figures 3F-3H). These results suggested that fully-reprogrammed iPSCs were generated more efficiently in the L507A condition. To evaluate the quality of iPSC clones generated with SeVdp vectors carrying KLF4 WT or L507A, the gene expression of NANOG, residual Sendai virus NP (Nucleocapsid Protein) RNA, and markers for differentiation-defective iPSCs (i.e., HERV-H and lincRNA-RoR) (Ohnuki et al., 2014) was examined by RT-qPCR (reverse transcription-quantitative polymerase chain reaction). RNA samples at first passage of more than 10 iPSC clones generated with WT or L507A conditions were collected. These iPSC clones generated with L507A mutant (n = 16) showed relatively homogeneous NANOG expression within a 2-fold range to a normal human iPSC line ( Figure 3I). On the other hand, these iPSC clones generated with WT (n = 13) showed a huge variation in NANOG expression. Some of them showed below half of the NANOG expression than a normal human iPSC line. In addition, 6 out of 13 clones generated with WT, which carried higher NP RNA amounts at more than 10,000 times than a normal hiPSC line, showed aberrantly higher NANOG expression (p = 0.0029 by Fisher's exact test in comparison of 0 out of 17 clones generated with L507A). There were no iPSC clones generated with L507A which showed high expression of HERV-H and/or lincRNA-RoR although some iPSC clones generated with WT showed higher expression of them ( Figure 3J). These results suggested that SeVdp vector carrying L507A mutant stably produced more homogeneous high-quality human iPSC clones without residual SeVdp vectors or the aberrantly high expression of differentiation-defective markers.
Screening all the natural amino acid residue variants at L507 position in reprogramming To determine how each type of amino acid residue in L507 position affected iPSC generation efficiency, we generated a series of retroviral vectors carrying every natural amino acid residue variant at L507 position.  iScience Article GFP-positive iPSC colonies were observed in the conditions of L507A, L507N, L507C, L507G, L507K, and L507S than in WT condition ( Figure 4B). On day 25 after transduction, more GFP-positive iPSC colonies were found in L507A, L507N, L507D, L507C, L507G, and L507S than in WT ( Figure 4C). No or few GFP-positive iPSC colonies were found in the conditions using L507R, L507Q, L507P, L507W, and L507Y. The ratio of Nanog-GFP-positive iPSC colonies to total colonies was also calculated on day 25 after transduction to evaluate the heterogeneity of iPSC generation. L507A, L507N, L507D, L507C, L507E, L507G, L507M, L507S, and L507V variants produced Nanog-GFP-positive colonies at a higher ratio than WT, whereas L507K variant produced Nanog-GFP-positive colonies at a lower ratio than WT ( Figure 4D). These results indicated that L507A, L507N, L507D, L507C, L507G, and L507S variants improved both the efficiency and speed of iPSC reprogramming from MEF. Interestingly, a negative correlation (R 2 = 0.5912) was identified between the numbers of Nanog-GFP-positive colonies of each KLF4 L507 variant on day 25 and the van der Waals volume of the amino acid residue of L507 position in each L507 variant ( Figure 4E). These results indicated that KLF4 variants with smaller amino acid residues in the L507 position acquired higher reprogramming activity.
Expression levels and protein stability of KLF4 L507A mutant are not causes of the effect on iPSC reprogramming To examine the molecular mechanisms of the enhancing effect of KLF4 L507A on iPSC reprogramming, we first asked whether KLF4 L507A might hold different expression levels and/or stability from WT or not. Previous studies focused on characterizing other domains of KLF4 for its posttranslational modifications and protein stability in pluripotency and reprogramming (Dhaliwal et al., 2019;Kim et al., 2012Kim et al., , 2015Reinhardt et al., 2020;Ye et al., 2018). KLF4 protein showed variable stability with a half-life of less than 2 h to more than 12 h depending on the different cellular status, which was regulated by protein-protein interaction, post-translational modification, and DNA-binding (Dhaliwal et al., 2019). Changes in KLF4 protein stability by the modulation of KLF4 N-terminal region regulated the efficiency of iPSC generation (Kim et al., 2015;Reinhardt et al., 2020). Thus, we examined the protein expression levels and stability of L507A in comparison with WT. First, L507A showed a similar protein expression level to WT transduced by retroviral vectors (Figures S1A and S1B). Next, we evaluated changes in protein stability by in silico analysis using an X-ray crystal structure of KLF4 ZnF and DNA complex. The L507A mutation was predicted to increase protein stability only slightly; however, some other mutations, which resulted in decreasing iPSC generation, were predicted to increase protein stability more ( Figures S3A and S3B iPSCs generation with pro-reprogramming factors dissects independent or shared functions with KLF4 L507A Then, we asked whether transcription traits of the KLF4 L507A mutant might be different from WT. First, to identify which known major pathways are related to enhanced reprogramming by KLF4 L507A, we used six putative pro-reprogramming factors capable of promoting iPSCs reprogramming when added to the conventional mixture of OSKM four reprogramming factors. Pro-reprogramming factors used in this assay were as follows: FOXH1 (Forkhead box H1) (Takahashi et al., 2014), GLIS1 (Glis Family Zinc Finger 1) (Maekawa et al., 2011;Worringer et al., 2014), Klf5 (Nakagawa et al., 2008), MB (microRNA 106a-363) (Liao et al., 2011), MC (microRNA Cluster 302-367) (Liao et al., 2011), NANOG (Yu et al., 2007), and TP53DD (p53 dominantnegative form) (Hong et al., 2009). In addition, mRFP1 (monomeric Red Fluorescent Protein 1) was used as a negative control (Hotta et al., 2009). We compared the efficiency of iPSCs generation between OSKM or OSK[L507A]M with each pro-reprogramming factor using the retroviral vector system in MEF. Overall, these pro-programming factors significantly increased the number and the ratio of Nanog-GFP-positive colonies in both WT and L507A conditions compared to mRFP conditions ( Figures 5A-5C and S4). Only in the conditions that GLIS1 or Klf5 was added, there were no detectable changes between WT and L507A. These results suggested that the effect of L507A in reprogramming was epistatic to GLIS1 and Klf5 and independent from other pro-programming factors. iScience Article iPSC reprogramming (Maekawa et al., 2011), these results suggested that the enhancing effect of L507A might be partially shared common functions and/or mechanisms with GLIS1 and Klf5.
Distinctive features of DNA binding patterns of KLF4 L507A in reprogramming Next, we examined the genome-wide DNA binding patterns of KLF4 L507A by ChIP-Seq. To obtain clear DNA-binding patterns of KLF4 during reprogramming, we used rapid and efficient iPSC generation from primary MEF using SeVdp in feeder-free conditions (Figure 2A). On days 2 and 10 after transduction in this induction method, ChIP DNA samples were collected from reprogramming MEF transduced with SeVdp carrying L507A or WT. ChIP-Seq was performed using an anti-human KLF4 antibody. The numbers of total peaks in ChIP-seq data were 2,257 in WT 2d sample, 10,064 in L507A 2d sample, 5,906 in WT 10d sample, and 12,617 in L507A 10d sample (determined as FDR <0.05). Because these differences of the peaks were not reflected by the number of mapped reads (17 million in WT 2d sample, 8 million in L507A 2d sample, 18 million in WT 10d sample, and 18 million in L507A 10d sample), these results indicated that L507A had a higher affinity to specific regions of DNA/chromatin during reprogramming. The distribution of all peaks iScience Article from these samples showed that binding sites were focused around TSS (transcription start site) more in day 10 samples than day 2 samples (Figures 6A and 6B). After extracting gene sets with specific binding peaks within 5 kilobase pairs from TSS from these ChIP-seq data, we compared these gene sets between KLF4 WT and L507A conditions ( Figure 6C and Table S4). We found that 128 genes and 312 genes (33% and 48% of total genes in the WT sample) overlapped between the L507A and WT samples. To evaluate which biological pathways were involved in L507A-specific gene sets, pathway enrichment analysis was performed using the WikiPathway database (Kelder et al., 2009;Pico et al., 2008;Slenter et al., 2018). From a gene set composed of all the genes identified in the L507A sample, a category, ''PuriNetWork (WP1763)'', was significantly ranked first ( Figure 6D) although no pathways were significantly enriched from other conditions with the same analysis methods (Adjusted p values < 0.05); Figures S5A-S5C). From these pluripotency-related genes, mapped ChIP peaks around TSS of Alpl (Alkaline phosphatase), Dppa5a (Esg1), Dsg2 (Desmoglein-2), and Klf5, showed that L507A bound to the promoter or enhancer region of these genes more frequently than WT did ( Figures 6E-6H). To identify what DNA sequence motifs are specifically bound by WT and L507A KLF4, motif discovery analysis was performed. Motif discovery program identified Klf1, a close family gene of Klf4, in the WT sample, confirming that WT sample preferentially bound to the consensus sequence of conventional KLF family proteins; however, Sox2, which is another reprogramming factor and pluripotency marker, was identified in the L507A sample ( Figures 6I and 6J).
Then we asked whether KLF4 L507A had stronger DNA binding activity to SOX2 protein than KLF4 WT did. By co-immunoprecipitation, SOX2 protein was more abundantly precipitated with KLF4 L507A protein than WT protein in the reprogramming cells ( Figures 6L and 6K). These results suggested that KLF4 L507A protein existed as a complex with SOX2 protein more frequently and that KLF4 L507A might show apparently distinct patterns of DNA binding, which were closely related to the binding patterns of another reprogramming factor, SOX2, in iPSC generation.

Distinctive features of transcriptional changes driven by KLF4 L507A in reprogramming
To evaluate global gene expression pattern changes between KLF4 L507A and WT during reprogramming, RNA-Seq was performed on the samples collected from the same reprogramming conditions as ChIP-Seq. Original MEF and resulting miPSCs were also used as samples. To visualize global gene expression patterns among these samples, we performed PCA (principal component analysis) ( Figure 7A). KLF4 L507A and WT samples on day 2 were situated closely in the two-dimensional map composed of two major principal components, and there were no genes which significantly changed their expression levels. These results suggested that L507A had no or little specific effects on gene expression patterns at the initial phase of reprogramming. Among day 10 samples, L507A samples in each experimental time were situated slightly closer to miPSCs on the PC1 axis. In the comparison of Day 10 samples between WT and L507A, 507 genes, of which 181 genes were downregulated and 326 genes were upregulated ( Figure 7B). To evaluate which biological pathways were involved in these L507A-specific gene sets, gene ontology (GO) enrichment analysis was performed. From a gene set composed of up-regulated genes in L507A samples, ''cell-cell junction'' and ''regulation of kinase activity'' were ranked first as GO cellular component and GO biological process, respectively ( Figures 7C and 7D). In addition, from a gene set composed of down-regulated genes in L507A samples, ''positive regulation of BMP signaling pathway'' was ranked first as GO biological process, and no significant GO terms in GO cellular components ( Figure 7E). Of note, in the ''cellcell junction'' category, epithelial marker genes, such as Cdh1, Dsg2, and CLDN4, were included. These results suggested that L507A specifically enhanced mesenchymal-to-epithelial transition during reprogramming. To identify direct target genes which were specifically regulated by KLF4 L507A, we combined the gene sets from ChIP-Seq and RNA-Seq data. Thirty-three genes were overlapped, and Alpl, Dppa5a, iScience Article Dsg2, Esrp1, and Klf5 were included in the overlapped genes ( Figure 7F). The expression levels of these genes were low or undetectable in MEF and were gradually up-regulated toward miPSCs (Figures 7G-7K). Because we have shown that KLF4 L507A was functionally epistatic to the addition of Klf5 in iPSC generation ( Figures 5B and 5C), Klf5 should be one of the direct and specific target genes of L507A during reprogramming. These results suggested that L507A mutant might enhance iPSC generation partly through amplifying Klf activity as a whole.
L507A mutant was predicted to acquire a unique conformation of protein-DNA complex with additional DNA interaction  Figure 8A). In contrast, the conformational distribution of the Klf4(L477A)-DNA complex has an additional peak, which represents a conformation unique to the Klf4(L477A)-DNA complex.
With the same methods of MD simulations on Klf4(L477R), Klf4(L477W), or Klf4(L477Y), which showed less or no iPSC generation activity ( Figures 5A-5C), this additional peak was not be generated ( Figures S6A-S6C). The population of the unique conformation (in the region below the black line; region 2) was about 33%. In this unique conformation, L477A triggered entire structural changes extended to three ZnFs ( Figures 8B-8D). The mode of protein-DNA interaction was also changed ( Figures 5E and F). Specifically, R449, R471, and H474 changed their DNA target base. Although S415 and S444 lost or decreased their interaction to DNA in the unique conformation, Y430, R452, R467, and S472 formed additional hydrogen bonds to the phosphate groups of DNA ( Figures 8G-8J). These additional hydrogen bonds were less observed in the MD simulations on Klf4(L477R), Klf4(L477W), or Klf4(L477Y) ( Figure S6D). These results suggest that the KLF4 L507A mutant should acquire an additional structural conformation that is stabilized by additional hydrogen bonds.

KLF4 L507A variant accelerates and stabilizes reprogramming to pluripotency
In this study, we identified that KLF4 L507A variant, which was discovered by alanine scanning mutagenesis methods on DNA-interacting amino acid residues in the KLF4 ZnF domain, improved iPSC generation. This KLF4 variant increased the number and the speed of iPSC generation in mouse and human somatic cells and enriched the fully-reprogrammed iPSCs among the transduced cell population. The benefits of using this variant in iPSC generation for regenerative medicine should be shortening the iPSC generation time and decreasing the required iPSC clones to be selected. While maintaining KLF4 function as a reprogramming factor, L507A variant additionally showed distinct DNA-binding patterns and downstream gene regulation from WT. Especially, we identified that KLF5 may be one of the direct transcriptional targets of KLF4 (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. Hydrogenbond occupancy was calculated for each possible atom pair between the protein and the DNA of each cluster. The ll OPEN ACCESS iScience 25, 103525, January 21, 2022 iScience Article L507A specifically by both ChIP-Seq and RNA-Seq analysis. As KLF5 has similar functions in pluripotency and reprogramming of pluripotent stem cells (Aksoy et al., 2014;Azami et al., 2018;Nakagawa et al., 2008;Parisi et al., 2010;Yamane et al., 2018), KLF4 L507A may amplify KLF activity as a whole during reprogramming. In addition, because the effects of L507A on DNA-binding pattern and transcriptome are evident in Day 10, but not in Day 2 samples of SeVdp reprogramming, L507A may promote reprogramming in the transition from partially-reprogrammed cells to fully-reprogrammed cells by up-regulating Dppa5a, Dsg2, Esrp1, and Klf5 transcription directly. This notion is supported by the previous findings that higher expression of Dppa5a in reprogramming cells results in fully-reprogrammed fate (Guo et al., 2019;Qian et al., 2016).
We also found that the enhancing effect of KLF4 L507A on iPSC reprogramming was partially masked by the additional GLIS1. GLIS1 was identified as one of the transcription factors which can induce Nanog-GFPpositive iPSCs without KLF4 although the efficiency was much lower than KLF4 (Maekawa et al., 2011). GLIS1 also promoted iPSC generation together with other reprogramming factors as retroviral vectors or synthetic replicative RNA formats (Lee et al., 2017;Wang et al., 2019;Yoshioka et al., 2013). Like KLF4, GLIS1 is a Kruppel-like C2H2 ZnF transcription factor (Kim et al., 2002) and should not be a downstream factor of KLF4 L507A variant as the expression of GLIS1 was low in reprogramming MEF induced by either WT or L507A variant KLF4 as well as in mESCs and miPSCs (Maekawa et al., 2011). KLF4 L507A variant shares the functional effects with GLIS1 in reprogramming a part at least, thereby may render GLIS1 unnecessary to be added as a pro-reprogramming factor.
KLF4 L507 position represents a ''molecular bump'' to regulate structural stability of the interaction between ZnF domain and DNA complex In iPSC generation experiments with all the amino acid residue variants of L507, we found that smaller amino acid residues tended to have higher iPSC reprogramming activity. In addition, in MD simulation analysis, L507A mutant is predicted to acquire a unique conformation of DNA-binding complex, which had additional hydrogen bonds between three other amino acid residues and phosphate groups of DNA. These results suggest that the L507 position represents a ''molecular bump'' in the KLF4 ZnF domain. Natural L507 seems to be tuned for mild interaction in this position. Our L507A and L507G variants may be tuned strongly for accomplishing herculean reprogramming tasks. Because L507 is conserved among three KLF family homologous members, we assume that the equivalents of L507 mutants in other KLF family genes possess improved reprogramming activity. It might be interesting to test how the same mutation will affect other KLF members in reprogramming and other biological contexts. This does not necessarily mean that this mutant simply might have a higher DNA-binding affinity overall, because we observed different DNA-binding patterns between L507A and WT. It might be interesting to test how this mutant could affect DNA binding affinity using biochemical analysis.
Coincidently, our previous study identified that an alanine-substituted mutant from leucine, L122A, in NANOG DNA-binding homeodomain showed enhanced properties to promote self-renewal and reprogramming of naive pluripotent stem cells (Hayashi et al., 2015). Although ZnF domain and homeodomain do not share the structural basis in DNA recognition and interaction, there might be some common features of these leucine residues in these DNA-binding domains.

Identification of critical amino acid residues of KLF4 ZnF domain in reprogramming
In addition, our results of alanine scanning experiments in the KLF4 ZnF domain identified functionally important residues in iPSC reprogramming. Specifically, K439, Y441, R473, E476, and D503 seemed critical because their alanine-substituted mutants were defective in iPSC reprogramming assays although these mutants were considerably expressed. These residues were reported to form critical interactions and/or to be located in the important places in the ZnF-DNA protein structure (Hashimoto et al., 2016; Liu  Schuetz et al., 2011). Thus, our study demonstrated the functional meaning of KLF4 ZnF domain and its DNA-binding interaction with regard to its pluripotency induction.
In summary, we have demonstrated the first generation of KLF4 ZnF variants with enhanced reprogramming activity. Only one amino acid modulation in a reprogramming factor can markedly improve natural transcription factor function to achieve faster and effective reprogramming, leading to cost-effective advantages in future scientific and medical applications. Moreover, as the protein bioengineering technology continues to proceed as ZnF proteins are used as versatile tools to bind any desired DNA sequences (Pabo et al., 2001), our study shares new insights regarding molecular mechanisms of transcription factors' DNAbinding with possible implications in designing highly engineered factors.

Limitations of the study
Our study aimed to focus on the improvement of reprogramming activity using KLF4 ZnF mutants.
Although we determined the effects of KLF4 ZnF mutants on iPSC reprogramming in this study, we cannot assure that these effects can be reproducible in other biological contexts. Because KLF4 has pleiotropic effects in a context-dependent manner, it might be interesting to investigate the function and the effect of these variants in other biological contexts in the future. It was especially difficult to express some of the mutants as we have seen in Figures S1 and S2. There are two possible reasons for the reduction. One may be a biological reason that mutant proteins had defects in their expression and stability. Another may be a technical reason that the vectors of the mutants had problems. Although we verified the sequence of the whole length of the KLF4 mutants, we could not rule out that these vectors of the mutant had defects in the sequence outside KLF4. In addition, although we have addressed the positive effects of the L507A on iPSC generation in this study, there might be potential negative side effects of this mutant because of a shifting in binding affinities and target sequences. We could develop better mutants that avoid such negative side effects by aiming to prolong protein half-life and/or improve expression levels in the future.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following: