Fig 2: KLF4K409Q activates FGF3 expression(A) Venn diagram showing the overlap of DEGs in HEK293 or A549 cells by RNA-seq analysis.(B) Scatter-plot of the log2 (fold change) of all genes called as significant in KLF4 or KLF4K409Q RNA-seq analysis. Positions of dots corresponding to FGF3, CALML5, ALPG, and TRH genes are marked by black arrows.(C) Time course of FGF3 and TRH mRNA expression in HEK293 cells transfected with KLF4- or KLF4K409Q-expressing plasmids by RT-qPCR analysis. Number of copies on the y-axis is presented as calculated copy number per 1,000 copies of GAPDH mRNA in the same sample (see also Figure S1).

Fig 3: FGF3 locus STRs bind KLF4K409Q and enhance KLF4K409Q-specific FGF3 promoter activity(A) Activity of luciferase (LUC) gene under the control of minimal FGF3 promoter (from -236 to +84 bp from TSS), alone or with different STRs positioned as enhancers. Reporter vectors were transiently co-transfected with KLF4- or KLF4K409Q-expressing plasmids into HEK293 cells. Luciferase activity was measured 48 h posttransfection. Data are represented as mean ± SD from at least four independent experiments.(B) Quantitative DNase I footprinting analysis of the 441 bp DNA fragment from STR-52 kb upstream of FGF3 TSS (FGF3-52 kb) with increasing amounts of recombinant KLF4 and KLF4K409Q DBD proteins as indicated. G + A ladder is shown as probe sequence marker. Open bars mark areas of KLF4 protein binding. The wide long grey box denotes the binding area of KLF4K409Q protein.(C) Quantitative DNase I footprinting analysis of the 463 bp DNA fragment from STR in FGF3 intron 2 (FGF3-IN2.1) was performed and marked as in (B). Both coding (+) and template (-) DNA strands were labeled and tested (as indicated) (see also Figure S4).

Fig 4: KLF4K409Q-binding regions in the FGF3 locus appear to be vast short tandem repeats (STRs)(A) ChIP-seq analysis of FGF3 locus. Bigwig tracks display log2 ratio of KLF4 ChIP-seq coverage relative to input. One representative replicate for each ChIP-seq condition (KLF4 in blue and KLF4K409Q in red) is labeled on the left side. Six independent biological replicates for each ChIP-seq condition were analyzed and are shown in Figure S3. Schematic position and direction of FGF3 gene transcription are shown below the tracks. Bottom track shows the FGF3 locus alignment with heatmap of RefSNPs database. Promoter and STR regions are depicted above the plots (see also Figures S3 and S6).(B) Sequences of FGF3 locus STRs. Schematic drawing (up-to-scale) of FGF3 locus shown as a thick grey line. FGF3 gene and its direction of transcription is shown by black arrow. Exons presented as thin black boxes. Promoter region and STRs are shown as wider open boxes. STR sequences are shown above and below the locus scheme in blow-out windows. Tandem copies of 19 bp repeats in -52 kb STR are labeled by alternating black and green letters. Nucleotides corresponding to the 10 bp KLF4K409Q consensus site are shown above the sequence window with bold letters (Y: pyrimidine, R: purine). Tandem copies of 4 bp repeats in IN2.1 and IN2.2 STRs from FGF3 intron 2 are marked by red, blue, and black letters. STR lengths (bp) and direction of FGF3 transcription are marked above each window with number and arrow (see also Figures S4 and S7).

Fig 5: KLF4 and KLF4K409Q exhibit distinct binding specificity in vitro and in vivo(A) Venn diagram showing the overlap of the peaks in KLF4 and KLF4K409Q sets by ChIP-seq analysis of HEK293 cells with ectopically expressed proteins.(B) Genomic (left) and epigenetic (right) context for the three ChIP-seq peak sets: KLF4, KLF4K409Q, and Shared. Categories for epigenetic context are defined by the ENCODE SCREEN project (https://screen.encodeproject.org/).(C) Heatmap of read density of KLF4 and KLF4K409Q ChIP-seq at ranges ±2 kb around consensus peak sets. The read depth was normalized across all six biological replicates for shared consensus peaks.(D) Scatter plot and boxplots of log2 average normalized read depth for peaks in each of three consensus sets: KLF4, KLF4K409Q, and Shared.(E ) Heatmap of normalized read depth per replicate for the top 10% most variable peaks in each set. Rows represent peaks. Columns represent replicates.(F) Motifs discovered de novo from differentially bound sequences within each set in (E). Sequence letter height is correlated with conservation.(G) EMSA analysis using FGF3-201 and TRH-122 sites and their mutants as probes and purified recombinant DBD of MBP-KLF4 or MBP-KLF4K409Q proteins (left panel) or BL21 E. coli lysates expressing MBP-KLF4 full-length proteins (right panel), as indicated. Free probes and DNA/protein complexes are marked by arrows.(H) Sequence of DNA probes used in EMSA in (G) and mutated nucleotides (in red) are aligned with the KLF4 DNA-binding consensus below. Nucleotides corresponding to the aligned consensus are bolded and nucleotide triplicates binding different ZFs are interspaced. N: any nucleotide; Y: pyrimidine (C or T); K: Keto (G or T). See also Tables S2 and S3.