Validation of FOXK2 targets identified by ChIP-seq. (A) Schematic illustration of the His-Flag (HF)-tagged FOXK2 construct. The Western blots show the expression of FOXK2-HF (using anti-Flag antibody) and total endogenous (Endo) and HF-tagged FOXK2 (using anti-FOXK2 antibody) in U2OS-HF (−) and U2OS-FOXK2-HF (FOXK2) cells. IB, immunoblotting. (B) MACS profiles of FOXK2 binding to the indicated genomic regions and input controls for the same regions. Where applicable, the transcriptional start site (TSS) is indicated. chr3, chromosome 3. (C and D) qPCR-ChIP validation of FOXK2 binding to genomic regions associated with the indicated genes using either anti-Flag antibody in U2OS-FOXK2-HF cells (C) or anti-FOXK2 antibody in U2OS cells (D). SRF intron 3 (int3) is a negative control (NC). Data are the averages plus standard deviations (error bars) of duplicate samples and are representative of 3 independent experiments and are shown relative to enrichment with Flag antibody in U2OS-HF cells (C) or nonspecific IgG (taken as 1) (D). The dotted line in panel represents the average of the negative controls (NC).

Coassociation of AP-1 binding events with FOXK2 binding regions. (A) Best octamer motif identified by Weeder analysis following masking out the GTAAACA motif. The pie chart indicates the percentage of binding regions that contain matches to this motif. (B) Occurrence of TGANTCA motifs depending on the location of FOXK2 binding regions or an equivalently sized set of random background regions. Significant differences in the distribution of the motifs between the indicated genomic locations and a background data set are indicated by asterisks as follows: ∗, P < 0.05; ∗∗, P < 0.0001. (C) Comparison of the genomic regions bound by FOXK2 in U2OS cells (red circles) and those bound by the indicated transcription factors in K562 cells (blue circles). Percent overlap values are relative to FOXK2. All 8,600 FOXK2 binding sites are used. (D) Overlap of ChIP-seq-derived binding regions for the indicated transcription factors and the 1,600 FOXK2 binding regions that contain TGANTCA motifs. Percent overlap is given relative to the FOXK2 data set.

Association of FOXK2-regulated genes with regions occupied by FOXK2. (A) FOXK2 was depleted from U2OS cells with siRNA, and genes whose expression changed by at least 25% (up or down) are compared to the presence of a FOXK2 binding event in their vicinity. siFOXK2, siRNA against FOXK2. (B) FOXK2 was overexpressed in HeLa cells by doxycycline treatment for 24 h, and genes whose expression changed by at least 1.25-fold (up or down) are compared to the presence of a FOXK2 binding event in their vicinity. Z-scores are calculated from comparing the FOXK2 ChIP-seq data with equivalent sized randomly chosen RefSeq genes. (C) The ratio between the numbers of genes going up and down by 1.25-fold following FOXK2 depletion is plotted for the indicated data sets. “All genes” represents all genes that change on the array, whereas the genes that also contain FOXK2-associated binding regions (gray bars) are shown in their entirety (all regions) or split into those that contain an AP-1 binding motif.

FOXK2 is required for AP-1 recruitment and maximal AP-1 activity. (A) Correlations between PMA-mediated gene expression in U2OS cells and FOXK2 regulatory effects. Gene expression changes (1.25-fold, P value < 0.05) caused by PMA stimulation for 2 h or by depletion of FOXK2 in the PMA-treated cells, were correlated, and the overlapping genes were compared to FOXK2 targets identified by ChIP-seq in the high-confidence data set. Significance was calculated by Z-score analysis compared to 10,000 sets of equivalently sized randomly selected genes. (B) qPCR analysis of the indicated genes in the presence of PMA stimulation for 2 h and depletion of FOXK2. Data are shown relative to each target gene in the presence of siRNA duplexes against GAPDH (taken as 1) and are the averages plus standard deviations (error bars) of three independent experiments. (C) Reporter gene analysis of the indicated promoter-reporter constructs in the presence (+) and absence (−) of PMA stimulation and/or FOXK2 overexpression. Data are the averages plus standard deviations (error bars) for three independent experiments. (D) Western blot analysis of the levels of the indicated proteins in cells treated for 2 h with PMA following depletion of GAPDH (control) or FOXK2. (E) ChIP analysis of FOS binding to FOXK2 binding regions in PMA-treated cells in the presence of siRNAs against GAPDH (control [Con]) and/or FOXK2. Data are shown relative to FOS binding in the presence of siRNA duplexes against GAPDH (taken as 1), are the averages of duplicate samples from two (for LOR) or four independent experiments. Values that are significantly different (P value of ≤0.05) from the control value are indicated by an asterisk.

Characterization of the FOXK2 binding events. (A) Genomic distribution of the FOXK2 binding events and a random set of genomic coordinates. 5′UTR, 5′ untranslated region. (B) Best octamer motif identified by Weeder analysis. The pie chart indicates the percentage of binding regions that contain matches to this motif. (C) Binding specificity of FOXK2. The occurrence of each of the indicated motifs in the FOXK2 binding regions compared to a background data set is shown. The dotted line represents the average number of motifs expected in the background data set based on an even distribution of each sequence. Statistically significant overrepresentation is indicated by asterisks as follows: ∗, P values < 0.05; ∗∗, P < 0.0001. A consensus binding region based on these preferences is shown below. (D) Occurrence of GTAAACA motifs depending on the locations of FOXK2 binding regions or an equivalently sized set of random background regions. A significant difference in the distribution of the motifs between the indicated genomic locations (P value = 0.0013) is indicated by the bracket and asterisk. (E) Comparison of the genomic regions bound by FOXK2 and those bound by the indicated FOX transcription factors. Percentage overlaps are relative to FOXK2. All 8,600 FOXK2 binding sites are used except for comparisons with FOXC2 ChIP-chip data where 1,603 sites are compared that are located within the same search space. (F) Top KEGG pathways identified as overrepresented among the genes associated with FOXK2 binding regions. MAPK, mitogen-activated protein kinase; TGF-beta, transforming growth factor β.

FOXK2 binding regions are associated with open chromatin. (A) qPCR analysis of FAIRE data. Data are normalized against uncross-linked genomic DNA for each primer pair (taken as 1). Signals obtained from FAIRE and input DNA are shown. FAIRE Chr18 represents a control region corresponding to a known FAIRE region found in pancreatic islet cells (13). (B) Overlap of FAIRE-seq regions from U2OS cells (P value < 1 × 10⁻⁵) and regions bound by FOXK2. The overlap with a comparable number of random genomic regions is shown. (C) The overlap of FAIRE-seq regions with regions bound by FOXK2 is further partitioned into FOXK2 regions that contain the AP-1 motif TGANTCA and those that do not contain the AP-1 motif. (D) Reporter gene analysis of genomic fragments containing FOXK2 binding regions. The name represents the closest gene to the particular fragment. The dotted line represents the activity of the empty pGL3 vector. Data are the averages plus standard deviations (error bars) of duplicate samples from three independent experiments.

FOXK2 and AP-1 coassociate on chromatin. (A) Western blot analysis of AP-1 component expression following stimulation of U2OS cells with PMA for the indicated times. Immunoblotting (IB) was performed with the indicated antibodies. (B to D) ChIP analysis of FOXK2 (B), JUN (C), and FOS (D) binding to FOXK2 binding regions. Cells were asynchronously growing except for the analysis of FOS binding, where cells were serum starved for 24 h followed by treatment with PMA for 2 h. Data are shown relative to binding to nonspecific IgG (taken as 1). Data are the averages plus standard deviations (error bars) of duplicate samples and are representative of 2 or 3 experiments. (E) Coimmunoprecipitation analysis of FOXK2 with JUN. U2OS cells were transfected with the indicated plasmids encoding Flag-tagged FOXK2 and HA-tagged JUN, followed by immunoprecipitation (IP) with anti-Flag antibody and immunoblotting (IB) with the indicated antibodies. GAPDH is a loading control. (F) Re-ChIP analysis using antibodies against FOS or JUN to reprecipitate the genomic regions associated with the indicated target genes from FOXK2 precipitated chromatin. Data are shown relative to binding to beads alone (control [Con]; taken as 1).