Fig 2: Comparing transcriptional targets and genomic binding sites of FOSL proteins with BATF. (A) Heatmap on the top shows logarithmic FC values for the DE genes that show opposite expression changes in FOSL DKD and BATF KD Th17 cells, at the indicated time points of differentiation. Heatmap in the bottom panel depicts the DE genes that show similar expression changes in FOSL DOE and BATF KD Th17 cells. Th17-related genes are highlighted in red. (B) ChIP-seq profiles of FOSL1, FOSL2 and BATF in Th17 cells. Graph (above) shows the overlay between the peak distribution profiles of the three TFs. Bar plot (below) depicts peak-annotation results for their identified binding sites. (C) Heatmap with k-means clustering shows the ChIP-seq signal intensities ± 2-kb around the centers of the genomic-binding regions of FOSL1, FOSL2 and BATF. (D) Venn diagram shows an overlap between the genomic binding sites of FOSL1, FOSL2 and BATF (overlap represents peaks sharing 200 bp or more). Adjoining heatmap depicts Log2FC values for the gene targets that are co-bound and oppositely regulated by FOSL proteins and BATF, at the given time points of Th17 differentiation. Genes showing shared occupancy of the three factors over promoter regions have been marked (*asterisk). Th17-related targets are highlighted. (E) IGV track snapshots illustrate the co-localization of FOSL1, FOSL2 and BATF over selected Th17-linked genes. The profile of H3K27ac histone mark around the shared binding sites of the three factors is shown. (F) Bar plot depicts immunoblot-based expression analysis of STAT4 in FOSL DKD (left) and BATF KD (right) Th17 cells, cultured for 72 h. Data shows mean ± SEM for three or four biological replicates, as indicated. Statistical significance is calculated using two-tailed Student's t test (*p < 0.05). Adjoining IGV track shows the binding overlap of FOSL1, FOSL2 and BATF, flanked by H3K27ac marks near the STAT4 locus.

Fig 3: Genome-wide occupancy profile of FOSL proteins in human Th17 cells. (A) Immunofluorescence images showing nuclear localization of FOSL1 (red, above panel) and FOSL2 (red, below panel) in 72 h-polarized Th17 cells. Lamin A/C (in green) marks the nuclear periphery, whereas phalloidin (in blue) stains the cytoplasmic actin. (B) ChIP-seq analysis was performed for FOSL1 and FOSL2 using 72 h-cultured Th17 cells. Figures on the left show distribution of FOSL1 and FOSL2 binding sites relative to the position of the closest transcription start site (TSS). TSS is defined to be at position zero. Figure on the right shows an overlay of the peak distribution profiles of the two factors. (C) The topmost consensus sequences for FOSL1 and FOSL2 genomic binding were identified using de-novo motif enrichment analysis by Homer. FOSL1 (left) and FOSL2 (right) peaks were further enriched for known TF motifs, and the top motifs identified by Homer are shown. Peaks with IDR p < 0.01 were used for motif discovery. (D) ChIPpeakAnno was used to determine the overlap in the genomic binding sites of FOSL1 and FOSL2 (overlap represents peaks sharing 200 bp or more). Genes neighbouring to these overlying sites and differentially expressed under DKD or DOE conditions (FDR ≤ 0.1, |FC| ≥ 1.5) were assigned as the shared-direct targets of FOSL1 and FOSL2. Adjoining volcano plots show the logarithmic fold changes for selected shared targets (DKD (left); DOE (right)). Downregulated genes are in blue, and upregulated ones are in red. Targets with FOSL occupancy over promoter regions (5-kb window around TSS) are marked with asterisk. (E) Integrative Genomics Viewer (IGV) track snapshots show the binding overlap of FOSL1 and FOSL2 over selected Th17-associated genes. (F) Heatmap depicts the shared direct targets that show opposite expression changes in FOSL DKD and DOE conditions, at the indicated time points of Th17 differentiation. Th17-relevant genes have been highlighted.

Fig 4: LINC00313 positively regulates FOSL2 by sponging miR-342-3p. Effects of LINC00313 on FOSL2 expression in U2OS and MG-63 cells were illuminated by transfection with siLINC00313/Scramble or co-transfection of siLINC00313 and anti-miR-342-3p/anti-NC. FOSL2 expression was determined using qPCR and Western blotting in U2OS (A and B) and MG-63 (C and D) cells. All experiments were carried out in triplicate. *p<0.05.

Fig 5: TF FOSL2 expression is up-regulated in PDAC mouse models.a Enrichment of all known motifs within each cluster of accessible regions. All motifs with an enrichment −(log P value) with a base of 10. TF categories are indicated on the right side. The motifs upon the −(log P value) with a base of 10 of motifs greater than 150 for clusters 1–5, separately, were shown. b–d Analysis of mRNA levels of TFs associated with clusters 1–5 in WT, KC, and KPC mice. The KC and KPC mice were compared with WT, respectively. *P < 0.05; **P < 0.01; ***P < 0.001. e Representative KPC-specific motifs and corresponding candidate TFs. f Genome browser view of ATAC-seq, H3K27ac ChIP-seq, and RNA-seq coverage at the Atf3, Junb, and Fosl2 loci in WT, KC, and KPC mice. g RT-qPCR assay was performed to detect the Fosl2 expression in WT, KC, and KPC mice. n = 4 for each group. The KC and KPC mice were compared with WT, respectively. **P < 0.01. h Immunoblot showing the abundance of FOSL2 in WT, KC, and KPC mice. i Representative images of IHC staining for FOSL2 in WT pancreas, KC, and KPC neoplastic tissues. Scale bar, 100 μm. ***P < 0.01.