Journal: Nucleic Acids Research

Article Title: A systematic comparison of FOSL1, FOSL2 and BATF-mediated transcriptional regulation during early human Th17 differentiation

doi: 10.1093/nar/gkac256

Figure Lengend Snippet: Expression of FOS-like proteins during human Th17 differentiation. ( A ) Rpkm values are plotted for FOSL1 (left) and FOSL2 (right) RNA at different time points of activation (Th0) or Th17-differentiation, using our published RNA-seq data . ( B ) Immunoblot images (lower panel) show FOSL1 (left) and FOSL2 (right) protein levels in Th0 and Th17-polarizing cells, over a time-course. Actin was used as loading control. Blots from three biological replicates were quantified using ImageJ and FOSL intensity values (normalized to actin) were plotted as a line graph in the above panel. ( C ) Flow cytometry analysis of FOSL1 (left) and FOSL2 (right) expression in naive CD4 + T-cells cultured for 24 h, under conditions of activation (Th0), Th17-differentiation, or activation in presence of the Th17-cytokines (used either alone or in combination). Bar plot shows median fluorescence intensity (MFI) values normalized to Th0, for three biological replicates. Statistical significance was calculated by comparing each condition to Th0. ( D ) Flow cytometry analysis of FOSL1 (left) and FOSL2 (right) protein levels in non-targeting (SCR) versus STAT3 KD cells, at 24 h of Th17 polarization. Graph shows MFI values normalized to SCR for three biological replicates. Adjoining histogram (flow cytometry analysis) confirms the depletion of STAT3 protein levels in the KD cells at 24 h of Th17 polarization. All graphs in panels B–D show mean ± standard error of the mean (SEM). Statistical significance is calculated using two-tailed Student's t test (* p < 0.05; ** p < 0.01, *** p < 0.001). ( E ) UCSC genome browser snapshots indicate the binding of STAT3 over the promoter of FOSL2 (above panel) and not FOSL1 (below panel), in Th17 cells cultured for 0.5 and 72 h. Figures were derived using bed files of STAT3 ChIP-seq data from our published study .

Article Snippet: To generate linearized vectors for the IVT reaction, the T7 promoter containing plasmids: empty pGEM-GFP64A, pCMV6-AC-GFP-FOSL1 (Origene, Cat. no. RG202104) and pCMV6-AC-GFP-FOSL2 (Origene, Cat. no. RG204146), were in vitro digested using the restriction enzymes Spe1 (NEB, Cat. no. R0133), Xma1 (NEB, Cat. no. R0180) and Ssp1 (NEB, Cat. no. R3132), respectively.

Techniques: Expressing, Activation Assay, RNA Sequencing Assay, Western Blot, Flow Cytometry, Cell Culture, Fluorescence, Two Tailed Test, Binding Assay, Derivative Assay, ChIP-sequencing

Journal: Nucleic Acids Research

Article Title: A systematic comparison of FOSL1, FOSL2 and BATF-mediated transcriptional regulation during early human Th17 differentiation

doi: 10.1093/nar/gkac256

Figure Lengend Snippet: FOSL1 and FOSL2 negatively regulate IL-17 expression. ( A ) Nucleofection workflow for FOSL1/FOSL2 knockdown (KD) is shown in the left panel. Naive CD4 + T-cells were treated with two different siRNAs targeting FOSL1 or FOSL2. Cells were rested at 37°C in RPMI-medium, and further cultured under Th17-polarizing conditions. At 24 h post differentiation, knockdown was analysed using immunoblotting (right panel). Representative blots for three biological replicates are shown. ( B ) ELISA was used to estimate IL-17A secretion in supernatants of FOSL1 (left) and FOSL2-silenced (right) Th17 cells, at 72 h of polarization. Values were first normalized for cell count (live), and then normalized to SCR control. Data represents four or five biological replicates, as indicated. ( C ) Naive CD4 + T-cells were silenced for FOSL1, FOSL2 or both factors in parallel (double KD; DKD) and cultured under Th17-polarizing conditions for 24 h. Total FOSL1 (left) or FOSL2 (right) protein was stained (Alexa-647) and analysed by flow cytometry. Representative histograms for four biological replicates are shown. ( D ) Bar plot shows ELISA results for secreted IL-17A levels in supernatants of FOSL KD/DKD Th17 cells, at 72 h of polarization. Values were first normalized for cell count (live), and then normalized to SCR control. Data represents four biological replicates. ( E ) qRT-PCR analysis for measurement of IL-17A (left) and IL-17F (right) RNA levels in FOSL KD/DKD Th17 cells (72 h). Fold-change normalized to the control was plotted for four biological replicates. ( F ) Naive CD4 + T-cells were treated with in-vitro transcribed GFP-FOSL1 RNA, GFP-FOSL2 RNA or both (double OE; DOE). GFP RNA was used as nucleofection control. After resting the cells for 18–20 h, total FOSL1 (left) or FOSL2 (right) protein was stained (Alexa-647) and analysed by flow cytometry. Representative histograms for four biological replicates are shown. ( G, H ) Graphs show IL-17 secretion (panel G) and IL-17A/F RNA expression (panel H) in FOSL OE/DOE Th17 cells at 72 h of polarization, as assessed by ELISA and qRT-PCR analyses, respectively. ELISA values were first normalized for cell count (live), and then normalized to GFP control. Panel H depicts fold change normalized to control. Data represents four biological replicates. Plots in figures B, D, E, G, H show mean ± SEM. Statistical significance is calculated using two-tailed Student's t test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: To generate linearized vectors for the IVT reaction, the T7 promoter containing plasmids: empty pGEM-GFP64A, pCMV6-AC-GFP-FOSL1 (Origene, Cat. no. RG202104) and pCMV6-AC-GFP-FOSL2 (Origene, Cat. no. RG204146), were in vitro digested using the restriction enzymes Spe1 (NEB, Cat. no. R0133), Xma1 (NEB, Cat. no. R0180) and Ssp1 (NEB, Cat. no. R3132), respectively.

Techniques: Expressing, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay, Cell Counting, Staining, Flow Cytometry, Quantitative RT-PCR, In Vitro, RNA Expression, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: A systematic comparison of FOSL1, FOSL2 and BATF-mediated transcriptional regulation during early human Th17 differentiation

doi: 10.1093/nar/gkac256

Figure Lengend Snippet: Transcriptional targets co-regulated by FOSL1 and FOSL2. ( A, B ) Heatmap in panel A shows the DE genes that are more profoundly altered in FOSL DKD Th17 cells, as compared to the single KD controls, at 24 h (above) and 72 h (below) of polarization. Panel B depicts the DE genes that show enhanced changes in FOSL DOE Th17 cells, as compared to the single OE controls, at 72 h of polarization. Fold-change (FC) was calculated relative to the respective control conditions (i.e. SCR or GFP). The FDR filtered (≤0.1) DE genes with |FC| ≥ 1.8 in DKD, and |FC| ≥ 2 in DOE, are included in the corresponding heatmaps. Th17-relevant, upregulated genes are depicted in red, and the downregulated ones are in blue. ( C, D ) Ingenuity pathway analysis (IPA) was used to identify signaling pathways that are altered upon FOSL DKD (panel C) or DOE (panel D). The top pathways related to T-cells and immune signaling are selectively shown. ( E, F ) Genome-wide expression analysis of FOSL DKD and DOE Th17 cells. Volcano plots in panel E highlight the Th17-associated transcripts that are differentially expressed upon co-depletion of FOSL1 and FOSL2, at 24 h (left) and 72 h (right) of Th17 polarization. Panel F shows the Th17-associated genes that are differentially expressed upon parallel over-expression of FOSL1 and FOSL2, at 72 h of Th17 polarization. Targets with FDR ≤ 0.1 and |FC| ≥ 1.8 have been plotted. Upregulated genes are in red, and the downregulated ones are in blue (for extended list of DKD and DOE targets, refer to ).

Article Snippet: To generate linearized vectors for the IVT reaction, the T7 promoter containing plasmids: empty pGEM-GFP64A, pCMV6-AC-GFP-FOSL1 (Origene, Cat. no. RG202104) and pCMV6-AC-GFP-FOSL2 (Origene, Cat. no. RG204146), were in vitro digested using the restriction enzymes Spe1 (NEB, Cat. no. R0133), Xma1 (NEB, Cat. no. R0180) and Ssp1 (NEB, Cat. no. R3132), respectively.

Techniques: Genome Wide, Expressing, Over Expression

Journal: Nucleic Acids Research

Article Title: A systematic comparison of FOSL1, FOSL2 and BATF-mediated transcriptional regulation during early human Th17 differentiation

doi: 10.1093/nar/gkac256

Figure Lengend Snippet: 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.

Article Snippet: To generate linearized vectors for the IVT reaction, the T7 promoter containing plasmids: empty pGEM-GFP64A, pCMV6-AC-GFP-FOSL1 (Origene, Cat. no. RG202104) and pCMV6-AC-GFP-FOSL2 (Origene, Cat. no. RG204146), were in vitro digested using the restriction enzymes Spe1 (NEB, Cat. no. R0133), Xma1 (NEB, Cat. no. R0180) and Ssp1 (NEB, Cat. no. R3132), respectively.

Techniques: Genome Wide, Immunofluorescence, ChIP-sequencing, Cell Culture, Binding Assay, Expressing

Journal: Nucleic Acids Research

Article Title: A systematic comparison of FOSL1, FOSL2 and BATF-mediated transcriptional regulation during early human Th17 differentiation

doi: 10.1093/nar/gkac256

Figure Lengend Snippet: 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.

Article Snippet: To generate linearized vectors for the IVT reaction, the T7 promoter containing plasmids: empty pGEM-GFP64A, pCMV6-AC-GFP-FOSL1 (Origene, Cat. no. RG202104) and pCMV6-AC-GFP-FOSL2 (Origene, Cat. no. RG204146), were in vitro digested using the restriction enzymes Spe1 (NEB, Cat. no. R0133), Xma1 (NEB, Cat. no. R0180) and Ssp1 (NEB, Cat. no. R3132), respectively.

Techniques: Binding Assay, Expressing, ChIP-sequencing, Western Blot, Cell Culture, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: A systematic comparison of FOSL1, FOSL2 and BATF-mediated transcriptional regulation during early human Th17 differentiation

doi: 10.1093/nar/gkac256

Figure Lengend Snippet: BATF and FOSL proteins show common interacting partners in Th17 cells. ( A ) Figure illustrates the common binding partners of FOSL1 and FOSL2 in Th17 cells (72 h), based on data acquired from our recent study . Interactors having reported roles in T-cell function are shown. ( B ) STRING network analysis of human BATF. Width of lines between the nodes indicate confidence values for each protein-protein association. Interactions with a minimum score of 0.7 are shown (high confidence). ( C ) BATF was immunoprecipitated using 72 h-polarized Th17 cultures. Immunoblotting was then used to analyse its interaction with selected (shared) binding partners of FOSL1 and FOSL2 (JUNB, SIRT-1, JUN and RUNX1). Data is shown for three biological replicates. Immunoblot for BATF confirms immunoprecipitation of the factor.

Article Snippet: To generate linearized vectors for the IVT reaction, the T7 promoter containing plasmids: empty pGEM-GFP64A, pCMV6-AC-GFP-FOSL1 (Origene, Cat. no. RG202104) and pCMV6-AC-GFP-FOSL2 (Origene, Cat. no. RG204146), were in vitro digested using the restriction enzymes Spe1 (NEB, Cat. no. R0133), Xma1 (NEB, Cat. no. R0180) and Ssp1 (NEB, Cat. no. R3132), respectively.

Techniques: Binding Assay, Cell Function Assay, Immunoprecipitation, Western Blot

Journal: Nucleic Acids Research

Article Title: A systematic comparison of FOSL1, FOSL2 and BATF-mediated transcriptional regulation during early human Th17 differentiation

doi: 10.1093/nar/gkac256

Figure Lengend Snippet: SNPs associated with autoimmune diseases localize within the genomic binding sites of FOSL1, FOSL2 and BATF. ( A ) Enrichment of disease-associated SNPs (or their proxies in Caucasian populations) within FOSL1, FOSL2 and BATF genomic-binding sites, relative to random sets of background SNPs. ( B ) SNPs relevant to the study were shortlisted . Of these, the SNPs that were functionally validated in DNA-affinity precipitation assays are shown. ( C, D ) DAPA followed by immunoblot analysis shows the SNPs that alter the binding of FOSL1, FOSL2 or BATF to their genomic sites (identified by ChIP-seq analysis). Wildtype (WT) oligonucleotides containing the binding motifs of these TFs (at different genomic loci), and mutant oligonucleotides harbouring a SNP within the binding motif, were used as baits for pull-down of the corresponding AP-1 factor from 72 h Th17-polarized cell lysates. For experimental controls, an oligonucleotide with a conserved binding sequence for BATF (BATF WT), and the corresponding mutated sequence which is known to disrupt BATF occupancy (BATF MUT) were used. Panel C includes SNPs affecting the binding of either FOSL1, FOSL2 or BATF. Those SNPs at the common binding sites of the three factors which also alter the binding affinities for all of them are shown in panel D. The common SNPs harboured within consensus AP-1 motifs are labelled. Data is representative of three biological replicates.

Article Snippet: To generate linearized vectors for the IVT reaction, the T7 promoter containing plasmids: empty pGEM-GFP64A, pCMV6-AC-GFP-FOSL1 (Origene, Cat. no. RG202104) and pCMV6-AC-GFP-FOSL2 (Origene, Cat. no. RG204146), were in vitro digested using the restriction enzymes Spe1 (NEB, Cat. no. R0133), Xma1 (NEB, Cat. no. R0180) and Ssp1 (NEB, Cat. no. R3132), respectively.

Techniques: Binding Assay, Affinity Precipitation, Western Blot, ChIP-sequencing, Mutagenesis, Sequencing