Journal: bioRxiv

Article Title: BATF controls IFN I production via DC-SCRIPT in plasmacytoid dendritic cells

doi: 10.1101/2024.01.11.574638

Figure Lengend Snippet: ( A ) Visualization of integrated RNA-Seq and ChIP-Seq data from FACS-sorted murine BM-derived Flt3L-cultured WT pDCs using Cytoscape. Gene shape represents gene groups (filled circles = IFN I, triangles = ISGs, Squares = IRFs), colour of the gene shape indicates expression (red: high expression, yellow: moderate expression, green: low expression, grey: no expression), and colour of the arrow shows interaction (blue) or no interaction (grey) with BATF according to ChIP-Seq data. ( B ) Integrated RNA-Seq, ChIP-Seq and ATAC-Seq data for all mouse transcription factors in untreated (left) and 2h CpG stimulated (right) FACS-sorted murine BM-derived Flt3L-cultured pDCs visualized in Venn diagrams. Total number of genes among all mouse transcription factors is shown for expression of genes between WT and Batf -/- pDCs (RNA-Seq, fold change ≥ |1.5| and FDR ≤ 0.05, EdgeR), direct BATF interaction with DNA (ChIP-Seq peaks called after MACS) in WT pDCs and a differentially opening of chromatin between WT and Batf -/- pDCs (ATAC-Seq, fold change ≥ |1.5| and FDR ≤ 0.05, DESEq2). Genes matching all three criteria are listed to the right of the Venn diagrams. ( C ) Quantitative RT-PCR (left) for the expression of Zfp366 in FACS-purified untreated and CpG-stimulated WT ( Batf +/+ ) and Batf -/- pDCs. Heatmap (right) showing the normalized counts per million (cpm) expression of Zfp366 in Batf +/+ and Batf -/- pDCs at steady state (untreated) and after CpG stimulation (2h, 6h, 12h) from RNA-Seq data. ( D ) Quantitative RT-PCR for the relative expression of Zfp366 mRNA in Ifnar -/- and WT ( Ifnar +/+ ) purified BM-derived pDCs, which were left untreated or stimulated with CpG or IFNα2 for 6h. ( E ) Histograms (upper panel) and bar chart (lower) showing the Zfp366-tdTomato expression as MFI in untreated (-) and for 24h CpG-stimulated (CpG 1668/CpG 2216) BM-pDCs from DC-SCRIPT-reporter or WT (control; grey bar) mice. ( F ) Top panel presents a screen shot from the ECR (evolutionary conserved regions) Browser web site of 5’ region of the mouse Zfp366 gene. Intronic regions are depicted in pink, UTRs in yellow and conserved non-coding sequence (CNS) in red. Bottom panels present BATF ChIP-Seq (green) in sorted Batf +/+ , untreated and CpG stimulated (2h) pDCs, as well as ATAC-Seq (red) peaks in Batf +/+ and Batf -/- , untreated and CpG stimulated (2h) pDCs for the Zfp366 gene visualized with Integrative Genomics Viewer (IGV). ( G ) Alignment of the BATF binding position at - 3464 of the transcription start site (TSS) of the Zfp366 gene in different mammalian species. Alignment of the indicated genomic regions was performed with Jalview with the blue colouring representing percentage identity. The position of the BATF binding to the AP-1 motif is marked with a red box. ( H ) qRT-PCR presenting time course of Batf and Zfp366 expression in FACS-sorted BM-derived Flt3L-cultured pDCs from C57BL/6 mice. Data represents relative expression of Zfp366 (blue line) and Batf (red line) mRNA in resting (0) or CpG stimulated pDCs. ( I ) Quantitative RT-PCR showing the expression of Zfp366 in untreated and 16h CpG-stimulated, GFP or Batf -GFP-overexpressing purified Hoxb8-Fl pDCs. Data shown are from one representative experiment performed in triplicate (A & B RNA-Seq, C, D & H) or duplicate (Chip-Seq & ATAC-Seq A, B & F) out of three independent experiments with comparable results (C, D & H) or are combined data from three independent experiments (E & I). Data shown in A are log 2 fold change expression, in C, D, H, and I are the mean expression ± SEM from 3 to 4 biological replicates (normalized to Actb in C left, D, H, and I). Statistical differences between the groups were analyzed by unpaired two tailed t test (C & E & I), multiple t-test using Bonferroni correction (D), or two-way ANOVA followed by multiple comparisons using Bonferroni corrections (H). *: p < 0.05, **: p < 0.002, ***: p < 0.001

Article Snippet: Genomic DNA regions of interest were isolated using 4μg of antibody against BATF (Clone: D7C5, Cell Signaling Technology, 8638BF).

Techniques: RNA Sequencing, ChIP-sequencing, Derivative Assay, Cell Culture, Expressing, Quantitative RT-PCR, Purification, Control, Sequencing, Binding Assay, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: CD8 + T cell-intrinsic IL-6 signaling promotes resistance to anti-PD-L1 immunotherapy

doi: 10.1016/j.xcrm.2022.100878

Figure Lengend Snippet: IL-6 regulates CTLs via STAT3-dependent BATF induction (A–C) RNA-seq analysis of WT naive CTLs stimulated with IL-6 +/− anti-CD3/CD28 antibodies for 4 h. (A) Differentially expressed genes (FDR <0.05 and absolute fold change >2). (B) IL-6-regulated genes with potential roles in CTL differentiation, and their functional categorization (C). (D) IFN-γ expression in WT or STAT3.ko CTLs (from CD4-Cre x Il6r loxP /loxP mice) activated with anti-CD3/CD28 +/− IL-6 or hyper-IL-6 for 3 days. Groups (mean ± SEM of n = 4 technical replicates) compared by t test; data representative of three independent experiments. (E) Batf mRNA expression (qRT-PCR) in WT or STAT3.ko CTLs activated +/− IL-6. ∗∗p < 0.01, ∗∗∗p < 0.001 (WT vs. STAT3.ko; t tests). Data points indicate mean +/− SEM of n = 4 technical replicates, from one of two independent experiments. (F) Western blot of BATF and p-STAT3 (Y705) in activated WT or STAT3.ko CTLs. Data represent one of two independent experiments. (G) BATF CRISPR-ko or control CTLs were activated +/− IL-6 and analyzed on day 3 (groups compared by t test; mean ± SEM of n = 4 technical replicates). Data representative of two independent experiments.

Article Snippet: Lysates were denatured with reducing sample buffer and dithiothreitol (Invitrogen, Waltham, MA) at 95°C for 10 min. Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis in a NuPAGE 4–12% gradient Bis-Tris gel and analyzed by western blotting with antibodies against phospho-STAT3 (clone D3A7, Cell Signaling Technology, Danvers, MA), total STAT3 (clone D1B2J, Cell Signaling Technology), BATF (clone D7C5, Cell Signaling Technology), and β-actin-HRP (Sigma-Aldrich, St Louis, MO).

Techniques: RNA Sequencing, Functional Assay, Expressing, Quantitative RT-PCR, Western Blot, CRISPR, Control

Journal: Cell Reports Medicine

Article Title: CD8 + T cell-intrinsic IL-6 signaling promotes resistance to anti-PD-L1 immunotherapy

doi: 10.1016/j.xcrm.2022.100878

Figure Lengend Snippet:

Article Snippet: Lysates were denatured with reducing sample buffer and dithiothreitol (Invitrogen, Waltham, MA) at 95°C for 10 min. Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis in a NuPAGE 4–12% gradient Bis-Tris gel and analyzed by western blotting with antibodies against phospho-STAT3 (clone D3A7, Cell Signaling Technology, Danvers, MA), total STAT3 (clone D1B2J, Cell Signaling Technology), BATF (clone D7C5, Cell Signaling Technology), and β-actin-HRP (Sigma-Aldrich, St Louis, MO).

Techniques: Control, Recombinant, Cell Stimulation, Staining, Cell Isolation, cDNA Synthesis, Expressing, CRISPR, Negative Control, In Situ Hybridization, Software

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: Loss of BATF impairs Th17 differentiation. ( A ) Rpkm values are plotted for BATF RNA at different time points of activation (Th0) or Th17-differentiation, using our published RNA-seq data . ( B ) Immunoblot (left) shows BATF protein levels in SCR versus BATF KD cells, at 24 h of Th17 polarization. Actin serves as loading control. Adjoining flow cytometry plots show percentage of CCR6 positive cells and the graph below shows ELISA analysis for IL-17 secretion in SCR versus BATF KD cells, at 72 h of Th17 polarization. ELISA values were first normalized for cell count (live), and then normalized to SCR control. Graph shows mean ± SEM for three biological replicates. Statistical significance was calculated using two-tailed Student's t test (** p < 0.01). ( C ) Volcano plots highlight the significantly upregulated (in red) and downregulated (in blue) genes in BATF-silenced Th17 cells at 24 h (left) and 72 h (right) of polarization (FDR ≤ 0.1, |FC| ≥ 1.8). DE genes with relevance to Th17 function are shown (extended DE gene list shown in ). ( D ) IPA was used to identify pathways altered upon silencing of BATF in Th17-polarized cells (24 h and 72 h). The top pathways related to T-cells and immune signaling are selectively shown. ( E ) Venn diagram shows the overlap between the genes that are altered upon BATF KD and the genes whose putative promoter regions (5-kb window around the TSS) are bound by BATF. The overlapping area represents the promoter-bound regulatory targets of BATF and the adjoining heatmap shows their corresponding expression changes in BATF KD Th17 cells. IGV images illustrate the occupancy of BATF over some of its Th17-associated targets. ( F ) Figure shows the topmost consensus sequence for genomic-binding of BATF, and the top six TF motifs enriched within BATF-bound sites, which were obtained using de-novo motif enrichment analysis by Homer. Peaks with IDR p value <0.01 were used for motif discovery.

Article Snippet: FOSL1, FOSL2 and BATF protein was analysed by western blotting using rabbit monoclonal FOSL1 antibody (D80B4), rabbit monoclonal FOSL2 antibody (D2F1E) and rabbit monoclonal BATF antibody (D7C5) from Cell Signaling Technology.

Techniques: Activation Assay, RNA Sequencing, Western Blot, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Cell Counting, Two Tailed Test, Expressing, Sequencing, Binding Assay

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: FOSL1, FOSL2 and BATF protein was analysed by western blotting using rabbit monoclonal FOSL1 antibody (D80B4), rabbit monoclonal FOSL2 antibody (D2F1E) and rabbit monoclonal BATF antibody (D7C5) from Cell Signaling Technology.

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: FOSL1, FOSL2 and BATF protein was analysed by western blotting using rabbit monoclonal FOSL1 antibody (D80B4), rabbit monoclonal FOSL2 antibody (D2F1E) and rabbit monoclonal BATF antibody (D7C5) from Cell Signaling Technology.

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: FOSL1, FOSL2 and BATF protein was analysed by western blotting using rabbit monoclonal FOSL1 antibody (D80B4), rabbit monoclonal FOSL2 antibody (D2F1E) and rabbit monoclonal BATF antibody (D7C5) from Cell Signaling Technology.

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