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
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