Nuclear Wiskott–Aldrich syndrome protein co-regulates T cell factor 1-mediated transcription in T cells

Figures

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  Fig. 1. ChIP-seq analysis of WASp enriched genes. a The F-8 anti-WASp Ab specifically recognizes WASp in total thymocyte lysates, nuclear extracts (NE), and cytosolic extracts (CE). Heavy chain of mouse IgG (IgGhc) is observed in eluted IP samples. GAPDH was used as a sample loading control for total lysates. b WASp is abundant in NE of WT thymocyte when compared to NE of WT spleen CD4+ T cells. Heavy chain of mouse IgG (IgGhc) is observed in eluted IP samples. GAPDH was used as a sample loading control for total lysates. These data are representative of four (thymus fractionation) and two (spleen CD4+ T cell fractionation) independent biological replicates. c ChIP-seq analysis of thymocytes and spleen CD4+ T cells flowchart. WASp-enriched genes by ChIP-seq analysis of WT thymocytes performed under high stringency condition. WASp-enriched genes by ChIP-seq analysis of spleen CD4+ T cells performed under low stringency conditions and compared to a ChIP-seq dataset from control Ab-free spleen CD4+ T cells. All common genes were subtracted from the spleen CD4+ T cells dataset. Both ChIP-seq datasets were then aligned and compared to identify common genes in thymocytes and spleen CD4+ T…

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  Fig. 2. Transcription matrix for selected WASp enriched genes. a Bioinformatic association of WASp ChIP-seq peaks from our datasets with activating (A) and/or repressive (R) epigenetic regulatory marks, RNA Polymerase II and TFs peaks and lamin B1 marks in thymocytes (T) and spleen CD4+ cells (S). b Association of WASp ChIP-seq peaks in thymocytes of Tcf12, Vav2, and Wasf2 with RNA Polymerase II ChIP-Seq signal by TFBS ENCODE/LICR resource at UCSC genome browser

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  Fig. 3. Variation of distances from WASp ChIP-Seq gene peaks to nearest exon(s). Selected examples of genes with (a) shorter distance range (0.5–5 kb) and (b) longer distance range (5–50 kb) between WASp ChIP-seq gene peak position and nearest exon. Additional file 1: Figure S4

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  Fig. 4. WASp functional status affects both basal and gene-specific transcription. a FUrd incorporation assays data for basal RNA polymerase II nascent transcripts in WT, WASp KO, and WASpI296T thymocytes as determined by intensity of FUrd-rich foci (red). The cell nucleus is labeled with DAPI (blue) and histone H3 (green). Note that mRNA transcripts can be seen in the cytoplasm already after 15 min. b Statistical analysis of FUrd assay results by nested ANOVA. Each dot represents one image. Top: WT n = 3, WASp KO n = 3 in one experiment; bottom: WT n = 4, XLNI296Tn = 4 in one experiment. c, d Gene expression profiling by RT-qPCR of WASp ChIP-seq genes (c) encoding TFs, other DNA- or RNA-binding proteins, and cytoskeletal network proteins performed on cDNA template of pooled samples: WASp KO n = 3 vs. WT n = 3 and (d) for common WASp-enriched genes in thymocytes and spleen CD4+ T cells. Expression in WASp KO thymocytes is shown and compared with expression level in WT thymocytes set to 1. Gene expression profiling by RT-qPCR was performed in three biological replicates: WASp KO n = 3 vs. WT n = 3 and in three technical replicates for each cDNA template sample with Ct variation between…

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  Fig. 5. Gene regulation by nuclear WASpI296T. a Functional groups of peak-corresponding genes in WASp ChIP-seq refined datasets. b Gene expression profiling by RT-qPCR of WASpI296T ChIP-seq genes in WASpI296T vs. WT thymocytes. Expression in WASpI296Tthymocytes is shown and compared with expression in WT thymocytes set to 1. Gene expression profiling by RT-qPCR was performed in three biological replicates: WASpI296T n = 3 vs. WT n = 3 and in three technical replicates for each cDNA template sample with Ct variation between triplicates less than cut-off ± 0.25. Resulted gene expression data: the mean of the technical triplicate, ΔCt, RQ value, and standard deviation between RQ of biological triplicates is indicated. Additional file 3: sheet 3

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  Fig. 6. The activation status of nuclear WASp affects TCF1-mediated Tcf12 expression. a TCF12 in NE and CE from WT, WASp KO, WASpI296T, and WASpL272P thymocytes. These data were repeated twice with independent biological replicates. Detection of GAPDH was used as a sample loading control for total lysate and for cytosolic extract. Detection of histone H3 was used as a sample loading control for total lysate and for nuclear extract. The quantification of nuclear and cytosolic TCF12 in the western blot is shown in the right panel. b Consensus DNA motif determined by WASp ChIP-seq data analysis and the canonical TCF1 DNA-binding site. c Proximity of WASp ChIP-seq gene peak positions and TCF1-binding sites in Tcf12 intron 3. d TCF1 in NE and CE from WT, WASp KO, WASpI296T, and WASpL272P thymocytes. These data were repeated twice with independent biological replicates. Detection of GAPDH was used as a sample loading control for total lysate and for cytosolic extract. Detection of histone H3 was used as a sample loading control for total lysate and for nuclear extract. The quantification of nuclear and cytosolic TCF1 in the western blot is shown in the right panel. e Co-IP of WASp and TCF1…