Figure 5. Nucleosomes can facilitate FoxP3 bridging of T3G repeats.
A. Total length of TnG repeats (in basepairs) within 1 kb window centering at the TnG repeat sites within (n=2,019) or outside FoxP3 CNR peaks (n=3,579). For TnG repeats outside FoxP3 CNR, only the sites within Treg ATAC peaks that are at least 10 kb away from the FoxP3 CNR peaks were considered. FoxP3 CNRs are the union of the Rudensky and Dixon-Zheng peaks as defined in Figure S1B. Statistical analysis was performed using two-tailed unpaired t-tests; ****P < 0.0001.
B. Distance from ATAC summits to the closest center of TnG region, ETS motif or CTCF motif within Foxp3 CNR peaks. Statistical analysis was performed using one-way ANOVA; ****P < 0.0001. Previously reported thymic Treg ATAC-seq data (PMID:27992401) were used.
C. Comparison of ATAC-seq intensity within 600 bp window, centered on TnG repeats within or outside Foxp3 CNR peaks, summits of ATAC peaks overlapping with Foxp3 CNR peaks, ETS or CTCF motifs within Foxp3 CNR peaks.
D. FoxP3 binding to free or nucleosomal DNA harboring (T3G)10-601-(T3G)10 as measured by native gel-shift assay. Free or nucleosomal DNA (50 nM each) was incubated with FoxP3 (0, 0.1,0.2,0.4, 0.6, 0.8, 1.6 μM), and complex formation was analyzed on native gel. While FoxP3-bound complexes did not enter the gel, FoxP3-unbound DNA could be quantitated by Sybrgold stain. Error bars represent standard deviations from three independent experiments.
E. FoxP3 binding to free or nucleosomal DNA harboring (T3G)10-601-random sequence as measured by native gel-shift assay as in (D).
F. Top: Representative negative stain electron micrographs of nucleosomal DNA without FoxP3 (left), nucleosome-free DNA with FoxP3 (middle), and nucleosomal DNA with FoxP3 (right). The same DNA harboring (T3G)10-601-(T3G)10 was used in all cases. Aggregate particles, more pronounced in nucleosomal DNA with FoxP3, were shown in black boxes. Particles in red circle were picked for 2D classification (middle). Bottom: structures of nucleosome (PDB:7OHC) and FoxP3-bound T3G repeat (PDB:8RSP) and a hypothetical model of nucleosomal DNA bound by FoxP3.
G. Trans effect of nucleosome on FoxP3’s interaction with Cy5-labeled (T3G)15 DNA. Unlabeled DNA harboring (T3G)10-601-(T3G)10 (free or nucleosomal, 50 nM) was incubated with FoxP3, and then with Cy5 labeled (T3G)15 DNA (20 nM), followed by native gel analysis. Sybrgold stain (left) and Cy5 fluorescence (middle) were used for visualization. The levels of Cy5 DNA in the FoxP3-bound and -unbound species were quantitated (right). Error bars represent standard deviations from three independent experiments.
H. Top: Dual luciferase assay schematic. Two types of plasmids were generated: an enhancer plasmid containing an enhancer element (UAS) which can be bound by Gal4 DBD, and reporter plasmid containing the firefly (FF) luciferase gene driven by a minimal promoter. Both plasmids contained (T3G)n repeats (n=12 or 24) or (T3G)12-601-(T3G)12, upstream of the promoter or enhancer. These two plasmids, along with the FoxP3-expressing plasmid and Renilla luciferase-encoding transfection control plasmid, were co-transfected into 293T cells expressing Gal4 DBD fused with a transcriptional activation domain (TAD) from the unrelated TF Aire (Gal4-TAD). Bottom: Relative levels of FF luciferase activities (normalized to Renilla) were shown. EV indicates an empty vector. Random indicates an enhancer plasmid with 48 nt random sequence in place of (T3G)12. Data are representative of three biological replicates and presented as mean ± s.d. p-values (one-way ANOVA with Tukey’s multiple comparisons test) were calculated for each comparison, **** p < 0.0001.
See also Figure S6.