Figure 4. Tcf1 recruits CTCF to the CD8⁺ T cell genome via direct interaction.

a–b. Co-immunoprecipitation of CTCF by an anti-Tcf1 antibody (a) and co-immunoprecipitation of Tcf1 by an anti-CTCF antibody (b) in the presence of EtBr in primary naïve CD8⁺ T cells. c. Co-immunoprecipitation of HA-tagged CTCF by FLAG-tagged Tcf1 full-length (FL) or mutant proteins (structures shown in diagram on the top) with an anti-FLAG antibody in the presence of EtBr after co-transfected into 293T cells. d. Co-immunoprecipitation of FLAG-tagged Tcf1 with HA-tagged CTCF full-length (FL) or mutant proteins (structures shown in diagram on the top) with an anti-FLAG antibody in the presence of EtBr after co-transfected into 293T cells. Data in a–d are representative from ≥2 independent experiments. e. Heatmaps showing changes in local chromatin features (including CTCF binding peaks, ChrAcc and H3K27ac) between naïve WT and dKO GFP⁺CD8⁺ T cells, at Tcf1+Lef1-dependent CTCF binding sites as detected by CUT&RUN and/or ChIP-seq methods, with aggregated profiles for each feature shown on the top. Color scale denotes relative strength of each molecular feature. f–g. Distribution of Tcf1⁺CTCF⁺ and Tcf1⁻CTCF⁺ sites (f) and Motif⁻ and motif⁺ CTCF sites (g) in Tcf1+Lef1-dependent CTCF binding peaks as detected by CUT&RUN and/or ChIP-seq methods, with 2,000 randomly selected non-differential CTCF binding sites between WT and dKO CD8⁺ T cells as negative controls. h. Sequencing tracks of Tcf1 ChIP-seq in naïve WT, CTCF CUT&RUN and CTCF ChIP-seq, DNase-seq and H3K27ac ChIP-seq in naïve WT and dKO GFP⁺CD8⁺ T cells at the Myb, Ccr7, and Prdm1 gene loci, with gene structure, transcription orientation, and genomic scale displayed on the top. Bars in yellow mark Tcf1+Lef1-dependent Tcf1⁺CTCF⁺ site(s) detected with both CUT&RUN and CTCF ChIP-seq methods, while open bars with cyan borders mark those determined with one method only.