Fig. 2. Ccl5 expression is regulated by two stage-specific enhancers.

a CBFβ ChIP-seq tracks in splenic total CD4⁺ and CD8⁺ T cells at one of the two CC chemokine clusters in which Ccl5 is located on chromosome 11. ATAC-seq tracks in naïve (Tnai) and effector (Teff) T cells and histone mark ChIP-seq tracks in memory CD8⁺ (Tmem) T cells obtained from public database are also indicated. Two significant CBFβ ChIP-seq peaks are indicated by rectangles, one of which (near Ccl5) is denoted as Ccl5-RBR. Ccl5-RBR has a 0.2 kb core enhancer region denoted as Ccl5-PE that harbors two Runx recognition sequences (RRSs). The genome region included in the BAC transgene construction is indicated by a dotted line. CBFβ ChIP-seq data are representative of two independent experiments. b Representative dot plots showing GFP expression in splenocytes of F0 transgenic founders. A Ccl5-GFP BAC transgene was constructed by replacing Ccl5 with eGFP. In the Ccl5-GFP-ΔRBR BAC transgene, a 1.2 kb RUNX binding region (RBR) near Ccl5 (Ccl5-RBR) was removed from the Ccl5-GFP BAC transgene. The graph on the right shows a summary of frequencies of GFP⁺ cells in splenic T cells of each F0 transgenic founder. Error bars indicate Mean ± SD and each dot represents a mouse examined over at least two independent experiments. Statistical significance is measured via unpaired two-tailed Student’s t tests and is presented as follows: ****p < 0.0001. c Representative dot plots show representative GFP expression in spleen T cells of the Ccl5-GFP BAC transgenic founders in the presence or absence of RRSs. Numbers in the dot plots indicate the percentage of cells in each quadrant. Error bars indicate Mean ± SD and each dot represents a mouse examined over at least two independent experiments. Statistical significance is measured via unpaired two-tailed Student’s t tests and is presented as follows: *p < 0.05. d Representative dot plots showing GFP expression in activated CD8⁺ T cells. The left and right plots are of the Ccl5-GFP BAC transgenic founder in the presence and absence of CBFβ, respectively. The bottom plot shows the GFP expression in one representative F0 founder line of the Ccl5-GFP-RBR^M BAC transgene, in which two RUNX recognition sequences (RRSs) within the Ccl5-RBR are mutated. The graph shows a summary of the frequencies of GFP⁺ cells in activated CD8⁺ T cells. Error bars indicate Mean ± SD and each dot represents a mouse examined over at least two independent experiments. Statistical significance is measured via unpaired two-tailed Student’s t tests and is presented as follows: **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file.