Figure 1.

XLOC_005968/ARIEL expression is associated with the activity of the ARID5B −135-kb enhancer. (A) ChIP-seq gene tracks displaying DNA binding of TAL1, and its regulatory partners, CBP, mediator 1 and RNA Pol II in Jurkat cells as well as various histone markers in CD4⁺ T cells (Th1, Th2, and Th17), normal thymus, Jurkat, and MOLT-4 cells at the XLOC_005968/ARIEL locus. RNA-seq data (positive strand) in Jurkat is shown. Superenhancer regions defined by the H3K27ac ChIP-seq analysis are indicated (red bars). A red arrowhead and a black arrowhead indicate the ARID5B −135-kb enhancer and promoter, respectively. (B-C) Jurkat cells were cotransduced with the catalytic-dead form of the dCas9-KRAB protein and an sgRNA (sgRNA1 or sgRNA2) or an empty vector (EV). ChIP analysis was performed using a specific antibody against H3K4me1 (B), H3K9me3 (C), or control IgG. The amount of genomic DNA in the ChIP and input samples was measured by qPCR using specific primers targeting the ARID5B −135-kb enhancer locus or a region +200 bp downstream from the center. The IGFBP3 promoter region was used as a negative control for ChIP-PCR. The percentage of the input is shown as the mean plus or minus standard deviation (SD) of triplicate experiments. (D) The RNA expression levels of XLOC_005968/ARIEL, ARID5B, and neighboring genes (CDK1 and JMJD1C) were analyzed by qRT-PCR. The expression was normalized to the ERCC RNA spike-in and is shown as the following values relative to the EV control: mean plus or minus SD of triplicate experiments. (E) Expression of ARIEL from the RNA-Seq data set reported by Liu et al³⁶ was first calculated and reported as transcripts per million (TPM). Expression of ARIEL between TAL1⁺ and TAL1⁻ patients were then analyzed using nonpaired Student t test. **P < .01.