Figure 1.

Design and generation of an inducible cell-based luciferase reporter assay for BACH2-mediated repression of AP-1-driven gene expression. (a) Analysis of known BACH2, JunD and p300 binding at the mouse Ifng locus as determined by ChIP-Sequencing of CD8⁺ and CD4⁺ T cells. A BACH2-bound putative enhancer of Ifng, Ifng + 18k, is indicated by the black triangle. (b) DNA sequence at Ifng + 18k containing a TPA response element (TRE; red letters). This sequence was concatenated three times and subcloned upstream of a minimal promoter sequence (minP, grey box) controlling expression of NlucP luciferase cDNA sequence in the pNL2.2 reporter vector. (c) Experimental schema for generation of clonally derived inducible-BACH2 reporter and control reporter lines. Jurkat cells stably transduced with pcDNA6/TR vector resulting in expression of the tetracycline repressor protein were co-transfected with luciferase reporter (pNL2.2 Ifng + 18k) and inducible expression (pcDNA4/BACH2) vectors. Stably transfected cells were selected with hygromycin and zeocin and then subjected to single-cell cloning resulting in the generation of a luciferase reporter line with the potential for inducible BACH2 expression and a control reporter line lacking BACH2-inducibility.