Charlotte J. Imianowski, Rahul Roychoudhuri, Jie Yang, Christopher Bot, Paula Kuo, Teresa Lozano, Simon Walker, Klaus Okkenhaug, Hanneke Okkenhaug, Panagiota Vardaka, Jonathan Ellery, Stuart Farrow, Sarah K. Whiteside, Apollo - University of Cambridge Repository, Walker, Simon [0000-0001-9185-4922], Yang, Jie [0000-0001-9157-0532], Okkenhaug, Klaus [0000-0002-9432-4051], Kuo, Paula [0000-0002-2476-0018], and Roychoudhuri, Rahul [0000-0002-5392-1853]
Whereas effector CD4+ and CD8+ T cells promote immune activation and can drive clearance of infections and cancer, CD4+ regulatory T (Treg) cells suppress their function, contributing to both immune homeostasis and cancer immunosuppression. The transcription factor BACH2 functions as a pervasive regulator of T cell differentiation, promoting development of CD4+ Treg cells and suppressing the effector functions of multiple effector T cell (Teff) lineages. Here, we report the development of a stable cell-based bioluminescence assay of the transcription factor activity of BACH2. Tetracycline-inducible BACH2 expression resulted in suppression of phorbol 12-myristate 13-acetate (PMA)/ionomycin-driven activation of a luciferase reporter containing BACH2/AP-1 target sequences from the mouse Ifng + 18k enhancer. BACH2 expression repressed the luciferase signal in a dose-dependent manner but this activity was abolished at high levels of AP-1 signalling, suggesting contextual regulation of AP-1 driven gene expression by BACH2. Finally, using the reporter assay developed, we find that the histone deacetylase 3 (HDAC3)-selective inhibitor, RGFP966, inhibits BACH2-mediated repression of signal-driven luciferase expression. In addition to enabling mechanistic studies, this cell-based reporter may enable identification of small molecule agonists or antagonists of BACH2 function for drug development.