A cell-based bioluminescence assay reveals dose-dependent and contextual repression of AP-1-driven gene expression by BACH2

Abstract

Whereas effector CD4⁺ and CD8⁺ T cells promote immune activation and can drive clearance of infections and cancer, CD4⁺ regulatory T (T_reg) 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⁺ T_reg cells and suppressing the effector functions of multiple effector T cell (T_eff) 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.

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.

Figure 2

BACH2-mediated repression of AP-1 driven luciferase expression using the inducible reporter system. (a) Inducible expression system. Tetracycline repressor (TR) protein in its active form (indicated as a circle) binds to the TetO₂ sequence upstream of BACH2 cDNA subcloned into the pcDNA4/BACH2 vector inhibiting transcription of BACH2. Tetracycline (Tet) addition changes the conformation and inactivates the tetracycline repressor (TR) protein (indicated as a square), which is subsequently not able to bind to TetO₂ sequence, allowing BACH2 transcription to commence. (b) Western blot for indicated proteins of total lysates isolated from the clonally derived inducible-BACH2 and control reporter cell lines with or without tetracycline treatment. (c) Luciferase activity in the inducible-BACH2 and control reporter lines after 6 h PMA/ionomycin stimulation with or without pre-treatment with tetracycline (1 μg/ml). Unpaired two-tailed Student’s t test (c). Data are representative of 3 independent experiments with 3 culture replicates per condition. Bars and error represent mean (SD); ns not significant; ***P < 0.001; ****P < 0.0001.

Figure 3

Dose-dependent repression of AP-1-driven gene expression by BACH2. (a) Luciferase activity of inducible-BACH2 reporter line after 6 h PMA/ionomycin stimulation with or without pre-treatment with indicated titrated doses of tetracycline. (b) Western blot analysis of the abundance of BACH2 protein within total protein lysates from cells in (a). Quantified and normalised to β-actin levels of BACH2 protein expression are displayed in the bar graph (top). (c) Positive correlation between BACH2 expression normalised to β-actin, and luciferase signal repression at the indicated in (a) tetracycline concentrations. Two-way ANOVA with Bonferroni correction (a) and linear regression analysis (c). Data are representative of 2 independently repeated experiments with 3 culture replicates per condition. Bars and error represent mean (SD); ns not significant; ***P < 0.001; ****P < 0.0001.

Figure 4

Bioluminescence imaging of BACH2-reporter cells. (a) Imaging of inducible-BACH2 reporter cells 6 h after PMA/ionomycin stimulation with or without tetracycline (1 μg/ml) treatment. Unstimulated cells were included (indicated as Vehicle). Images were captured after luciferase substrate addition using brightfield (left) and luminescence (right) channels. Each panel is a representative 300 μm × 300 μm cropped area from the overview image. (b) Frequency of positive luminescent cells in stimulated inducible-BACH2 reporter cells following pre-treatment with the indicated doses of tetracycline.

Figure 5

Contextual signal-responsive repression of luciferase expression by BACH2. (a) Luciferase activity in inducible-BACH2 reporter line after 6 h stimulation with the indicated concentrations of PMA/ionomycin, with or without pre-treatment with titrated tetracycline doses. Concentrations of tetracycline are indicated in the figure legend. (b) Luciferase signal repression at the indicated PMA/ionomycin concentrations with or without tetracycline (1 μg/ml) pre-treatment. (a, b) Data are representative of 2 independently repeated experiments with 3 culture replicates per condition. Bars and error represent mean (SD).

Figure 6

BACH2-mediated repression of luciferase expression is inhibited by the HDAC3 inhibitor molecule RGFP966. (a, b), Luciferase activity in inducible-BACH2 reporter line after 6 h stimulation with PMA/ionomycin and pre-treatment with or without 1 μg/ml tetracycline and with 12.5 μM of RGFP966 or without (indicated as Veh). (c) Western blot analysis of the abundance of BACH2 within total protein lysates from cells in (a, b) treated with or without 12.5 µM RGFP966. Unpaired two-tailed Student’s t test (a, b). (a, b) Data are representative of 2 independently repeated experiments with 3 culture replicates per condition. Bars and error represent mean (SD); ns not significant; ****P < 0.0001.