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

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.

Scientific Reports | (2020) 10:18902 | https://doi.org/10.1038/s41598-020-75732-z www.nature.com/scientificreports/ contain bZip domains enabling them to form heterodimeric complexes at palindromic 12-O-Tetradecanoylphorbol-13-acetate (TPA) response elements (TRE; 5′-TGA(C/G)TCA-3′) within regulatory DNA 9,10 . Upon TCR-signalling, AP-1 complexes translocate to the nucleus where they bind to TRE of genes associated with T eff cell differentiation and function 11 . BACH2 is a 92 kDa transcriptional repressor of the bZip TF family and is predominantly expressed in lymphocytes 12 . It functions as an important regulator of immune activation and transcriptional repression. BACH2 intrinsically regulates the differentiation and function of multiple conventional T cell lineages and is required for efficient development of T reg cells. Deficiency of BACH2 results in a cell-intrinsic defect in T reg cell differentiation, such that C57BL/6 syngenic mice lacking BACH2 protein expression develop lethal inflammation 13 . In addition, BACH2 promotes tumour immunosuppression in a T reg -dependent manner 11 . Genetic deletion of Bach2 in mice results in increased clearance of subcutaneously syngeneic B16 melanoma tumours. Furthermore, the BACH2 gene in humans is a prominent risk locus for multiple autoimmune and allergic diseases 12 .
The DNA-binding bZip domain of BACH2 is located at the C-terminus of the protein and is required for its repressive activity. In T cells, BACH2 binds to DNA sequences which embed TRE 14 . Through shared possession of bZip domains, BACH2 and AP-1 competitively bind to the same sites within enhancers 11,15 . It has been proposed that such competitive interactions by BACH2 allow it to repress effector-associated gene expression. IFN-γ, encoded by the Ifng gene, is an inflammatory cytokine that contributes to antiviral and anti-tumour immunity and can contribute to inflammation and immunopathology 16 . Ifng expression is markedly elevated in mouse Bach2-deficient CD4 + and CD8 + T cells 11,17 . In addition, repression of IFN-γ expression is partially required for BACH2 to promote induced T reg (iT reg ) cell induction 13 . These results suggest that repression of IFN-γ expression is a critical biological function of BACH2, but whether these results derive from direct transcriptional repression of the Ifng gene has not been formally established. Moreover, the immunoregulatory function of BACH2 and its predominantly lymphocyte-specific gene expression profile make it a potential target in development of therapies for autoimmune diseases and cancer.
In this work, we have developed a cell-based assay system to report the transcription factor activity of BACH2, wherein tetracycline-inducible BACH2 expression represses AP-1-driven luciferase activity. 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 control of AP-1 driven gene expression by BACH2. In addition to enabling mechanistic studies, we propose that this cell-based reporter will enable identification of small molecule agonists or antagonists of BACH2 function for drug development.

Results
Generation of a cell line-based luciferase reporter assay of BACH2 repressor function. A putative enhancer of the mouse Ifng gene (Ifng + 18k), containing a canonical TRE and bound by p300, BACH2, and the AP-1 factor JunD in CD4 + and CD8 + T cells was identified (Fig. 1a) 11 . A short concatenated DNA sequence surrounding the TRE at Ifng + 18 k was subcloned upstream of a minimal promoter (minP) and a luciferaseencoding cDNA sequence (Fig. 1b). We additionally subcloned a human BACH2 cDNA inducible expression vector containing a CMV promoter and control elements from the bacterial tetracycline (Tet) resistance operon. We verified the insert and surrounding vector sequences in both constructed plasmids using Sanger sequencing (Supplementary Fig. 1 and 2). The luciferase reporter and inducible BACH2 expression vectors were co-transfected into Jurkat cells constitutively expressing the Tet repressor protein. Transfected cells were selected using antibiotic selection. Stably transfected single-cell clones were isolated using limiting dilution. A tetracyclineinducible BACH2 functional reporter assay was established in addition to a control reporter lacking inducible BACH2 expression (Fig. 1c). In the developed system, the Tet repressor binds to a specific sequence upstream of BACH2 cDNA, inhibiting BACH2 protein expression. The addition of tetracycline results in a conformational change of the Tet repressor protein, preventing its binding, and allowing BACH2 to be expressed (Fig. 2a).
We first examined whether BACH2 protein expression is inducible using this system. Cells were treated with or without 1 μg/ml tetracycline, to induce BACH2 expression. Lysates were resolved using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and proteins were detected by western blotting. We observed inducible expression of BACH2 protein upon tetracycline treatment of the inducible-BACH2 reporter line but not of the control reporter line, whereas expression of the AP-1 factors JunB, c-Jun and JunD in total cellular lysates was unchanged (Fig. 2b).
The specificity of anti-BACH2 antibody reactivity was tested by adding a blocking peptide during antibody staining of membranes, which resulted in abolishment of the signal ( Supplementary Fig. 3). The inducible-BACH2 reporter line treated with and without tetracycline was stimulated with PMA/ionomycin to cause Ifng + 18k enhancer-driven luciferase expression. A ~ 40% reduction of luciferase signal was observed in tetracycline-treated cells, which was not observed in the control reporter line (Fig. 2c). These results indicate that tetracycline-inducible BACH2 protein represses signal-driven luciferase gene expression controlled by a sequence from the consensus Ifng + 18k enhancer.

Dose-dependent repression of AP-1-driven gene expression by BACH2.
To examine the dosedependency of BACH2-mediated AP-1-driven signal repression, we performed tetracycline titration experiments. Inducible-BACH2 cells were treated with titrated doses of tetracycline and their luciferase activity was determined following PMA/ionomycin stimulation ( Fig. 3a and Supplementary Fig. 4). BACH2 protein expression was also examined by SDS-PAGE and western blotting (Fig. 3b). Luciferase activity was negatively correlated with tetracycline concentration (Supplementary Fig. 5) and a significant positive linear correlation between Scientific Reports | (2020) 10:18902 | https://doi.org/10.1038/s41598-020-75732-z www.nature.com/scientificreports/ repression of signal-driven luciferase induction and BACH2 protein expression was observed (Fig. 3c). Imaging of inducible-BACH2 reporter cells after 6 h of PMA/ionomycin stimulation also revealed dose-dependent repression of signal-driven luminescence following treatment of cells with tetracycline ( Fig. 4a, b). These data suggest that BACH2 functions as a dose-dependent repressor of AP-1-driven gene expression regulated by sequences derived from the + 18k enhancer of Ifng.
Contextual dose-dependency of AP-1-driven signal repression by BACH2. BACH2 restrains TCR-driven effector differentiation programmes within CD8 + T cells 11 . However, despite possessing high levels of BACH2 expression, naïve T cells are able to differentiate into effector cells in the presence of strong levels of TCR stimulation. We asked whether BACH2-mediated repression of AP-1-driven gene expression occurs to the same extent at any level of AP-1 activation or whether its repressor function is limited at saturating levels of AP-1 activation. We therefore stimulated cells with titrated doses of PMA/ionomycin using a single concentration of tetracycline per titration. Importantly, we observed a loss of BACH2-mediated luciferase signal repression at higher levels of PMA/ionomycin stimulation (Fig. 5a, b). These results suggest that BACH2 capacity to mediate AP-1-driven gene expression repression is reduced in the presence of strong activating signals. Thus, dosedependent AP-1 signal repression by BACH2 is contextual and regulated by the strength of activation signalling in the system.

BACH2-mediated repression of signal-driven luciferase induction is inhibited by the HDAC3 inhibitor RGFP966.
There are no known direct activators or inhibitors of BACH2 function. However, it was recently shown that BACH2-mediated repression of the Prdm1 gene in B cells is partially dependent upon corecruitment of a complex containing histone deacetylase 3 enzyme (HDAC3). Thus, its repressor function at this locus is inhibited by the HDAC3-selective inhibitor RGFP966 18,19 . We therefore examined whether pre-treatment of cells with RGFP966 would result in inhibition of BACH2-mediated repression in the developed reporter assay. We observed near-complete loss of BACH2-repression of PMA/ionomycin-driven luciferase expression when cells were pre-treated with 12.5 μM RGFP966 (Fig. 6a, b). Importantly, RGFP966 treatment did not affect BACH2 expression in the assay (Fig. 6c). These results provide a positive control for pharmacological inhibition of BACH2 activity in the reporter system developed and shed light on potential mechanisms by which BACH2 represses Ifng expression in T cells.

Discussion
In this study, we have generated a cell-based luciferase reporter assay system to facilitate analysis of the transcriptional repressor function of BACH2 in vitro. Sequences derived from the mouse Ifng + 18k enhancer sequence bound by both BACH2 and Jun family AP-1 factors in T cells were used to drive luciferase gene expression. Its signal-driven induction was repressed by tetracycline-inducible BACH2 expression 11 . Our inducible BACH2 reporter system suggests that BACH2-mediated repression of AP-1-driven gene expression is dose-dependent and limited at the highest levels of AP-1 signalling. Previous work has shown that BACH2 represses IFN-γ expression, but whether this was the result of direct control of regulatory elements of the Ifng gene had not been tested. These findings suggest that BACH2 represses AP-1-driven induction of Ifng through regulatory interactions with AP-1 factors at the Ifng + 18k enhancer ( Supplementary Fig. 6).
In this study, we examined BACH2-mediated regulation of Ifng regulatory elements in a Foxp3-negative conventional T cell line. Repression of IFN-γ expression is a critical biological function of BACH2 not only in conventional CD4 + Th1 cells and CD8 + T cells, but also during early iT reg cell development, where BACH2mediated repression of IFN-γ is required for stabilization of iT reg differentiation prior to Foxp3 induction [11][12][13] . Moreover, we and others have shown that within lineage-committed Foxp3 + T reg cells, BACH2 is re-purposed and is not required to maintain Foxp3 expression or suppress IFN-γ expression, but rather blocks the TCR-driven transition between resting T reg (rT reg ) and activated T reg (aT reg ) states 20,21 . Given these observations, we chose to study the regulation of Ifng expression by BACH2 in the Foxp3-negative Jurkat cell line. It would be useful in Stable transfection of the reporter system allowed for the effect of BACH2 on a chromatinized reporter to be determined, as opposed to commonly utilized transfected plasmid luciferase reporters which are not integrated into the host genome and therefore exist as non-chromatinized plasmid DNA. This system provided the opportunity to examine whether BACH2-mediated repression of gene expression in T cells is in part dependent upon regulation of chromatin. Histone deacetylase 3 enzyme (HDAC3) is found in specific complexes containing NCoR1 and NCoR2 and can be recruited to chromatin by transcriptional repressors 23,24 . In B cells, BACH2 has been shown to interact with NCoR1 and NCoR2 resulting in recruitment of HDAC3 to the Prdm1 gene. As a result, repression of Prdm1 by BACH2 is dependent upon the activity of HDAC3 18 . Consistent with these findings, we observed that repression of signal-driven luciferase expression by BACH2 was inhibited upon pre-treatment of cells with the HDAC3-specific inhibitor RGFP966. These findings provide an important positive control for inhibition of BACH2-mediated repressor activity in the developed assay system, relevant to development and validation of high-throughput screening assays. It will also be important in future studies to test the extent to which BACH2-mediated repression of Ifng expression in primary cells requires the histone deacetylase function of HDAC3.
A functional relationship between BACH2 and AP-1 factors underlies T cell memory formation 11,12 . BACH2 inhibits both effector and terminal effector differentiation programmes under conditions of weak TCR-signalling, contributing to differentiation of memory CD8 + T cells and long-lived responses following viral infection 11 . In our assays, loss of BACH2-mediated repression at high levels of stimulation suggests that BACH2-mediated www.nature.com/scientificreports/ repression is itself regulated by the strength of activating signals that cells receive. This is consistent with a requirement for T cells expressing high levels of BACH2 to nevertheless be able to differentiate into effector cells in the presence of strong TCR and inflammatory signalling. Indeed, a number of regulatory pathways are known to affect the post-translational stability, localisation and function of BACH2 and an opportunity to further interrogate their role in a reductionist system is provided by this assay. However, such investigations would need to be complemented by corresponding assays using more physiological systems, including in primary T cells. Finally, this cell-based reporter provides an opportunity for identification of small molecule agonists or antagonists of BACH2 function using high-throughput screening. Such assays may enable identification of novel therapeutic compounds to either augment or inhibit the suppressive function of BACH2 in immune activation.    Luciferase assay. Clonally derived cell lines were treated with or without tetracycline (T8032, Sigma-Aldrich) for 18 h. Subsequently, cells were stimulated with phorbol 12-myristate 13-acetate (PMA) (P1585, Sigma-Aldrich) and ionomycin (I0634, Sigma-Aldrich) at 25 ng/ml and 1.25 μg/ml respectively, if not otherwise stated, for 6 h in replenished culture medium containing tetracycline. Luciferase expression was acquired using the Nano-Glo Luciferase Assay System kit (N1130, Promega) following the manufacturer's instructions. Luciferase signal was measured using a PHERAstar FS spectrophotometer. Data were analysed using GraphPad Prism 8 software.

Plasmids and generation of inducible-BACH2 and control reporter cell-based lines.
Western blotting. Selected clones were treated with or without tetracycline for 18 h. The cells were harvested and washed twice in phosphate-buffered saline (PBS). Cells were lysed in RIPA buffer (89901, Thermo Scientific) containing protease inhibitors (11836170001, Sigma-Aldrich). Total protein concentration was quantified using BCA assay (23225, Thermo Scientific) and normalised protein amount was loaded on SDS-PAGE gels followed by semi-dry western blotting. BACH2 protein was detected using BACH2-specific antibody (D3T3G Rabbit mAb, 80775S, Cell Signalling Technology). Detection of Jun family members was conducted with primary anti-c-Jun antibody (N, clone sc-45, J1713, Santa Cruz Biotechnology), anti-JunB antibody (210, clone sc-73, J1813, Santa Cruz Biotechnology) and anti-JunD antibody (329, clone sc-74, A3113, Santa Cruz Biotechnology). As a loading control β-actin protein was stained using anti-β-actin antibody (clone AC-74, A5316, Sigma-Aldrich). The specificity of anti-BACH2 antibody reactivity was tested by adding a BACH2 specific blocking peptide (38475S, Cell Signalling Technology) during primary antibody staining. Stripping of primary and secondary antibodies was performed by incubating the membrane in Restore Western Blot Stripping Buffer (21059, Thermo Scientific) followed by re-probing as described above. Protein quantification was conducted using ImageJ software 25 .

RGFP966 inhibitor treatment.
Inducible-BACH2 reporter cells were plated and pre-treated with or without 1 μg/ml tetracycline for 5 h prior to RGFP966 (16917, Cayman Chemical Company) inhibitor addition. The tetracycline pre-treated cells were additionally treated with 12.5 μM or without RGFP966 for 12 h following protein extraction or PMA/ionomycin stimulation for 6 h as described previously. Subsequently, BACH2 protein level detection with western blotting or luciferase activity measurements were performed using methods outlined above.
Imaging. Cells from the inducible-BACH2 reporter line were pre-treated with titrated concentrations of tetracycline (namely 0.0024 μg/ml, 0.012 μg/ml, 0.5 μg/ml and 1 μg/ml) or without for 18 h. Stimulation of cells with PMA/ionomycin at above concentrations followed for 5 h. Cells were imaged prior and subsequently to luciferase substrate (Nano-Glo Live Cell Assay System kit (N2011, Promega)) addition following the manufacturer's instructions. Luminescence and brightfield images were captured using a Nikon Ti-E microscope, Andor iXon Ultra EM-CCD camera, Nikon 20 × 0.8 NA objective, OKO lab environment chamber at 36 °C with 5% CO2 and Nikon Elements with JOBS module software. A 3 × 3 montage of images was acquired in each well with the camera set to maximum sensitivity (300 EM gain, 5.1 amplifier gain) using 10 s and 50 ms exposure times for luminescence and brightfield channels respectively. Images were processed and quantified with FIJI 26 using the PureDenoise plug-in 27 to improve the background of the luminescence images and the StarDist plug-in 28 to create cell segmentation masks.
Statistical analysis. Statistical tests of luciferase assays were performed using unpaired two-tailed Student's Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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