Activation of AhR promotes phosphorylation of ARNT isoform 1 as a switch for optimal AhR activity in human T cell malignancies.

The aryl hydrocarbon receptor nuclear translocator (ARNT) is a transcription factor present in immune cells as a long and short isoform, referred to as isoform 1 and 3, respectively. However, investigation into potential ARNT isoform-specific immune functions is lacking despite the well-established heterodimerization requirement of ARNT with, and for the activity of, the aryl hydrocarbon receptor (AhR), a critical mediator of immune homeostasis. Here, using global and targeted transcriptomics analyses we show that the relative ARNT isoform 1:3 ratio in human T cell lymphoma cells dictates the amplitude and direction of AhR target gene regulation. Specifically, shifting the ARNT isoform 1:3 ratio lower by suppressing isoform 1 enhances, or higher by suppressing isoform 3 abrogates AhR responsiveness to ligand activation through preprograming a cellular genetic background that directs explicit gene expression patterns. Moreover, the fluctuations in gene expression patterns that accompany a decrease or increase in the ARNT isoform 1:3 ratio are associated with inflammation or immunosuppression, respectively. Molecular studies identified the unique casein kinase 2 (CK2) phosphorylation site within isoform 1 as an essential parameter to the mechanism of ARNT isoform-specific regulation of AhR signaling. Notably, CK2-mediated phosphorylation of ARNT isoform 1 is dependent on ligand-induced AhR nuclear translocation and is required for optimal AhR target gene regulation. These observations reveal ARNT as a central modulator of AhR activity predicated on the status of the ARNT isoform ratio and suggest that ARNT-based therapies are a viable option for tuning the immune system to target immune disorders. Karpas 299 cells were propagated in RPMI-1640 medium (Corning, 15-041-CV) complete with 10% FBS (Atlanta Biologicals, S11550) and 2 mM GlutaMAX (Gibco, 35050-061) at 37 °C, 5% CO2. Hepa-1c1c7 cells were cultured in MEM Alpha (1X) + 2 mM GlutaMAX (Gibco, 32561-037) with 10% FBS at 37 °C, 5% CO2. Stable BpRc1 cells, an Arnt-null variant of Hepa-1c1c7 cells, were cultured in DMEM (Corning, 15-018-CV) with 10% FBS, 2 mM GlutaMAX, and 2 mg/mL puromycin (Invivogen, ant-pr) at 37 °C, 5% CO2. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) (Cerilliant, ED-901-C), a high affinity AhR ligand, was used to activate the AhR pathway in samples that include TCDD in the label. TCDD was added to a final concentration of 10 nM and incubated for 2 hours, followed by total RNA collection.