As an essential basis to identify and characterize TLX ligands we established a hybrid reporter gene assay to capture TLX activity in a bidirectional fashion by combining Gal4-TLX with the potent transcriptional inducer Gal4-VP16 (see Supporting Information and Figure ). This artificial system reflected the repressor activity of TLX and simultaneously enabled potent control experiments to confirm TLX modulation. We also demonstrated that this VP16/TLX assay was representative of cellular TLX function. When we co-transfected Gal4-TLX with constitutively active NRs (Nurr1 or RORα resembling VP16 as ligand-independent transcriptional inducer) or NRs with low intrinsic activity in Gal4 format, a dose-dependent repression of reporter activity by TLX could be observed (Figure ) corroborating the VP16/TLX assay setting as suitable to discover and profile small molecule modulators of TLX activity. These cellular experiments indicated TLX-mediated effects on the activity of a variety of NRs. To confirm this observation in a cell-free setting, and to determine whether direct interactions are involved, we probed dimerization of TLX with RXR by titrating Tb 3+ -cryptate-labeled TLX LBD with GFP-labeled RXRα LBD in a homogenous time-resolved fluorescence resonance energy transfer (HTRF) assay. We detected strong RXR-TLX heterodimerization with an EC50 value of 120 nM (Figure ) suggesting an unprecedented direct repressor activity of TLX on nuclear receptors and further validating the VP16/TLX assay setting as highly suitable to study TLX modulation by small molecules.