ERR alpha (estrogen receptor-related receptor alpha) is a member of the nuclear receptor superfamily. To further our understanding of the detailed molecular mechanism of transcriptional regulation by ERR alpha, we searched for ERR alpha-interacting proteins using a yeast two-hybrid system by screening a human mammary gland cDNA expression library with the ligand-binding domain (LBD) of ERR alpha as the "bait". Fast skeletal muscle troponin I (TNNI2), along with several known nuclear receptor co-activators, were isolated. We demonstrated that TNNI2 localizes to the cell nucleus and interacts with ERR alpha in co-immunoprecipitation experiments. GST pull-down assays also revealed that TNNI2 interacts directly with ERR alpha. Through luciferase reporter gene assays, TNNI2 was found to enhance the transactivity of ERR alpha. Combining mutagenesis and yeast two-hybrid assays, we mapped the ERR alpha-interacting domain on TNNI2 to a region encompassing amino acids 1-128. These findings reveal a new function for TNNI2 as a co-activator of ERR alpha. (C) 2008 Elsevier Inc. All rights reserved.