RORgamma is a nuclear receptor that binds to DNA motifs as a monomer to constitutively activate target genes. RORgamma plays an important role in thyrnocyte development and lymph node organogenesis, while the regulation of RORgamma-mediated transcriptional activation is currently unclear. The purpose of this study was to identify other nuclear proteins that interact with RORgamma. A yeast two-hybrid screen with Y190 yeast cells under stringent conditions resulted in the identification of CHD4, also known as Mi-2beta, as a RORgamma-interacting protein. This interaction was confirmed by GST pull-down assays. This interaction occurred within the middle regulatory region (amino acids 719-1164) of Mi-2beta. Transfection of Ga14-RORgamma into HeLa cells resulted in constitutive transactivation of the MH100-tk-luc reporter. The addition of Mi-2beta resulted in a dramatic 50% decrease in Ga14-RORgamma-mediated transactivation. These data demonstrate that RORgamma forms a protein-protein interaction with the regulatory region of Mi-2beta, resulting in inhibition of RORgamma transcriptional activity. These results may provide evidence as to how RORgamma-mediated transactivation is regulated by other nuclear proteins. (C) 2004 Elsevier Inc. All rights reserved.