During their development in the thymus, T lymphocytes of the adaptive immune system undergo a process of selection in which self-reactive cells are deleted. This ensures that the mature T cell repertoire does not contain autoreactive T cells capable of attacking self tissues in the periphery. Central to this process is a protein called Aire, for autoimmune regulator. Patients with mutations in Aire develop a syndrome characterized by various autoimmune manifestations, called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) or autoimmune polyendocrinopathy syndrome Type-1 (APS1). Likewise, mice lacking Aire protein develop autoimmunity, indicative of a breakdown in central tolerance. But despite compelling genetic data indicating an essential role for Aire in directing expression of antigens that are normally tissue restricted, little is known of the molecular mechanism by which Aire can promote this promiscuous gene expression (PGE). The experiments proposed here will elucidate the mechanisms of Aire function. First, we will purify Aire and identify associated proteins by mass spectrometry. This should provide important clues to the mechanism of Aire function. The second objective is to assay the Aire complex for histone-modifying activities. Finally, we will develop a system to study Aire function, by transducing a cell line with a promoter shown previously to be regulated in an Aire-dependent manner. By introducing various Aire mutants, we will be able to determine which regions of Aire are critical for transactivation. This system will also enable us to map the region of the promoter necessary for Aire-dependent activity, and identify a minimal Aire-responsive element. ? ? ?
