Evidence that ARNT plays a role in the regulation of the immunoglobulin heavy chain enhancer and identification of a putative ARNT ligand

Basic helix-loop-helix (bHLH) proteins are involved in the regulation of a multitude of developmental processes including cellular differentiation, cellular proliferation and xenobiotic metabolism. Among the members of the bHLH protein family are the products of the Pan gene Pan-1, Pan-2 and ITF -1. Pan proteins have been demonstrated to be required for proper B cell development, suggesting a unique role for Pan proteins during B cell formation. In our study we tested the function of ARNT (Ah receptor nuclear translocator) at the IgH (immunoglobulin heavy chain) enhancer. We were able to determine that ARNT appears to partially down-regulate activation at the IgH enhancer by Pan-1 in transient transfection assays by cotransfection of the multimerized murine form of the IgH enhancer elements 1-1E2, !-LE3 , and 1-1ES upstream of a luciferase reporter gene, a rodent Pan-1 (human homolog E47) expression vector, and an ARNT expression vector. Furthermore, during our investigation we discovered a putative ARNT -binding ligand that increases DNA-binding activity of the ARNT homodimer. This ligand was partially characterized by UV crosslinking studies and a variety of biochemical studies using electrophoretic mobility-shift assays. Preliminary data suggests that it is hydrophilic, heat-stable, small, and non-protein