THE MURINE SIM-2 GENE-PRODUCT INHIBITS TRANSCRIPTION BY ACTIVE REPRESSION AND FUNCTIONAL INTERFERENCE

The Drosophila single-minded (Dsim) gene encodes a master regulatory p rotein involved in cell fate determination during midline development. This protein is a member of a rapidly expanding family of gene produc ts possessing basic helix-loop-helix (bHLH) and hydrophobic PAS (desig nated a conserved region among PER, ARNT [aryl hydrocarbon receptor nu clear translocator] and SIM) protein association domains, Members of t his family function as central transcriptional regulators in cellular differentiation and in the response to environmental stimuli such as x enobiotics and hypoxia, We have previously identified a murine member of this family, called mSim-2, showing sequence homology to the bHLH a nd PAS domains of Dsim. Immunoprecipitation experiments with recombina nt proteins indicate that mSIM-2 associates with the arnt gene product , In the present work, by using fine-structure mapping we found that t he HLH and PAS motifs of both proteins are required for optimal associ ation, Forced expression of GAL4/mSIM-2 fusion constructs in mammalian cells demonstrated the presence of two separable repression domains w ithin the carboxy terminus of mSIM-2. We found that mSIM-2 is capable of repressing ARNT-mediated transcriptional activation in a mammalian two-hybrid system. This effect (i) is dependent on the ability of mSIM -2 and ARNT to heterodimerize, (ii) is dependent on the presence of th e mSIM-2 carboxy-terminal repression domain, and (iii) is not specific to the ARNT activation domain, These results suggest that mSIM-2 repr ession activity can dominantly override the activation potential of ad jacent transcription factors. We also demonstrated that mSIM-2 can fun ctionally interfere with hypoxia-inducible factor 1 alpha (HIF-1 alpha )/ARNT transcription complexes, providing a second mechanism by which mSIM-2 may inhibit transcription.