Abstracts Biological Sciences

Characterisation of the transcriptional properties of the mSim gene products

by Peter. Moffett

The basic-Helix-Loop-Helix PAS (bHLH-PAS) protein family is a growing family of transcription factors. Included in this family is the Drosophila single-minded (DSim) gene, which is critical for the development of the CNS midline cells. We have isolated a murine homologue of the Drosophila Sim gene, mSim-2. The murine and Drosophila gene products share a high degree of homology in the bHLH and PAS regions but not within the carboxy terminus. Northern blot and RT-PCR analysis of adult murine tissues revealed that the mSim-2 is expressed at the highest levels in the kidneys and at lower levels is present in skeletal muscle, lung, testis, brain, and heart. In situ hybridisation experiments demonstrate that mSim-2 is also expressed in early fetal development in the central nervous system and in cartilage primordia. We investigated the ability of mSIM-2 to associate with the Arnt gene product, a common dimerisation partner of a number of bHLH-PAS proteins. We found that the HLH and PAS motifs of both proteins are required for optimal association. We demonstrated the presence of two separable repression domains within the carboxy terminus of mSIM-2, in contrast to dSIM, which is a transactivator. We find that mSIM-2 is also capable of repressing activation by its binding partner, ARNT. We also demonstrate that mSIM-2 can functionally interfere with another bHLH-PAS transcription factor, HIF-1alpha, by competing for ARNT binding, providing a second mechanism by which mSIM-2 may inhibit transcription. We also investigated the effects of mSIM-2 and its close paralogue mSIM-1, as heterodimers with ARNT, on reporter constructs containing native DNA binding sites. We find that mSIM-1 can effect transcriptional activation through it's association with ARNT. In contrast, mSIM-2/ARNT does not activate, as the mSIM-2 repression domains quench ARNT transactivation. We also find that the mSIM-2 can interfere with mSIM-1 mediated transactivation by competing for dimerisation with ARNT and for DNA binding site occupation. Our results suggest that mSIM-1 and mSIM-2 have similar dimerisation and DNA binding properties but different transcriptional effects and may therefore antagonise each other which may in turn be a mechanism of gene regulation by these two proteins.