Differential activities of murine single minded 1 (SIM1) and SIM2 on a hypoxic response element - Cross-talk between basic helix-loop-helix/Per-Arnt-Sim homology transcription factors

Abstract

The basic-helix-loop-helix/Per-Arnt-Sim homology (bHLH/PAS) protein family comprise a group of transcriptional regulators that often respond to a variety of developmental and environmental stimuli. Two murine members of this family, Single Minded 1 (SIM1) and Single Minded 2 (SIM2), are essential for post-natal survival but differ from other prototypical family members such as the dioxin receptor (DR) and hypoxia inducible factors, in that they behave as transcriptional repressors in mammalian one hybrid experiments and have yet to be ascribed a regulating signal. In cell lines engineered to stably express SIM1 and SIM2, we show that both are nuclear proteins that constitutively complex with the general bHLH/PAS partner factor, ARNT. We report that the SIM factors, in combination with ARNT, attenuate transcription from the hypoxia inducible Erythropoietin (EPO) enhancer during hypoxia. Such cross talk between coexpressed bHLH/PAS factors can occur through competition for ARNT, which we find evident in SIM repression of DR induced transcription from a xenobiotic response element reporter gene. However, SIM1/ARNT, but not SIM2/ARNT, can activate transcription from the EPO enhancer at normoxia, implying the SIM proteins have the ability to bind hypoxia response elements and affect either activation or repression of transcription. This notion is supported by co-immunoprecipitation of EPO enhancer sequences with the SIM2 protein. SIM protein levels decrease with hypoxia treatment in our stable cell lines, although levels of the transcripts encoding SIM1 and SIM2 and the approximate 2h half lives of each protein are unchanged during hypoxia. Inhibition of protein synthesis, known to occur in cells during hypoxic stress in order to decrease ATP utilisation, appears to account for the fall in SIM levels. Our data suggests the existence of a hypoxic switch mechanism in cells which coexpress HIF and SIM proteins, where upregulation and activation of HIF-1a is concomitant with attenuation of SIM activities.