Cross-talk between SIM and other bHLH/PAS factors and the search for SIM2 target genes.
Date
2004
Authors
Woods, Susan Lesley
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Abstract
This project aimed to investigate how the murine Simgle minded (mSIM) proteins interact with other active bHLH/PAS transcription factors and provide the first study to identify direct target genes of mSIM2. We show that the murine SIM (mSIM) factors are constitutively nuclear proteins in cells and that the presence of an mSIM protein outcompetes the dioxin receptor (DR) for aryl hydrocarbon nuclear translocator 1 (ARNT1) binding, resulting in attenuation of transcription of a reporter gene induced by a constitutively active form of the DR. In contrast, the hypoxia inducible factor-1a (HIF-1a) and mSIM proteins appear equally competent to sequester ARNT1. Expression of either mSIM protein repressed, but did not totally prevent, the hypoxic activation of a reporter containing hypoxic response element (HRE) sequences from the erythropoietin (EPO) enhancer and endogenous hypoxic target genes in 293T cells. This repression occurs through competition of the HIF-1a and theSIM proteins for binding to ARNT1 but also for DNA binding sites in the HRE-reporter plasmid. In contrast to HIF-1a, which rapidly accumulates in low oxygen conditions, SIM protein levels decrease in hypoxia, most likely as a result of general translational inhibition in hypoxia stressed cells. Cross-talk between the mSIM and HIF-a or DR proteins may occur to enable the cell to adapt to multiple environmental and developmental signals and is particularly relevant in pathological states such as Down’s Syndrome (DS) or tumour types recently recognized to contain elevated levels of a short isoform of human SIM2 (hSIM2). Microarray experiments identified a large number of transcripts that are differentially regulated by mSIM2 and one of thes predominantly expressed in muscle, Myomesin2 (Myom2), was shown to potentially be the first identified direct target gene of mSIM2/ARNT1. Bacterially expressed and partially purified N-terminal portions of mSIM2/ARNT1 bound a 40bp probe derived from the Myom2 promoter sequence but not an identical probe with the 5’-AACGTG-3’ site mutated, and mutation of the putative mSIM2/ARNT1 binding site in the Myom2 promoter led to a decreased reporter induction with the mSIM2AD chimeric protein, in which the mSIM2 repression region is replaced with the activation domain from the DR. Evidence that mSIM2 regulates the transcription of endogenous Myom2 awaits the generation of a specific anti-mSIM2 antibody to allow examination of the Myom2 promoter occupation by mSIM2/ARNT1 using chromatin immunoprecipitation and the effect of mSim2 knockdown on Myom2 transcript levels. The transcriptional activity of the mSIM proteins appears to be dependent on the promoter context and cell type examined, as surprisingly we have observed the mSIM1/ARNT1 dimer activating transcription of the HRE-reporter, whilst the mSIM2/ARNT1 dimer does not activate the Myom2-reporter in 293T cells, but does activate it in 293 cells. Both SIM1 and SIM2 were previously described as harbouring repression regions in their C-termini, however, when dimerised with ARNT1, activation is sometimes observed which is dependent on the presence of the ARNT1 transactivation domain.
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School of Molecular and Biomedical Science
Dissertation Note
Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2004
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