Investigating mechanisms and biological functions of the bHLH/PAS transcription factors SIM1 and ARNT2
Date
2021
Authors
Gerassimou, Alexis Georgia
Editors
Advisors
Whitelaw, Murray
Peet, Dan
Peet, Dan
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Thesis
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Abstract
The basic Helix-Loop-Helix/PER-ARNT-SIM (bHLH/PAS) family of transcription factors are involved in gene regulation during both embryonic development and throughout adulthood to maintain cellular homeostasis. Members of this family must heterodimerise to activate or repress target gene expression. ARNT and ARNT2 are homologues that are common dimerisation partners for many other bHLH/PAS transcription factors. Previous attempts to generate knockout mice of ARNT and ARNT2 transcription factors have resulted in embryonic or perinatal lethality due to the important developmental roles of both factors. These studies have also indicated unique as well as overlapping functions of ARNT and ARNT2 during embryonic development. Due to the lethality of homozygous null mice, there is limited knowledge of unique and overlapping functions of these two proteins in adult tissues. The development of a doxycycline (Dox) inducible short hairpin RNA (shRNA) system is an attractive approach to deplete gene expression without irreversibly damaging a gene and can allow normal embryonic development to occur prior to studying effects of reduced protein levels in adult tissues. In this study, we evaluated the efficiency of Dox inducible shRNA sequences to result in global knockdown of ARNT and ARNT2 in a mouse model. While we were able to show mosaic shRNA knockdown in embryonic shRNA mice, we did not observe any knockdown of either ARNT or ARNT2 in adult mice. Genetic mouse models have revealed one key partner protein of ARNT2 to be Single-Minded 1 (SIM1), a bHLH/PAS factor predominately expressed in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus. SIM1 has been established to be essential in the developing hypothalamus and has a physiological role in appetite control during adulthood. Haploinsufficiency of Sim1 in mice results in increased weight gain due to hyperphagia and increased linear growth, and non-synonymous point mutants of SIM1 can be key drivers of obesity in humans. Several studies indicate that SIM1 plays an important role in the appetite controlling Leptin-Melanocortin signalling pathway in the hypothalamus, however the target genes of SIM1 and its up- and downstream regulatory pathways have yet to be defined. To further our understanding of the role of SIM1 in appetite control, we generated a mouse model harbouring a weakly functioning mutant of SIM1 to demonstrate that a single amino acid change in SIM1 could underpin monogenetic obesity. Composite with a transgene expressing GFP under the control of the Sim1 promoter, this mouse model, in combination with cell based assays, was used to investigate mechanisms of dysfunction that result from SIM1 mutations. We were able to show that the mutation did not decrease SIM1 protein stability or the ability of SIM1 to dimerise with interacting proteins, nor did it have an apparent effect on PVN development. There is currently a lack of structural data for the bHLH/PAS family of transcription factors, including SIM1. Using the mammalian expression system Expi293F cells, we developed a system to express and purify a truncated SIM1/ARNT2 heterodimer for use in structural studies. A SIM1 structure will allow further understanding into how SIM1 functions and will aid development of targeted drug design.
School/Discipline
School of Biological Sciences
Dissertation Note
Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 2021
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