Function and Mechanism of the Single-Minded 2 gene

Abstract

Single-Minded 2 (SIM2) is a member of the basic Helix-Loop-Helix PER-ARNT-SIM (bHLH/PAS) family of transcription factors which are known to play diverse roles in development, cellular homeostasis, and disease. The amino-terminal halves of bHLH/PAS transcription factors contain a basic DNA binding region followed by a helix-loop-helix dimerisation domain. This is then followed by a PAS domain consisting of two PAS repeats, PAS-A and PAS-B which also function in dimerisation and protein:protein interaction and ligand binding. Their carboxy-terminal halves contain transactivation or transrepression domains for target gene regulation. In mice Sim2 is essential for normal development. Mice lacking Sim2 have been found to have multiple abnormal phenotypes, including abnormal skeletal structures and overgrowth of gut bacteria. SIM2 has also been implicated in the progression of several cancers, with its function appearing to be highly context dependent. Upregulation of SIM2 in prostate, pancreatic and colon cancers favours tumour progression, whereas downregulation of SIM2 in breast cancer favours tumour progression. There is still much to discover regarding the function and target genes of SIM2 during development and in human disease. Therefore, the aim of this thesis was to further investigate the functions and mechanisms of action of SIM2 both in developmental and disease contexts. To investigate whether SIM2 may contribute to the pathogenesis of human developmental disorders, SIM2 non-synonymous gene variants identified in patients with neurological phenotypes were functionally assessed to determine their impact on activity of SIM2 as a transcription factor. This study identified five variants that caused a significant reduction in the transcriptional activating potency of SIM2 and were further characterised to determine the mechanism associated with the deficiency. This work identified a set of residues that are important for the function of SIM2 as a transcription factor and may contribute to human pathology. To investigate the function of SIM2 in breast cancer, the weak SIM2 expressing MDAMB- 231 cell lines was modified to enable inducible upregulation of SIM2 and subsequently subjected to RNA-sequencing. This study found a set of genes that were significantly differentially expressed upon upregulation of SIM2, culminating in a proposed mechanism whereby SIM2 crosstalks with other bHLH/PAS transcription factors to modulate their protumourigenic functions. To investigate the function of Sim2 during development, a conditional Sim2 knockout mouse model was generated to selectively remove Sim2 expression from the brain. These mice underwent feeding and behavioural studies to assess the impact of Sim2 knockout in the brain, however the tests did not identify any significant differences between the conditional knockout animals and their normal litter mate controls. An epitope tagged SIM2 mouse line was generated that will provide a valuable tool for assessing endogenous SIM2 protein functions and interactions in vivo. In addition, attempts were made to generate a reporter mouse in which expression of Sim2 would be replaced with a fluorescent protein. While various CRISPR based attempts were not successful in replacing a Sim2 allele with fluorescent Tomato coding sequence in zygotes, cultured mouse embryonic stem cells were successfully targeted. These Sim2- Tomato ES cells can be used in future to generate the desired Sim2-Tomato reporter mouse line.