Please use this identifier to cite or link to this item: http://hdl.handle.net/1946/25680
Introduction: Cells have adaptive responses to tolerate changing environments. It is likely that some of these responses involve epigenetic regulation of adaptive gene expression changes. By understanding these responses one may be able to better understand the mechanistic basis of therapeutic hypothermia. Here we explore whether the H3K4me3 system (a Trithorax ortholog), previously implicated in environmental responses in plants, mediates a similar response in humans. Furthermore we investigated whether known cold-responsive genes (CIRP and SP1), show cell type specific responses to cold stimuli. Finally, we developed an unbiased strategy to map the factors that play a role in the (epigenetic) upregulation of genes known to upregulate in response to cold stress.
Materials and methods: Here we employed two reporter alleles that measure global activity of the H4Ac and H3K4me3 machineries to test the hypothesis that global changes in H3K4me3 act as a switch to help human cells respond to cold stress. Furthermore, we developed several novel fluorescence based indicator constructs for two genes known to be upregulated with cold temperature stimuli (SP1 and CIRP), to rapidly and robustly explore responses to cold stress in multiple cell types. Finally, one of these constructs (SP1-Short indicator) was stably integrated into HEK293 genome after preliminary studies and will be used in an unbiased forward mutagenesis screen to identify upstream regulators of SP1.
Results: We have developed an initial tool-kit for the exploration of cold stimulated adaptive responses in human cells. Furthermore, through our use of fluorescence indicators we demonstrate variation in expression of SP1 among different cancer cell lines when exposed to cold temperature stimuli.
Discussion and Conclusion: We hypothesize that these changes could either be influenced by cell type of origin or the genetic complement of the individual lines. We are in the process of using a non-biased forward mutational screen that has the potential of uncovering novel regulators of cold adaptation. A better understanding of cold adaption may yield insight into the therapeutic benefit of hypothermia, a poorly understood therapeutic strategy that is widely used in clinical medicine. This opens up the possibility to develop a pharmacological strategy to capture some of the positive effects of hypothermia without the need to anesthestize the patient.