Kevin Ostacolo, Kijin Jang, Kimberley Anderson and Hans Tomas Bjornsson
Introduction: Physicians commonly actively lower core temperature using a strategy called targeted temperature management. This strategy is utilized to prevent neurological damage following events such as neonatal hypoxic-ischemic brain injury and cardiac arrest. However, the precise factors that play a role in the mild hypothermia response (MHR) remain unclear. Here we seek to elucidate and describe additional factors participating in this response.
Methods: To identify additional factors of the MHR, we employed diverse approaches for a comprehensive understanding. Initially, using HEK293 cells, we conducted a Genome-scale CRISPR-Cas9 Knockout (GeCKO) screen, systematically targeting all genes in the genome. Effects were assessed through SP1 or RBM3 activation, known as key MHR genes. RNA-Seq was performed on the same cell model and in NPCs under normothermic and hypothermic conditions to evaluate transcriptional effects. ATAC-Seq will be conducted at both temperatures to elucidate transcription factor usage. Lastly, unbiased proteomics will be conducted under normothermia and hypothermia to identify translational changes.
Results: Our GeCKO screen identified 556 candidate regulators of SP1. We have already validated one regulator, SMYD5, which appears to regulate levels of SP1. Of genes identified in the screen, 140 show significantly differential expression levels in one of our RNA-Seq experiments. We then plan to identify transcription factors from the ATAC-seq data and complement these results by determining expression pathways through proteomics analysis.
Conclusion: Using a multi-omics approach, we believe we can delineate the full MHR, potentially providing insights for therapeutic strategies aimed at reducing neurological damage without cooling.
