SMYD5 is a regulator of the mild hypothermia response [CRISPR-Cas9 screen]

Organisms have homeostatic mechanisms to respond to cold temperature to ensure survival including the activation of the mammalian neuroprotective mild hypothermia response (MHR) at 32°C. Utilizing a forward CRISPR-Cas9 mutagenesis screen, we identify the histone lysine methyltransferase SMYD5 as an epigenetic gatekeeper of the MHR. SMYD5 represses the key MHR gene SP1 at euthermia but not at 32°C. This repression is mirrored by temperature-dependent levels of H3K36me3 at the SP1-locus and globally indicating that the mammalian MHR is regulated at the level of histone modifications. We identified additional SMYD5-temperature dependent genes suggesting a broader MHR-related role for SMYD5. Our study provides an example of how the epigenetic machinery integrates environmental cues into the genetic circuitry of mammalian cells and suggests novel therapeutic avenues for neuroprotection after catastrophic events. Overall design: To find additional regulators of the mild hypothermic response we performed a genome wide CRISPR-Cas9 screen using the SP1-MHI (HEK293+Cas9+SP1-MHI). We selected for stable sgRNAs with puromycin selection for 6 days and 8 hours and exposesd to 32°C the last 16h before FACS. We sorted our 5% most and least fluorescent cells. Next generation sequencing (NGS) was performed with single read, 80 cycles and 8 indexing cycles with PhiX spike in of 20% on NovaSeq S4 at deCODE genetics for HEK293WT+Cas9+SP1 and HEK293WT+Cas9(control). We did this experiment in 4 biological replicates.