SMYD5 is a regulator of the mild hypothermia response [Cut & Run]

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 look further at epigenetic modifications both in SMYD5 KD and WT cells and SMYD5 binding at 37°C and 32°C we decided to do CUT&RUN. We followed the Epicypher CUTANA protocol (v.1.7) on 300.000 cells per sample. We permeabilized the cells with 0.01% digitonin (Sigma, D141). For normalization we spiked in E. Coli DNA (Epicypher, 23618-1401) at the final concentration of 0.2 ng per sample. The library preparation was performed with TrueSeq-Chip Sample preparation Kit (Illumina, IP-202-9001). The following antibodies were used for the CUT&RUN: CTCF antibody (Cell Signaling, 2899S), H3K36me3 (Thermo Fisher Scientific, MA5-24687), H4K20me3 (EpiCypher, 13-0054), H3K9me3 (Abcam, ab176916), H3K4me3 (EpiCypher, 13-0041), H3K27me3 (Thermo Fisher Scientific, MA5-11198), SMYD5 (custom), and Rabbit IgG Antibody (Epicypher, 23613-0042). The sequencing was performed on a NovaSeq 6000 S4 at deCODE genetics, paired-end 8 cycles of index1, 8 cycles of index 2, 150 cycles read 1 and, 150 cycles read 2. Some samples were sequenced using a NextSeq 550 using the NextSeq 500/550 Mid Output Kit v2.5 (20024904) with 75 bp paired-end sequencing. For all samples we aimed for 10 million reads per sample.