SMYD5 is a regulator of the mild hypothermia response

Abstract

The mild hypothermia response (MHR) maintains organismal homeostasis during cold exposure and is thought to be critical for the neuroprotection documented with therapeutic hypothermia. To date, little is known about the transcriptional regulation of the MHR. We utilize a forward CRISPR-Cas9 mutagenesis screen to identify the histone lysine methyltransferase SMYD5 as a regulator of the MHR. SMYD5 represses the key MHR gene SP1 at euthermia. This repression correlates with 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 have identified 37 additional SMYD5 regulated temperature-dependent genes, suggesting a broader MHR-related role for SMYD5. Our study provides an example of how histone modifications integrate environmental cues into the genetic circuitry of mammalian cells and provides insights that may yield therapeutic avenues for neuroprotection after catastrophic events.

Fig. 1.. MHIs allow single cell fluorescent quantification of MHR activation.

(A) A schematic of MHI structures. (B-C) Representative fluorescent images of SP1-Lenti-MHIs and CIRBP-Lenti-MHI, respectively, at 37°C and 32°C. (D) A Western blot demonstrating increased SP1 after 16 h at 32°C compared to 37°C. Each data point is a biological replicate (n=2), and their mean is depicted. Significance levels were calculated with an unpaired two-tailed t-test. (E) geometric Mean Fluorescence Intensity (gMFI, flow cytometry) after varying lengths of hypothermia exposure for SP1-MHI. Each data point is a technical replicate (n=2), with mean and standard deviation (SD) where applicable. Significance levels were calculated with an unpaired one-tailed t-test. (F-G) Mean fluorescence (MFI, flow cytometry) of SP1-MHI and CIRBP-MHI, respectively (16 h at 32°C, 37°C, 40°C). Each point is a technical replicate (n=3), with mean and SD where applicable. Significance levels were calculated with Šidák’s multiple comparison test. (H) gMFI of RBM3-MHI (16 h at 32°C and 37°C). Each point is a technical replicate (n=4–5), with mean and SD where applicable. Significance levels were calculated with an unpaired one-tailed t-test. All significance levels in this figure were calculated in GraphPad Prism, * = P<0.05, ** = P<0.01, *** = P<0.001, **** = P<0.0001.

Fig. 2.. Genome-scale CRISPR-Cas9 knock out screen on SP1-MHI reveals multiple potential inhibitors and activators of the MHR.

(A) Overview of the genome-scale CRISPR-Cas9 knock out approach for the HEK293WT+Cas9+SP1 cell line. (B) Fluorescence measurements and sort gates of the 4 replicates of transduced HEK293WT+Cas9+SP1 cells (green), negative control (HEK293WT, black) and positive control (HEK293WT+Cas9+SP1, grey). (C-D) Genes marked in red are transcription regulators that have a −Log10(RRA) score >3.5 and a known repressive function. Genes marked in green are transcription regulators that have a known activating function and a −Log10(RRA) score >3.5. Colored dots represent genes that have a −Log10(RRA) score >3.5, where the orange dots indicate genes that have a positive log fold change (LFC) and the blue dots indicate genes that have a negative LFC, from either the SP1 activator (C) or repressor (D) screen. −Log10(RRA) score and LFC was calculated with MAGeCK.

Fig. 3.. SMYD5 is a direct repressor of SP1 at 37°C.

(A) Overexpressed FLAG-tagged SMYD5 (n=2) in mESc binds at promoters of Sp1 and Cirbp but not of Rbm3. H3K36me3 peaks over Sp1, Cirbp and Rbm3 promoter and gene body regions. Data from Zhang et. al. (B) Venn graph showing both up- and downregulated SMYD5-bound genes in an RNASeq of Smyd5-KO cells (n=2). Statistical analysis was done with GeneOverlap package in R using Fisher’s Exact test. (C) Smyd5-KO leads to increased mRNA expression of SP1 but not RBM3 or CIRBP in mESCs. (D) SMYD5-KD by siRNA yields higher levels of fluorescence of SP1-MHI at 32°C and 37°C compared to empty vector control. Each data point is a biological replicate (n=3), that has been normalized against the same non-transfected HEK293WT+Cas9+SP1-MHI cell line, mean and SD are depicted where applicable. Significance levels were calculated with Šidák’s multiple comparisons test in GraphPad Prism. (E) Relative expression of SMYD5 mRNA compared to GAPDH from SMYD5-KO cells, measured by RT-qPCR at 37°C, with or without rescue with Flag-SMYD5 sgRNAres plasmid (labelled SMYD5 sgRNA#6res). Each data point is a biological replicate (n=4), with mean and SD where applicable. Significance levels were calculated with an unpaired one-tailed t-test in GraphPad Prism. (F) Western blot using antibodies against SMYD5 and Lamin B in a SMYD5-KO HEK293 cell line at 37°C with and without rescue with Flag-SMYD5 sgRNAres plasmid (labelled SMYD5 sgRNA#6res). SMYD5-KO was successful at the protein level at 37°C, (n=2–3). Data shown as in (E). (G) Relative expression of SP1 mRNA compared to GAPDH from SMYD5-KO cells, measured by RT-qPCR at 37°C, with or without rescue with Flag-SMYD5 sgRNAres plasmid (labelled SMYD5 sgRNA#6res), (n=4). Data shown as in (E). (H-J) Western blot quantification with and without 16 h incubation at 32°C and representative examples using antibodies against SP1, CIRBP and RBM3, respectively, in SMYD5-KO cells. Each data point is a biological replicate (n=2–3), with mean and SD where applicable. Significance levels were calculated with an unpaired one-tailed t-test in GraphPad Prism. * = P<0.05, ** = P<0.01, *** = P<0.001, **** = P<0.0001.

Fig. 4.. SMYD5 is depleted at 32°C in vitro by the proteasome and in vivo.

(A) Representative image of intensity of endogenous SMYD5 at 37°C (top, a) and 6 h incubation at 32°C (bottom, b). Nuclei are marked with a dotted line. Other images can be found in Fig. S6. (B) Quantification of cellular endogenous SMYD5 mean intensity levels from immunocytochemistry, mean fold change between conditions is depicted. Each data point is a biological replicate (n=3), mean and SD shown where applicable, significance levels were calculated with an unpaired one-tailed t-test. (C) The ratio of nuclear to whole cell SMYD5 mean intensity, (n=3). Data shown as in (B) (D) Western blot using a SMYD5 antibody at 37°C and 6 h incubation at 32°C, (n=3). Significance levels calculated with an unpaired two-tailed t-test otherwise data shown as in (B). (E) qRT-PCR results for SMYD5 expression at 37°C and 32°C after 6 h incubation, (n=3). Significance levels calculated with an unpaired one-tailed t-test. (F) SMYD5 levels at 37°C and at 32°C with and without the exposure of the proteasomal inhibitor MG132, (n=3). Data shown as in B. (G) A schematic overview of sagittal sections of a P10 mouse brain. Boxes indicate areas that were used in (H-J). (H) Representative SMYD5, DAPI and NeuN staining in brain sections of mice kept at euthermia (37°C; a-d) after neonatal hypoxic-ischemic injury compared to those treated with cooling at 32°C, for 6 h (e-h). Sagittal brain sections are from the Sub (a and e), CA1 (b and f), CA3 (c and g) and somatosensory cortex (d and h).⁺ Background for SMYD5 in merged images has been adjusted to emphasize the specific staining of SMYD5. (I-J) Quantification of NeuN and SMYD5 staining from (H) (n=2), where hippocampus included Sub, CA1 and CA3 regions. Each data point is a biological replicate, that is an average of two technical replicates, the mean of the replicates is shown. Mean fold change between conditions is depicted. Significance level calculated with an unpaired one-tailed t-test. All significance levels for this figure were calculated in GraphPad Prism, * = P<0.05, ** = P<0.01, *** = P<0.001, **** = P<0.0001.

Fig. 5.. SMYD5 is a regulator of the MHR.

(A) Overlap of upregulated DEGs in vitro and in vivo RNASeq datasets from mNPCs (n=3 at 37°C, n=4 at 32°C), hippocampal (n=6 at 37°C, n=6 at 32°C), and cortical cells (n=6 at 37°C, n=6 at 32°C), at 32°C compared to 37°C. (B) Overlap of downregulated DEGs in vitro and in vivo RNASeq datasets from (n=3 at 37°C, n=4 at 32°C), hippocampal (n=6 at 37°C, n=6 at 32°C), and cortical cells (n=6 at 37°C, n=6 at 32°C), at 32°C compared to 37°C. (C) Overlap of upregulated DEGs from in vitro and in vivo RNASeq datasets (mNPCs, hippocampal and cortical cells), at 32°C with SMYD5-bound and repressed genes in mESC. Statistical analysis was done with GeneOverlap package in R using Fisher’s Exact test. (D) Heatmap for the 37 genes from (C), depicting all normalized counts >10 converted to z-scores, the transformation was done with variance stabilization in DESeq2. *DEG in each dataset that have p. adjusted < 0.1 calculated with DESeq2. (E) Mean Log₂FC of H3K36me3 peaks per gene over promoter sites and distal intergenic sites. Each datapoint depicted is a gene that had p value < 0.05 for both datasets and Log₂FC < −1 for H3K36me3 modification when comparing 37°C to 32°C in SMYD5-WT cells (SMYD5-WT; n=6, SMYD5-KD; n=3). Red colored dots represent genes that are upregulated at 32°C in one of three RNASeq datasets presented in (A). Blue colored dots represent genes that are downregulated at 32°C in one of three RNASeq datasets presented in (B). Gray colored dots represent genes that are not upregulated at 32°C in any of the three RNASeq datasets presented in (A). (F-G) Overlap of genes from (E) and human orthologs of up- or downregulated genes at 32°C from at least one of the three RNASeq datasets from (A) or (B), at promoter sites and distal intergenic sites, respectively. Data shown as in (C).