Genetic Screen Identified Prmt5 as a Neuroprotection Target against Cerebral Ischemia [ChIP-seq]

Epigenetic regulators present novel opportunities for both ischemic stroke research and therapeutic interventions. While previous work has implicated that they may provide neuroprotection by potentially influencing coordinated sets of genes and pathways, most of them remains largely uncharacterized in ischemic conditions. In this study, we used the oxygen-glucose deprivation (OGD) model in the immortalized mouse hippocampal neuronal cell line HT-22 and carried out an RNAi screen on epigenetic regulators. We identified Prmt5 as a novel negative regulator of neuronal cell survival after OGD, which presented a phenotype of translocation from the cytosol to the nucleus upon oxygen and energy depletion both in vitro and in vivo. Prmt5 bound to the chromatin and a large number of promoter regions to repress downstream gene expression. Silencing Prmt5 significantly dampened the OGD-induced changes for a large-scale of genes, and gene ontology analysis showed that Prmt5-target genes were highly enriched for Hedgehog signaling. Encouraged by the above observation, we treated mice with middle cerebral artery occlusion (MCAO) with the Prmt5 inhibitor EPZ015666 and found that Prmt5 inhibition sustain protection against neuronal death in vivo. Together, our findings revealed a novel epigenetic mechanism of Prmt5 in cerebral ischemia and uncovered a potential target for neuroprotection. Overall design: HT-22 cells were subjected to oxygen-glucose deprivation (OGD) treatment and crosslinked with 1% formaldehyde for 10 min at room temperature. Formaldehyde was quenched by 200 mM glycine and cells were rinsed twice with ice-cold PBS. Cells were transferred to 15 ml conical tubes and collected by centrifugation. Cells were lysed with lysis buffer A (50 mM HEPES-KOH (pH 7.5), 140 mM NaCl, 1 mM EDTA, 0.5% NP-40, 0.25% Triton X-100, 10% Glycerol and protease inhibitor cocktail (Roche)), incubated at 4°C for 10 min and collected by spinning at 1300 × g for 5 min at 4°C. Cells were then resuspended in lysis buffer B (10 mM Tris-Cl (pH 8), 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA and protease inhibitor cocktail), incubated at room temperature for 10 min. Nuclei were pelleted by spinning at 1300 × g for 5 min at 4°C. The pellet was suspended with lysis buffer B (10 mM Tris-Cl (pH 8), 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine and protease inhibitor cocktail) and incubated for 15 min on ice. Chromatin shearing was conducted with cells on ice, using a microtip attached to Misonix 3000 sonicator. Sonicate 8-12 cycles of 30 s ON and 90 s OFF around 30-watt power-output. A final concentration of 1% Triton X-100 was added and gently mixed by pipetting. The chromatin solution was clarified by spinning at 20 000 g at 4°C for 30 min. Chromatin immunoprecipitation was performed with 50 ul Dynabeads protein G (Life technology) conjugated preliminary antibodies antibody overnight at 4°C. The immunoprecipitated material was washed five times with wash buffer (10 mM Tris-Cl (pH 8), 1 mM EDTA, 0.5% NP40, 0.5M LiCl, 0.5% Na-Deoxycholate) and once with TE buffer (PH 8.0), then, eluted by heating for 30 min at 65°C with elution buffer (50 mM Tris-Cl (pH 7.5), 10 mM EDTA, 1% sodium dodecyl sulphate). To reverse the crosslinks, samples were incubated at 65°C overnight, then the eluted was digested with a final concentration of 0.5 µg/ml RNasesA at 37°C, followed with a final concentrated of 0.5 µg/ml Proteinase at 55°C for 2 h. The immunoprecipitated DNA were then purified using the DNA clean and concentrator 5 column (Zymo Research). The ChIP DNA was used for qPCRs using indicated primers and data were plotted as the percentage of input. All experiments were performed three or more times, and representative results were shown in the figures. For ChIP-seq, 1 ng precipitated DNA or input was used to generate sequencing libraries using the Nextera XT DNA sample preparation Kit (Illumina). The resulting libraries were sequenced on Next-Seq (Illumina). Two biological replicates were performed, and combined reads were used for further analysis.