Methyltransferase SETD4 mediates macrophages proliferation through EGFR signaling

Abstract Purpose: Epigenetic modifications have important impacts on the biological functions of cells. This study explored the effects of SET domain-containing protein (SETD)4, a histone lysine methyltransferase, on the general biological functions of bone marrow-derived macrophages (BMMs). Materials and Methods: We employed a CRISPR/Cas9 technology-edited mouse model. After isolated from SETD4 knockout (KO) and Wild-type (WT) mice, BMMs were maintained and identified. Then, serials in vitro experiments were performed to test the levels of methylation of histone lysine sites and the abilities in proliferation, cell cycle, migration, inducing vascular tube-formation and polarity. The phosphorylation pathway profiling arrays focused on the Mitogen-Activated Protein Kinase (MAPK), AKT, Janus Kinase/signal Transducer and Activator of Transcription(JAK/STAT), Nuclear Factor-κB (NF-κB), and Transforming Growth Factor β (TGF-β) signaling pathways were performed. After the Differentially Expressed Proteins (DEPs) were screened, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were adopted on those DEPs. Finally, CI-1033, an inhibitor for pan Epidermal Growth Factor Receptor (EGFR) was administrated to treat the BMMs, the cell viability and expression of phosphorylated(p)-EGFR(Ser1070) were assessed. Results: BMMs from SETD4 KO mice exhibited significant inhibition of monomethylation, dimethylation or trimethylation of histones H3K4, H3K36, H3K79 and H4K20; significant enhancement of the proliferation, EdU-positive cell ratio, Ki67 mRNA levels, and ability to form vascular structures; and attenuation of cell migration ability. After induction by lipopolysaccharide + interferon-γ or interleukin (IL)-4, a majority of the markers that were tested, including pro-inflammatory Interleukin-1β , Interleukin-6 , I nducible nitric oxide synthase and tumor necrosis factor-α and anti-inflammatory Fizz-1 , Arginase 1 and Mannose receptor C-type 1 were all downregulated, while TGF-β1 was upregulated. The phosphorylation pathway profiling arrays revealed 32 DEPs, six that were upregulated and 26 that were downregulated in BMMs from SETD4 KO mice versus WT mice. These DEPs were involved in 1365 terms from the GO and 137 signals from the KEGG database. Inhibition of the most significantly upregulated protein, EGFR(Ser1070), by CI-1033 significantly reduced p-EGFR(Ser1070), and was accompanied by a decrease in the cell viability of BMMs, particularly in the SETD4 KO cells. Conclusion: This is the first study to implicate the involvement of SETD4-mediated EGFR signaling in the proliferation of BMMs. SETD4 mediates the methylation of multiple histone lysine sites in BMMs, in turn affecting cell biology.