SET domain-containing protein 4: biological functions and role in genomic methylation profiles of bone marrow mesenchymal stem cells

Background: Epigenetic modification is a crucial mechanism affecting the biological function of stem cells. SETD4 is a histone methyltransferase, and its biological role in bone marrow mesenchymal stem cells (BMSCs) is currently unknown. This work was aimed to reveal the SETD4 biological role as well as its impacts on the genomic methylation profiles in BMSCs. Methods: BMSCs were isolated form SETD4 knockout (KO) and wild type (WT) mice that established by CRISPR/Cas9 technology. The cell proliferation, migration, myogenic differentiation and angiogenesis were tested according to appropriate biology techniques. And the Reduced Representation Bisulfite Sequencing (RRBS) method was adopted to analyze the global genomic methylation profiles of BMSCs, following bioinformatics analysis of GO functions and KEGG signaling of differential methylated CpG sites and differential methylation regions (DMRs). Finally, validation experiments were conducted to examine the expression of histone lysine methyltransferase and some representative genes. Results: SETD4 KO significantly promoted BMSCs proliferation, which was characterized by enhanced cell viability and increased expression of PCNA, Cyclin A2, Cyclin E1, CDK2, CDK6, Bcl2 and decreased the expression of P16, P21 and Caspase3. SETD4 deficiency impaired BMSCs migration and myogenic differentiation potentials, and even the angiogenesis via paracrine of VEGF. Compared with WT control, the overall genomic methylation of BMSCs in the SETD4 KO group only was decreased by 0.47%. However, the changed genomic methylation covers a total of 96,331 differential methylated CpG sites and 8692 DMR, with part of them settled in promoter regions. GO and KEGG analysis revealed that differential CpG islands and DMRs in promotes impacted 270 GO functions and 34 KEGG signaling pathways, with some closely related to stem cell biology. SETD4 KO inhibited sets of monomethylases and dimethylases for histone lysine, along with significant changes in some factors including Nkx2.5, Gata4, Gli2, Grem2, E2f7, Map7, Nr2f2 and Shox2 that associated with stem cell biology. Conclusions: These results are the first to reveal that even though SETD4 changes the genome’s overall methylation to a limited extent in BMSCs, it still affects the numerous cellular functions and signaling pathways, implying SETD4-altered genomic methylation serves a crucial molecular role in BMSCs’ biological functions.