m6A hypomethylation of DNMT3B regulated by ALKBH5 promotes intervertebral disc degeneration via E4F1 deficiency

The intervertebral disc (IVD) degeneration is the leading cause of low back pain, which accounts for a main cause of disability. N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic messenger RNAs and is involved in various diseases and cellular processes by modulating mRNA fate. However, the critical role of m6A regulation in IVD degeneration remains unclear. Nucleus pulposus cell (NPC) senescence is critical for the progression of IVD degeneration. Here, we uncovered the role and explored the regulatory mechanism of m6A in NPC senescence during IVD degeneration.Identification of NPC senescence during IVD degeneration was based on the analysis of tissue samples and the cellular model. ALKBH5 upregulation inducing cellular senescence was confirmed by functional experiments in vivo and in vitro. ChIP-qPCR and DNA-Pulldown were used to reveal increased ALKBH5 was regulated by KDM4A-mediated H3K9me3. Furthermore, Me-RIP-seq was performed to identify m6A hypomethylation of DNMT3B transcripts in senescent NPCs. Stability analysis showed that DNMT3B expression was enhanced for less YTHDF2 recognition and increased DNMT3B promoted NPC senescence and IVD degeneration via E4F1 methylation by in vivo and in vitro analyses.Expression of ALKBH5 is enhanced during IVD degeneration and NPC senescence, due to decreased KDM4A-mediated H3K9me3 modification. Functionally, ALKBH5 causes NPC senescence by demethylating DNMT3B transcripts and in turn promoting its expression via less YTHDF2 recognition and following degradation due to transcript hypomethylation in vitro and in vivo. Increased DNMT3B promotes the development of IVD degeneration and NPC senescence, mechanistically by methylating CpG islands of E4F1 at the promoter region and thus restraining its transcription and expression.Collectively, our findings reveal an epigenetic interplay mechanism in NPC senescence and IVD degeneration, presenting a critical pro-senescence role of ALKBH5 and m6A hypomethylation, highlighting the therapeutic potential of targeting the m6A/DNMT3B/E4F1 axis for treating IVD degeneration.