NF-κB-mediated enhancement of H3K27me3 through EZH2: a mechanism to suppress pyocyanin-induced autophagy in macrophages.

Background: Histone modification is a key mechanism of epigenetic regulation. Our previous study demonstrated that histone H3 acetylation at lysine 27 (H3K27ac) promotes pyocyanin (PYO)-induced autophagy in macrophages. However, the regulatory role of H3K27 trimethylation (H3K27me3) in this process remains unclear.

Methods: THP-1 macrophages were treated with PYO, and autophagy was assessed by evaluating LC3B II expression and autophagosome formation. The expression of EZH2 and JMJD3 was analyzed to identify the key enzyme responsible for regulating H3K27me3. Nuclear-cytoplasmic fractionation and co-immunoprecipitation were performed to determine the distribution of NF-κB and its interaction with H3K27me3. To explore the role of H3K27me3 in PYO-induced autophagy, cells were co-treated with PYO and EZH2 inhibitors (EI1 or CPI-169), and the transcription of ULK1, BECN1, and MAP1LC3B was analyzed using ChIP-qPCR. Similarly, to assess the role of NF-κB, cells were co-treated with PYO and the NF-κB nuclear translocation inhibitor curcumin, followed by ChIP-qPCR analysis. Finally, the reciprocal transcriptional regulation between NF-κB and H3K27me3 was further investigated.

Results: PYO increases LC3B II expression and autophagosome formation in THP-1 macrophages. It also elevates H3K27me3 levels by upregulating EZH2 expression, while JMJD3 remains unchanged. Co-treatment with EZH2 inhibitors reduces H3K27me3 levels, leading to increased LC3B II expression and enhanced autophagosome formation. ChIP-qPCR analysis shows that H3K27me3 enrichment at the ULK1 and MAP1LC3B promoters correlates with reduced transcription, whereas BECN1 remains unaffected. PYO promotes nuclear translocation of NF-κB and enhances its interaction with H3K27me3. ChIP-qPCR further reveals that NF-κB represses the transcription of ULK1 and MAP1LC3B and upregulates EZH2 transcription, which contributes to increased H3K27me3 levels and further suppression of autophagy-related gene expression.

Conclusions: The NF-κB/EZH2/H3K27me3 axis plays a pivotal role in suppressing PYO-induced autophagy in macrophages by repressing the transcription of ULK1 and MAP1LC3B.