Investigation Of The Molecular Mechanism Of Autophagy Induced By BIX-01294,An Inhibitor For Histone H3K9 Methylation

Autophagy is an important biological process that is highly conserved in eukaryotic cells and can degrade large molecules such as proteins and organelles.Although the classic autophagy pathway induced by starvation has been thoroughly studied,the type of autophagy regulated by the alteration of epigenetic modifications in the nucleus still needs to be further understood.In order to investigate the effect of histone modifications on the occurrence of autophagy,we screened and obtained several small molecule inhibitors of epitopemodifying enzymes that can induce autophagy in mammalian cells.BIX-01294 is an inhibitor of the histone methylase G9 A,which can totally reduce the levels of H3K9 dimethylation and trimethylation modification in cells.The molecular mechanism by which BIX-01294 induces autophagy is unclear.We used CRISPR /Cas9 gene knockout technology to get some autophagy genes-knockout cell lines.These genes are considered essential in the classic autophagy pathway.We found that BIX-01294-induced autophagy is dependent on ATG5 and ATG7.The deletion of ULK1 and BECN1 genes did not affect the autophagy induced by BIX-01294.This suggests that this type of autophagy is not exactly the same as the classic autophagy pathway.In addition,we found that BIX-01294 can also reduce the m RNA and protein levels of the nuclear lamina protein LMNB1,promote proteasome-dependent degradation of histone variant H1.2,the formation of phosphorylation of histone variant H2AX(?-H2AX)and the activation of innate immune response.We found that BIX-01294 can induce autophagic degradation of LMNB1 protein expressed exogenously,which indicates that the protein level of LMNB1 is co-regulated by gene expression and post-translational modification.Autophagic inhibition can slightly attenuate the decrease of LMNB1 gene expression induced by BIX-01294,and down-regulate BIX-01294-induced ?-H2 AX modification.Combining the results of Ch IP-seq of BIX-01294 processing with SETDB1 knock-out,we found that BIX-01294 may promote the expression of the retrotransposons and thereby activate the innate immune response by repressing the H3K9me3 signal enrichment on the SINE.Autophagic inhibition can promote further activation of the innate immune response induced by BIX-01294.Our results showed that autophagic inhibition may promote the higher expression of cellular inflammatory factors by down-regulating the activation of AP1 transcription factor.In summary,our research has initially explored the molecular mechanism of autophagy induced by alterations of histone modifications,providing a new perspective on the role of autophagy in nuclear homeostasis and innate immune responses.