氧化应激通过DNA羟甲基化作用于DCAF8-WDR76-LSH轴调控铁死亡相关基因转录的分子机制研究

铁死亡是由铁依赖的脂质过氧化累积导致的较新型细胞死亡方式。目前铁死亡的研究集中在核外直接参与维持氧化还原稳态的通路，相关基因的转录调控研究比较滞后。通过前期系列生化实验，我们发现CRL4-DCAF8泛素连接酶以染色质重塑因子LSH为底物，促进其经蛋白酶体的降解；而另一个WD40蛋白WDR76可以抑制CRL4-DCAF8的促LSH降解作用。非常有趣，铁死亡激活剂可以调节DCAF8-WDR76对LSH降解的竞争性拮抗，并以此影响下游相关基因的转录。基于这些结果以及WDR76/LSH对5hmC的选择性结合，深入探索DCAF8-WDR76-LSH轴的分子界面和内在作用机制，明确氧化应激条下5hmC丰度和基因组定位变化与DCAF8-WDR76介导的LSH稳定性调节之间的因果关系，解析该轴调控氧化应激基因转录依赖的辅助因子，将推动我们深入理解铁死亡的表观遗传机制，并为基于铁死亡的肿瘤治疗提供新靶点。

Accumulation of overproduced reactive oxygen species (ROS) is implicated in a range of pathologies, including cancer, degenerative disease, and premature aging. ROS caused macromolecular damages have been extensively studied under the topics of DNA base mutation and protein conformational aberrance, yet mounting evidences have pointed to the function of lipid peroxidation in attacking membrane polyunsaturated lipids and inducing iron-dependent, nonapoptotic, and regulated form of cell death, ferroptosis. Despite the emerging evidence on ferroptosis implicated in diverse pathologies, molecular linkage between oxidative inducers and chromatin as epigenetic memory carrier for its propagation remains elusive. Lymphoid Specific Helicase (LSH, also known as HELLS or PASG), a member of SNF2 family chromatin remodeling ATPase, possessing essential epigenetic functions for the maintenance of global DNA methylation and guarding repetitive elements of mammalian genome, is critically implicated in carcinogenesis and premature aging. However, the molecular mechanisms and biological consequences for the stability control over LSH in cancer and senescence remain elusive. In our pilot experiment, we identified the core components CUL4, DDB1, and DCAF8, a substrate binding adaptor of Cullin4 ubiquitin (CRL4) E3 ligase complex, in LSH interactome. Degradation analysis and CRL4-DCAF8 complex reconstitution demonstrate that CRL4DCAF8 is a bona fide E3 ligase for LSH. In contrast, WDR76 antagonizes DCAF8-targeted LSH proteolysis. Importantly, this opposing regulatory strategy is utilized in lipid hydroperoxide induced ferroptosis, where we identify key redox homeostasis genes significantly regulated by the DCAF8/WDR76/LSH axis through transcriptomic epistasis analysis. With these results and the fact that LSH/WDR76 have been identified as specific readers of 5-hydroxymethylcytosine (5hmC) and the global 5hmC abundance is decreased in hypoxic environment along with decreased TET (ten-eleven translocation) enzymatic activities in tumors, it will be of great interest to understand the detailed molecular interface within CRL4-DCAF8-WDR76-LSH system, and the molecular mechanisms of DCAF8-WDR76-antagonized LSH degradation. We will also probe the interaction between CRL4-DCAF8-WDR76-mediated LSH degradation and other epigenomic variables, especially genome-wide DNA hydroxymethylation and histone modification patterns, owing to the reported nucleosome remodeling activities of LSH and the possible connection between oxidative context associated with ferroptosis and decreased hydroxymethylation in ROS-overproduced environment. These mechanistic insights could then be combined with downstream phenotypic and functional analysis to give a comprehensive set of data for the roles of CRL4-DCAF8-WDR76-LSH regulation at protein and epigenetic levels in carcinogenesis and senescence. The identification of CRL4-DCAF8-WDR76-mediated stability control over LSH and the elucidation of underlying molecular mechanisms would finally contribute to the establishment of a full picture for the epigenetic regulation network driving ferroptosis and its clinical applications as anti-tumor therapies.