Discrete chromatin alterations and deregulated gene expression upon PROTAC-induced rapid loss of the trithorax protein ASH2L [Click-it]. Discrete chromatin alterations and deregulated gene expression upon PROTAC-induced rapid loss of the trithorax protein ASH2L [Click-it]

The trithorax protein ASH2L is essential for organismal and tissue development and for cell proliferation. ASH2L is a subunit of KMT2/COMPASS methyltransferase complexes that catalyze the methylation of histone H3 lysine 4 (H3K4). Tri- and mono-methylation of H3K4 (H3K4me3 and H3K4me1) are associated with active promoters and enhancers, respectively. The molecular relevance of these modifications is not fully understood. We have used mouse embryo cells with a PROTAC-sensitive, degradable ASH2L to assess the functional consequences of KMT2 complex inactivation. The rapid loss of ASH2L resulted in a sequential alterations of histone marks at promoters, first a decrease of H3K4me3, then an increase of H3K4me1, and a decrease of H3K27ac during the first 16 hrs, while an increase in H3K27me3 was very slow. These consequences were most prominent at CpG island promoters within a window of ±1 kb of the transcription start sites. Despite the the rapid loss of ASH2L, the effect on transcription in the first 8 hrs was minimal. This was accompanied with an alterations in gene expression and associated proliferation stop and cell cycle arrest. These findings suggest an order series of events upon loss of ASH2L that requires considerable amount of time to unfold. Overall design: We have generated a conditional knockout mouse, in which we can delete exon 4 of Ash2l. This prevents production of Ash2l protein, however due to the long half-life of Ash2l, it requires days before the protein is depleted. From these animals we established mouse embryo fibroblasts (MEF). Upon knockout of the endogenous alleles, the cells stop proliferating and enter senescence. We have now introduced into these cells a construct that expresses an FKBP-ASH2L fusion protein. Upon knockout of the endogenous alleles, the MEF cells proliferate dependent on FKBP-ASH2L. We find that this fusion protein is sensitive to PROTACs (Proteolysis Targeting Chimeras) and is degraded by more than 99% within 30 minutes after addition of a PROTAC.

三胸蛋白（trithorax protein）ASH2L对机体与组织发育及细胞增殖至关重要。ASH2L是KMT2/COMPASS甲基转移酶复合物的亚基，该复合物可催化组蛋白H3赖氨酸4（histone H3 lysine 4, H3K4）的甲基化修饰。H3K4的三甲基化（H3K4me3）与单甲基化（H3K4me1）分别对应活跃启动子与增强子的表观遗传标记。目前，此类修饰的分子调控机制尚未完全阐明。本研究采用携带对蛋白降解靶向嵌合体（Proteolysis Targeting Chimeras, PROTAC）敏感的可降解ASH2L的小鼠胚胎细胞，以评估KMT2复合物失活所产生的功能效应。ASH2L的快速缺失会引发启动子区域组蛋白修饰的时序性改变：在最初16小时内，H3K4me3水平率先下降，随后H3K4me1水平升高，同时H3K27乙酰化（H3K27ac）水平降低；而H3K27三甲基化（H3K27me3）的水平升高则极为缓慢。上述变化在转录起始位点（transcription start sites, TSS）±1 kb范围内的CpG岛启动子区域最为显著。尽管ASH2L的缺失过程十分迅速，但在最初8小时内对细胞转录的影响微乎其微。该现象伴随基因表达谱改变、增殖停滞与细胞周期阻滞出现。本研究结果表明，ASH2L缺失后会触发一系列有序的生物学事件，且该过程需要较长时间才能充分展开。
整体实验设计：我们构建了Ash2l基因条件性敲除（conditional knockout）小鼠，可通过敲除Ash2l的外显子4（exon 4）阻断Ash2l蛋白的合成；但由于Ash2l蛋白半衰期较长，需历经数天才能实现蛋白耗竭。我们从该小鼠品系中分离得到小鼠胚胎成纤维细胞（mouse embryo fibroblasts, MEF）。在内源等位基因被敲除后，此类细胞会停止增殖并进入衰老状态。我们随后向这些细胞中转入表达FKBP-ASH2L融合蛋白的载体。在内源等位基因被敲除后，MEF细胞的增殖依赖于FKBP-ASH2L融合蛋白。我们发现该融合蛋白对PROTAC敏感，在添加PROTAC后30分钟内即可被降解99%以上。