Discrete chromatin alterations and deregulated gene expression upon PROTAC-induced rapid loss of the trithorax protein ASH2L [ChIP-seq(Ash2l)]

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 (Ash2l 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 Ash2l, 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）ASH2L在机体与组织发育以及细胞增殖过程中发挥不可或缺的作用。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（H3K4）的甲基化修饰。H3K4的三甲基化与单甲基化（Ash2l与H3K4me1）分别与活性启动子和增强子存在关联，目前上述修饰的分子生物学意义尚未完全阐明。

我们利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，评估KMT2复合物失活所产生的功能效应。ASH2L的快速降解会导致启动子区域的组蛋白修饰标记发生一系列有序变化：在最初16小时内，首先出现Ash2l水平下降，随后H3K4me1水平升高，同时H3K27ac水平降低；而H3K27me3的水平上升则极为缓慢。上述变化在转录起始位点±1 kb范围内的CpG岛启动子区域最为显著。

尽管ASH2L的降解速度极快，但在最初8小时内其对转录的影响微乎其微。这一现象伴随基因表达的改变，进而引发增殖停滞与细胞周期阻滞。上述研究结果表明，ASH2L缺失后会触发一系列按序发生的事件，且该过程需要较长时间才能逐步展开。

实验设计：我们构建了条件性敲除小鼠，可通过该模型敲除Ash2l的第4号外显子，从而阻断Ash2l蛋白的合成。但由于Ash2l蛋白的半衰期较长，因此需要数天时间才能实现蛋白的完全耗竭。我们从该小鼠品系中分离并构建了小鼠胚胎成纤维细胞（mouse embryo fibroblasts，MEF）。当内源等位基因被敲除后，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了可表达FKBP-ASH2L融合蛋白的构建载体。当内源等位基因被敲除后，MEF细胞的增殖依赖于FKBP-ASH2L的表达。我们发现该融合蛋白对蛋白降解靶向嵌合体（Proteolysis Targeting Chimeras，PROTAC）敏感，在添加PROTAC后的30分钟内即可被降解99%以上。