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. 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ASH2L对于生物体及组织发育、细胞增殖均至关重要。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（H3K4）的甲基化修饰。H3K4的三甲基化与单甲基化（分别对应H3K4me3与H3K4me1）分别与活跃的启动子及增强子相关联，目前学界尚未完全阐明这类修饰的分子生物学意义。我们利用携带PROTAC（蛋白水解靶向嵌合体，Proteolysis Targeting Chimeras）敏感型可降解ASH2L的小鼠胚胎细胞，评估了KMT2复合物失活后的功能后果。在最初的16小时内，ASH2L的快速缺失引发了启动子区域组蛋白修饰的一系列动态变化：首先是ASH2L蛋白水平下降，随后H3K4me1水平升高，而H3K27ac水平降低；与此同时，H3K27me3的水平上升则极为缓慢。这类变化在转录起始位点上下游±1 kb范围内的CpG岛启动子区域最为显著。尽管ASH2L快速缺失，但在最初8小时内对转录的影响微乎其微，后续则伴随基因表达改变、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有序事件，且需要较长时间才能逐步显现。我们构建了条件性敲除小鼠，可靶向敲除Ash2l基因的第4外显子，从而阻断Ash2l蛋白的合成，但由于Ash2l蛋白半衰期较长，需耗时数日才能实现蛋白完全耗竭。我们从该小鼠中分离获得了小鼠胚胎成纤维细胞（MEF，Mouse Embryo Fibroblasts），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。我们随后向这类细胞中导入了表达FKBP-ASH2L融合蛋白的重组载体，当内源等位基因被敲除后，MEF细胞的增殖依赖于FKBP-ASH2L的存在。我们发现该融合蛋白对PROTACs敏感，在添加PROTAC后30分钟内即可被降解99%以上。