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

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.