Functional characterization of the trithorax protein Ash2l, a core component of histone H3 lysine 4 methyltransferase complexes

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Differential gene expression is essential for cell identity and to control cell physiology. Modulating chromatin, in particular nucleosome remodeling and core histone modifications, contribute to the regulation of gene expression. One of these modifications is methylation of histone H3 at lysine 4 (H3K4). Its mono-methylation and tri-methylation (H3K4me3) is associated with accessible and open enhancers and promoters, respectively, with transcribed genes carrying typically H3K4me3 in their promoter regions. This modification is transferred by the KMT2 family of methyltransferases, including MLL1-4, SET1A and SET1B. They require a core complex of WDR5, RBBP5, ASH2L and DPY30 (aka WRAD complex) for catalytic activity. We are studying ASH2L as we identified this trithorax protein as an interactor of the proto-oncoprotein c-MYC. We find that the knockout (KO) of Ash2l in the liver or in the hematopoietic system in the mouse is lethal, accompanied by a loss of H3K4 methylation and deregulated gene expression. From these and other findings, it is evident that methylation at H3K4 is necessary for normal cell physiology, but whether H3K4me3 has a more general effect on chromatin or a more instructive role in gene transcription remains open. Therefore, the main objectives are (i) to learn more about the function of Ash2l itself together with its associated H3K4 methyltransferase activity and (ii) to address the function of H3K4 methylation at promoters. We will use our mouse embryo fibroblasts with floxed Ash2l alleles that express regulatable Cre-ERTM2 recombinase. We will perform a structure-function analysis of ASH2L to determine relevant regions and domains by measuring the ability to complement proliferation, gene transcription and H3K4 methylation in the KO mouse embryo fibroblasts. Moreover, we will screen for novel interaction partners using an ASH2L mutant that is unable to bind to other WRAD complex components to address novel functions of ASH2L. Because the Ash2l KO is rather slow, we will establish a system in mouse and human cell lines that allows short-term regulation of ASH2L by using fusions with an auxin inducible degron. This will be used for short-term inactivation and reactivation of ASH2L to determine direct effects on chromatin and gene regulation. We will probe chromatin compaction dependent on Ash2l using nucleosomal mapping and ATAC-seq. This will be complemented by targeting ASH2L and mutants as well as other epigenetic regulators to specific genes using dCas9 fusions to evaluate H3K4 methylation at promoters and cooperativities. With deep sequencing and chromatin and gene expression analyses we will be able to evaluate the consequences of H3K4 methylation and cooperating activities. Finally, we will expand on signaling processes that control gene expression dependent on H3K4 methylation. In summary, the proposal addresses fundamental aspects of ASH2L function and the role of H3K4 methylation in gene transcription.