Catalytic and Non-Catalytic Functions of COMPASS Family H3K4 Methyltransferases in Flies and Humans

Rickels, Ryan A

doi:https://doi.org/10.21985/n2-3k34-d368

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Catalytic and Non-Catalytic Functions of COMPASS Family H3K4 Methyltransferases in Flies and Humans

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Methylation of histone 3 lysine 4 (H3K4) catalyzed by the COMPASS family of lysine methyltransferases is universally associated with eukaryotic transcription. However, despite thousands of published studies examining the deposition, dynamics, and genomic positions of this chromatin modification, there is no clear consensus as to the molecular function or biological significance of H3K4-methylation. Furthermore, deletion of any COMPASS family member causes severe developmental abnormalities in metazoans, whereas catalytic- inactivating mutations are typically non-lethal and produce remarkably milder phenotypes. While only a limited number of studies have attempted to deconvolute catalytic-dependent from catalytic–independent functions for COMPASS, the findings are consistent thus far with a growing body of work highlighting a functional disconnect between histone modifications and the enzymes that catalyze them. My thesis work focuses on using Drosophila as a tool to disentangle enzymatic from non-enzymatic functions for COMPASS members Trithorax (Trx) and Trithorax-related (Trr), and applying my findings in a mammalian system to test for conservation of function as well as medical relevance. I performed the first genome-wide assessment of Trx-dependent H3K4-methylation and found H3K4 dimethylation (H3K4me2) deposited by Trx is highly predictive of Polycomb Response Elements (PREs) in Drosophila. Although I was unsuccessful in determining whether or not Trx- dependent H3K4me2 is necessary for PRE function, I demonstrate a conserved role for the mammalian homolog, MLL1, in catalyzing H3K4me2 at CpG-rich sequences that functionally resemble PREs in the human genome. This study establishes that, in a given cell type, several hundred developmentally regulated genes require MLL1, not for transcriptional activation, but to maintain activation by blocking silencing by Polycomb Repressive Complex 2 (PRC2). These results identify a subset of genes whose expression levels are balanced by MLL1 and PRC2, and challenges a passive model of PRC2 recruitment to transcriptionally silent promoters. Lastly, my thesis work culminates with the discovery that enhancer-associated H3K4- monomethylation (H3K4me1) is not essential for Drosophila development, and is generally dispensable for enhancer function in mammalian cells as well. While trr deletion is recessive lethal, flies harboring a Trr catalytic-inactivating mutation develop to productive adulthood and only display mild wing-vein phenotypes when reared at higher temperatures, suggesting enhancer-associated H3K4me1 might play a role in buffering enhancer-promoter communication under environmental stress. Consistent with my findings in Drosophila, similar experiments in mouse embryonic stem cells demonstrate thousands of gene expression changes in the absence of Trr mammalian homologs, MLL3/MLL4; however, catalytic- inactivating mutations result in relatively few expression changes, suggesting a non-enzymatic function for Trr/MLL3/MLL4 is essential for their role in facilitating enhancer-mediated gene activation during development. Genetic complementation assays in Drosophila identified a Trr fragment of unknown function was able to rescue Trr-null lethality, and this fragment was shown to bind and stabilize Utx, an H3K27 demethylase known to promote enhancer activation. The Utx-binding domain is conserved in mammalian MLL3/4 and expression of an 80 residue ‘minimal’ peptide was demonstrated to bind UTX and block its degradation in the absence of full-length MLL3/4. Utx and Kdm6a are essential genes in flies and mice, respectively, suggesting that stabilizing this important chromatin modifier represents at least one important non-enzymatic function of Trr/MLL3/MLL4 that is critical for life. Taken together, this thesis on the catalytic and non-catalytic functions of COMPASS provides a more nuanced view of epigenetic regulation and, by testing assumptions, serves as a guide from which to contemplate the role of chromatin modifiers in the process of transcription.

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