Chromatin Regulation of Tissue Regeneration and Stem Cell Function

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Genomic regulators such as transcription factors and chromatin-modifying enzymes work together to instruct cell fate during developmental processes, including both the routine cell turnover that occurs during homeostasis and the major tissue replacement and repatterning that occurs during regeneration. One specific group of chromatin-modifying complexes whose activity is strongly associated with gene expression is the Set1/MLL family of histone methyltransferases. These enzyme complexes share a core of several essential subunits, yet each particular enzyme (Set1, MLL1, MLL2, etc.) is functionally non-redundant in multicellular animals i.e., individual null mutations are lethal. These data and others, including some from my laboratory, indicate that each Set1/MLL complex regulates its own distinct set of genes. However, many of the molecular and mechanistic details driving this biochemical and functional specificity remain unknown. To uncover the fundamental molecular mechanisms underlying this process, my laboratory studies the planarian model of animal regeneration. Planarians are free-living flatworms with incredible regenerative capacities. They are also amenable to genetic perturbation through RNAi, easily dissociated for single cell analyses, and encode chromatin modifying proteins with strong homology to those of other organisms. For example, planarian Set1 and MLL1/2 enzymes show strong sequence homology to their mammalian counterparts and my previous research has shown that loss of either enzyme leads to loss of their conserved histone modifying activity, trimethylation of histone H3 at lysine 4 (H3K4me3). Moreover, we find that each enzyme targets a specific set of genes that correlate with their phenotype after enzyme loss. Specifically, loss of MLL1/2 through RNAi of its transcript leads to loss of H3K4me3 at highly conserved cilia genes as well as loss of cilia and ciliated cells. However, we do not know how MLL1/2 targets these cilia genes specifically in vivo. To begin identifying the molecular and mechanistic basis of this targeting, we first need to characterize the planarian MLL1/2 complex in terms of its biochemical composition and function. Notably, the phenotype induced in planarians after RNAi of a core Set1/MLL complex subunit, dpy-30, may provide an opportunity to uncover the mechanism that distinguishes Set1 from MLL1/2 function in vivo. To test this idea, I have recruited a promising undergraduate student to identify planarian DPY-30 interacting proteins. He will begin by cloning the two planarian versions of DPY-30 with an epitope tag. He will then express these planarian proteins in mammalian cells and ask if they stably interact with mammalian MLL1/2 proteins and form a functional histone methyltransferase complex. These studies will inform whether planarian DPY-30 is required MLL1/2, but not Set1, assembly and/or function as a histone methyltransferase complex. This project will also allow Ethan to gain the experience and skills needed to build a career in biomedical research.