DEFINING REGULATORY ROLES FOR HISTONE H3 METHYLATION IN DEVELOPMENT

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Project Title: Defining regulatory roles for histone H3 methylation in development Principle Investigator: Justin Brumbaugh, University of Colorado Boulder I am submitting this document is to apply for an administrative supplement to my NIH R35 (R35GM142884) award entitled, “Defining regulatory roles for histone H3 methylation in development.” This request is in response to NOT-GM-22-017 (PA-20-272). Financial status of current grant. I was awarded an early-stage investigator R35/MIRA grant in July 2021. I am currently in the first year of the funding period. As a result, we do not yet have unobligated funds to report; however, based on our current spending rate, we anticipate that we will have approximately $20,000-$40,000 of unobligated funds to carry into year 2, most of which are a result of COVID-related lab restrictions. We plan to use these funds to sequence large-scale genomics samples that we have collected over the past months and are awaiting analysis. Parent Award Summary Project summary. Pluripotent stem cells hold tremendous scientific and therapeutic potential because they have the capacity to differentiate into any cell in the adult body. Mounting evidence suggests that differentiation is driven, in part, by epigenetic mechanisms such as histone modifications that help to establish and subsequently maintain cell identity. However, demonstrating a direct role for an individual histone modification is challenging via traditional mutagenesis approaches because multiple copies of canonical histone genes are present in the mammalian genome. Moreover, many histone marks are regulated by several, redundant enzymes, which are difficult to perturb simultaneously and in a physiological context. The long-term goal of our research is to resolve the role of histone modifications in directing cell fate, both in vivo and in tissue culture systems. Our approach is innovative because it overcomes current limitations in the field by taking advantage of lysine-to-methionine (K-to-M) mutations on histone H3, which act as dominant negative inhibitors of methylation at their respective sites. The objective of this grant is to characterize the function of methylation on residues H3K9 and H3K36, which change dramatically during differentiation and development. Our central hypothesis is that H3K9 and H3K36 methylation have distinct and crucial roles in developmental transitions. To test this hypothesis, we will express mutant histones, H3K9M and H3K36M, in early embryos and pluripotent stem cells. Specifically, we will track the maternal to zygotic transition and early lineage decisions following suppression of H3K9 and H3K36 methylation in embryos. We will then apply in vitro cell culture systems to investigate the molecular basis for the effects of K-to-M mutants on chromatin and gene expression. A key feature of our approach is that expression of the mutant histones is doxycycline-inducible, which permits induction or withdrawal of our histone mutants in a tissue- and time-specific manner. Using this tool, we will ask whether cells are capable of reestablishing histone modifications to rescue proper differentiation after mutant histone withdrawal. Collectively, this work is significant because it will provide valuable insight into the functional role of histone modifications in cell fate change. Understanding the regulatory mechanisms that control stem cell function is a crucial step in realizing their tremendous potential. We therefore anticipate that the proposed work will have important implications for both basic science and medicine. Brief progress to date (current status). The primary aims of our parent grant are to clarify how particular