DEFINING REGULATORY ROLES FOR HISTONE H3 METHYLATION IN DEVELOPMENT

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 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 (Project 1). 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 (Project 2). 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 (Project 3).
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.

Grant Number: 5R35GM142884-05
NIH Institute/Center: NIH
Principal Investigator: Justin Brumbaugh