PROJECT SUMMARY/ABSTRACT
The long term goal of this project is to elucidate the composition, architecture, and biophysical properties of
heterochromatin, and to understand how they contribute to nuclear functions. Heterochromatin is enriched in
repeated DNAs, is concentrated in pericentromeric and telomeric regions, and forms a distinct and dynamic 3D
domain inside nuclei. Heterochromatin is required for normal sister chromosome pairing and segregation,
nuclear architecture, recombination suppression, transposon silencing, and gene silencing. Heterochromatin
recruitment is regulated by epigenetic components and mechanisms, specifically di- and tri- methylation of
histone H3 lysine 9 (H3K9me2/3) by specific methyltransferases. Heterochromatin Protein 1 (HP1) binds this
`mark' and recruits many proteins and complexes to the heterochromatin. We currently lack a clear
understanding of the fine structure and organization of the heterochromatin domain, and the biophysical
properties responsible for its functions and behaviors. Our preliminary studies in Drosophila have revealed
unexpected structural complexity and biophysical properties of heterochromatin that raise questions about our
current understanding of the structure and function of this domain, and suggest that heterochromatin may form
and function through biophysical mechanisms that have not been associated with chromatin structure and
function. In particular, our findings led to the novel hypothesis that the heterochromatin domain forms through a
phase separation mechanism, which has recently been shown to compartmentalize functional molecular
networks into structures that lack constraining membranes, but has not until now been applied to chromatin
domains.
We will capitalize on these novel findings and apply advanced imaging, epigenomics, biochemical and
biophysical approaches to elucidate: 1) the structural, biochemical and biophysical properties of the
heterochromatin domain, 2) the components and mechanisms responsible for heterochromatin formation, and
3) the ways that heterochromatin substructure and biophysical properties contribute to nuclear and organismal
functions. Testing the phase separation hypothesis will elucidate important information about the organization
and function of heterochromatin in cells and animals, offering the potential of providing a paradigm-shifting
foundation for understanding how other chromatin domains form and function. In addition, defective
heterochromatin produces genome instability and altered gene expression, contributing to cancer, birth
defects, and aging. Understanding how human diseases and conditions alter the biophysical properties that
underlie heterochromatin formation and function will ultimately impact the approaches to their diagnosis and
treatment.