PROJECT SUMMARY/ABSTRACT
Goals: Peri-Centromeric Heterochromatin (PCH) is required for genome stability/DNA repair, chromosome
pairing, nuclear architecture, and transposon and gene silencing. Previous studies suggested that histone H3
lysine 9 methylation (H3K9me2/3), Heterochromatin Protein 1 (HP1) binding, HP1-interacting protein
recruitment and chromatin compaction are sufficient to explain PCH formation and function. In 2017, my lab
and the Narlikar lab published complementary studies suggesting that 3D PCH domains form via liquid-liquid
phase separation (LLPS), generating membrane-less condensates with an immobile HP1a core surrounded by
a liquid. We proposed that novel properties associated with highly networked, phase separated systems (e.g.
liquidity) are critical to understand how PCH, and other chromatin domains, form and regulate essential nuclear
functions. However, we lack a mechanistic understanding of the organization, dynamics and
biophysical/material properties of PCH components and condensates in a cellular and organismal context. In
addition, we need to determine if and how biophysical properties regulate genome functions such as repair,
replication and transcription, a current major challenge for the whole field of condensate biology.
Approach: This MIRA will interrogate how LLPS and biophysical properties impact the in vivo organization
and function of heterochromatin and other associated nuclear bodies. We will capitalize on our preliminary
results and knowledge of PCH biology, combined with advanced imaging, biochemical, and experimental and
theoretical biophysical approaches, to elucidate 1) the molecular interactions responsible for PCH domain
formation; 2) the architectural, biophysical and chemical properties of the domain; and 3) whether or not phase
separation and liquidity regulate PCH functions and interplay with other nuclear bodies.
Innovation: Although LLPS and biological condensates have become a popular topic for study and
discussion in recent years, we know little about in vivo mechanisms and relevance to function in the complex
but important cellular and organismal contexts. This is an emerging field, with unique challenges, and an
interdisciplinary approach is required to address these key questions. Thus in this MIRA proposal we will
combine our decades of experience in PCH biology with the expertise of collaborators in experimental and
theoretical biophysics, and advanced bioimaging. Testing our hypothesis will elucidate important information
about the organization and function of heterochromatin in cells and animals, potentially providing a paradigm-
shifting foundation for understanding how chromatin domains in general form and function.
Health Relatedness: Defective PCH causes genome instability and altered gene expression, contributing
to cancer, birth defects, and aging. Understanding how biophysical properties that underlie PCH formation and
function are altered in human diseases will likely result in novel approaches to diagnosis and treatment.