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.
We have discovered that the same principles that cause mixtures of oil and water to spontaneously separate into distinct liquid `droplets', known as phase separation, play a key role in how genomes are organized and regulated. Here we propose to advance our basic knowledge of how phase separation and other biophysical mechanisms and properties organize and regulate a mysterious part of our genomes, known as heterochromatin. Ultimately, we expect that the results of these and related studies will have enormous impact on our understanding of the causes of human diseases such as dementia and cancer, and could generate novel and effective approaches to their diagnosis and treatment.
