53BP1 regulates genome biology and cellular physiology through liquid phase separation

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The function of nuclear DNA is not only determined by its sequence, but also depends on its three dimensional (3D) structure. A particular type of DNA in eukaryotes is called heterochromatin, which refers to as tightly packed DNA structure in the nucleus. Heterochromatin plays a critical role in genome function including DNA structural maintenance, chromosome segregation, epigenetic inheritance, DNA replication, repair and transcription. Recently, increasing evidence suggests the involvement of a new biological process called liquid- liquid phase separation in the formation and function of heterochromatin. Liquid-liquid phase separation, whose concept was borrowed from polymer sciences, is a unique process that involves the formation of membraneless liquid droplets by proteins and nucleotides when their concentration have reached a threshold. These liquid droplets enable the assembly and disassembly of functional protein-based organelles within a cellular compartment following environmental cues. Hence, liquid-liquid phase separation has facilitated our understanding of fundamental cellular information processing, cellular homeostasis, and cellular physiology. Recently, we unexpectedly identified a new player, human 53BP1, in regulating the heterochromatin integrity through liquid-liquid phase separation. 53BP1 was previously known as a critical player in regulating the DNA double strand break repair. However, we discovered that the protective role of 53BP1 in both the structure and the function of heterochromatin is distinct from its canonical activity in DNA double strand break repair. Hence, our studies opened a new research paradigm for this important protein in signaling, biology and cellular physiology. The goal of this proposal is to establish this new research field by addressing several unanswered important questions regarding this new function of 53BP1. These include the molecular basis by which 53BP1 forms the liquid droplets and its significance in biology, genome biology, and cellular physiology. We have used mass spectrometry to identify components in the liquid droplets formed by 53BP1. We will interrogate their functions in this application. By assembling an interdisciplinary team consisting of experts on molecular and cellular biology, proteomics, computational bioinformatics, and next generation sequencing, we will use a combination of cell biological, molecular, biochemical, genetic, morphological, and chemical approaches to answer these questions. Our studies will illustrate a previously uncharacterized function of 53BP1 and a novel interplay between 53BP1 and heterochromatin and determine their impact on genome stability, facilitating our understanding of fundamental cellular information processing, cellular homeostasis, and cellular physiology.