Nuclear actin filaments and phase separation: new drivers of heterochromatin repair - SUMMARY DSBs occur in every cell because of exposure to environmental mutagens, such ionizing radiation, chemotherapeutic drugs, UV light, toxic pollutants, and smoking, as well as in response to normal cell metabolism, such as DNA replication. Repairing DSBs is particularly challenging in pericentromeric heterochromatin, a poorly characterized region of the genome where the abundance of repeated sequences can trigger aberrant recombination and widespread genomic instability. And yet, heterochromatin repair mechanisms remain poorly understood. We discovered a specialized pathway that promotes faithful homologous recombination (HR) repair in heterochromatin while preventing aberrant recombination. HR starts inside the heterochromatin “domain” with resection, but it continues only after a striking relocalization of repair sites to the nuclear periphery. Relocalization likely prevents aberrant recombination by isolating DSBs and their repair templates away from ectopic sequences before strand invasion. We have recently discovered that the movement occurs in two phases. Repair sites initially diffuse from the center to the periphery of the heterochromatin domain, and this requires Nup98 and its phase separation properties. Next, nuclear actin filaments (F-actin) and myosins drive the directed motion of repair sites to the nuclear periphery. How condensates contribute to these dynamics is not clear. The mechanisms targeting condensate formation and F-actin assembly specifically to heterochromatic DSBs are also unknown. Dysregulation of heterochromatin repair is likely one of the most underestimated and powerful sources of tumorigenesis and identifying the mechanisms involved is essential for understanding cancer etiology. Our central hypotheses are that immiscibility between Nup98 and HP1 condensates generate capillary forces that mobilize repair sites inside the heterochromatin domain, while DNA damage response proteins work in concert with HP1 to target Nup98 and actin nucleators specifically to heterochromatic DSBs. We will combine a wealth of imaging, genetic and biochemical approaches to investigate the molecular mechanisms involved in this process. Expected positive outcomes of this research include the systematic identification of the molecular machinery that protects heterochromatin from massive genome rearrangements, enabling successful completion of HR repair. These studies are also expected to illuminate missing links between nuclear architecture and dynamics, phase separation, repair progression, and the stability of repeated DNA sequences. These results will have an important positive impact by identifying crucial safeguard mechanisms used by normal cells to protect their genome from a variety of environmental threats. Dysregulation of these pathways result in genome instability and tumorigenesis. Thus, we expect that the proposed studies and future research will trigger exciting advancements in the prevention, early detection, and treatment of cancer and other genome-instability disorders.
