Chromatin Nano-Organization in Peripheral Blood Mononuclear Cells After In-Solution Irradiation with the Beta-Emitter...

Area of Science:

• Nuclear Medicine and Radiation Biology
• Molecular and Cellular Biology
• Genetics and Epigenetics

Background:

• Nuclear medicine utilizes radiopharmaceuticals, such as beta-emitting Lutetium-177 (Lu-177), for cancer treatment via internal irradiation.
• Low-linear energy transfer (low-LET) beta irradiation typically induces isolated DNA double-strand breaks (DSBs), contrasting with high-LET radiation's clustered damage.
• Understanding the nanoscale organization of DNA damage and repair is crucial for optimizing radiotherapy efficacy and minimizing side effects.

Purpose of the Study:

• To investigate the nanoscale spatial organization of chromatin, DNA damage response (DDR) proteins, and heterochromatin marks following Lu-177 beta irradiation.
• To analyze the co-localization patterns of DDR markers (γH2AX, 53BP1, MRE11, pATM) and heterochromatin (H3K9me3) at DSB sites.
• To evaluate the impact of low-dose beta irradiation on chromatin structure and its implications for DNA repair mechanisms.

Main Methods:

• In-solution beta irradiation of peripheral blood mononuclear cells (PBMCs) with Lu-177 at an absorbed dose of approximately 100 mGy.
• Single-molecule localization microscopy (SMLM) to visualize the nanoscale distribution of fluorescently tagged DDR proteins and H3K9me3.
• Mathematical analysis including Ripley distance statistics, DBScan clustering, and persistent homology to quantify spatial organization and co-localization.

Main Results:

• SMLM revealed characteristic clustering of DDR markers (γH2AX, 53BP1, MRE11, pATM) around one or two DSB foci per nucleus.
• MRE11 concentrated within γH2AX clusters, while 53BP1 was distributed throughout, indicating distinct roles in DSB repair.
• Significant co-localization was observed between γH2AX and the heterochromatin mark H3K9me3, even at a strict 20 nm threshold, suggesting minimal chromatin relaxation around DSBs.

Conclusions:

• Low-dose Lu-177 beta irradiation induces DSBs that trigger localized DDR protein clustering.
• The observed co-localization of DSB markers with H3K9me3 suggests that heterochromatin remains largely intact, potentially influencing DNA repair dynamics.
• These findings support the hypothesis that single DSBs from low-dose, low-LET irradiation may not lead to significant heterochromatin relaxation, impacting repair outcomes in nuclear medicine.