Idoya Mª Martínez Vicente¹, Sonia Cerdido Ochoa², Marta Abrisqueta González¹, Ana Lambertos Escudero¹, Cecilia María Herraiz Serrano¹, María Concepción Olivares Sánchez¹, CELIA JIMENEZ-CERVANTES FRIGOLS¹, JOSE CARLOS GARCIA-BORRON MARTINEZ¹

(1) CONTROL MOLECULAR DE LA PROLIFERACIÓN Y DIFERENCIACIÓN, IMIB-Arrixaca, España
(2) Universidad de Murcia, 30204, España (Región de Murcia)

The mouse mahoganoid mutation abrogates expression of the E3 ubiquitin ligase Mahogunin Ring Finger-1 (Mgrn1) and leads to a complex phenotype with hyperpigmentation, congenital heart defects and neurodegeneration. We showed that knockdown of Mgrn1 in mouse melanocytes decreased the proliferation rate and increased the percentage of cells in the S phase of the cell cycle. Progress throughout the S phase is dependent on occurrence of DNA damage, which activates a DNA damage response (DDR) that delays or stops cell cycle progression.

Since DDR and cell cycle progression are linked, we investigated the biological effects of Mgrn1 deficiency on DDR using Mgrn1-KO melanocytes (mahoganoid melan-md1 cells and CRISPR-Cas9 Mgrn1-KO cells). We compared DNA integrity in control and Mgrn1-null cells using comet assays and γH2AX staining. Additionally, we analyzed the kinetics of repair of DSBs generated by an intense oxidative challenge (mediated by H2O2) and prolonged replicative stress caused by long hydroxyurea (HU) treatment. Furthermore, metaphase chromosome spreads preparation were employed to investigate chromosomal alterations.

. Higher amounts of DNA breaks were detected in Mgrn1-null cells. In agreement, the lack of Mgrn1 induced stronger γH2AX labelling compared to control cells. We also found a significant increase in the percentage of aneuploid cells in Mgrn1-null cells. Steady-state levels of DNA lesions can result from higher rates of formation and/or lower rates of clearance. We demonstrated that the rate of break clearance was lower in Mgrn1-KO melanocytes upon oxidative challenge. In addition, melan-a6 melanocytes cleared most of the ensuing DSBs over a 24 h recovery period from HU-induced lesions, but Mgrn1-KO cells did not achieve a comparable clearance.

Therefore, lack of Mgrn1 increased genomic instability and decreased the ability of melanocytes to cope with DSBs generated by oxidizing agents or HU-induced replicative stress, suggesting that genomic instability might contribute to the pathologic mahoganoid phenotype.