HMGB1 during the transition from naive to primed pluripotency
Embryo implantation is characterized by the process of naive-to-primed pluripotency transition in epiblast cells, involving an anabolic boost, mitochondrial remodeling, and increased proliferation. Yet, the molecular mechanisms underlying these extensive changes remain poorly understood. High mobility group box 1 (HMGB1) is a non-histone, redox-sensitive chromatin protein involved in diverse cellular processes, however its role in pluripotency control has not been fully characterized.To determine the function of HMGB1 in mouse embryonic stem cells (ESCs), Hmgb1-knockout (KO) ESCs were generated using CRISPR/Cas9 system. KO ESCs were analyzed for cell proliferation, cell cycle progression, and apoptosis, as well as for levels of active mitochondria, mitochondrial membrane potential, and reactive oxygen species (ROS) using fluorescent-based reagents and flow cytometry. Pluripotency was assessed by analyzing the expression of pluripotency markers with immunocytochemistry, Western blotting, qRT-PCR, as well as by teratoma formation assay. Naive-to-primed pluripotency transition was investigated by in vitro culture. Molecular analysis was performed with RNA sequencing, bioinformatics, qRT-PCR, and Western blotting. The ultrastructure of mitochondria was examined using transmission electron microscopy.We first successfully generated HMGB1 KO in mouse ESCs and showed that HMGB1 function is dispensable for cell viability and pluripotency maintenance, while it is required for the cell proliferation boost during the naive-to-primed pluripotency transition. Molecular and transcriptomic analysis identified the involvement of HMGB1 in the regulation of energy metabolism processes by regulating mitochondrial structure and function, and ROS homeostasis. Loss of HMGB1 function in mouse ESCs results in altered mitochondrial structure and excessive ROS production. HMGB1-dependent elevated ROS levels negatively regulate cell proliferation during the transition from naive to primed pluripotency in vitro.While HMGB1 deficiency does not impair self-renewal in the naive state, it causes a marked reduction in proliferation as cells advance to primed pluripotency. Our findings thus identify HMGB1 as a key regulator of mitochondrial integrity and ROS homeostasis during the naive-to-primed pluripotency transition.