Abstract
BackgroundOct4 is a specific marker of embryonic stem cell (ESC) pluripotency. However, little is known regarding how Oct4 responds to DNA damage. Here, we investigated whether Oct4 recognizes damaged chromatin in mouse ESCs stably expressing GFP-Oct4. These experiments should contribute to the knowledge of how ESC genomic integrity is maintained, which is crucial for potential application of human ESCs in regenerative medicine.Methodology/Principal FindingsWe used time-lapse confocal microscopy, microirradiation by UV laser (355 nm), induction of DNA lesions by specific agents, and GFP technology to study the Oct4 response to DNA damage. We found that Oct4 accumulates in UV-damaged regions immediately after irradiation in an adenosine triphosphate-dependent manner. Intriguingly, this event was not accompanied by pronounced Nanog and c-MYC recruitment to the UV-damaged sites. The accumulation of Oct4 to UV-damaged chromatin occurred simultaneously with H3K9 deacetylation and H2AX phosphorylation (γH2AX). Moreover, we observed an ESC-specific nuclear distribution of γH2AX after interference to cellular processes, including histone acetylation, transcription, and cell metabolism. Inhibition of histone deacetylases mostly prevented pronounced Oct4 accumulation at UV-irradiated chromatin.Conclusions/SignificanceOur studies demonstrate pluripotency-specific events that accompany DNA damage responses. Here, we discuss how ESCs might respond to DNA damage caused by genotoxic injury that might lead to unwanted genomic instability.
Highlights
Embryonic stem cells (ESCs) display different sensitivities to DNA damage compared with differentiated cells, including mouse embryonic fibroblasts (MEFs) that are used as a feeder layer for embryonic stem cell (ESC) cultivation
The GFP-Oct4 fusion protein is functional in GOWT1 mouse ESCs (mESCs) [32], we verified that endogenous Oct4 can recognize UV-damaged chromatin (Fig. 1B)
ESCs are interesting because proper recognition of DNA lesions and their repair is crucial for normal embryonic development
Summary
Embryonic stem cells (ESCs) display different sensitivities to DNA damage compared with differentiated cells, including mouse embryonic fibroblasts (MEFs) that are used as a feeder layer for ESC cultivation. Mouse ESCs (mESCs) are more sensitive to treatment with UV or c-ray irradiation than differentiated MEFs [1] These differences can be ascribed to the more open chromatin configuration in ESCs [1,2]. Oct collaborates with Sox and Nanog to maintain ESC pluripotency, and Oct, Nanog, and Sox form an interconnected autoregulatory network [7,8]. We investigated whether Oct recognizes damaged chromatin in mouse ESCs stably expressing GFP-Oct. We investigated whether Oct recognizes damaged chromatin in mouse ESCs stably expressing GFP-Oct4 These experiments should contribute to the knowledge of how ESC genomic integrity is maintained, which is crucial for potential application of human ESCs in regenerative medicine
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