Recruitment of Oct4 Protein to UV-Damaged Chromatin in Embryonic Stem Cells

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.

Determination of Protein Interactome of Transcription Factor Sox2 in Embryonic Stem Cells Engineered for Inducible Expression of Four Reprogramming Factors

“…Interestingly, Sox2 was found to associate in ESC with Rad23 in two of three MudPIT analyses and Cetn2 in one of three analyses (data not shown). In support of the reciprocal possibility, Oct4 has recently been reported to associate with UV-damage chromatin in ESC (36).…”

Histone H3 Lysine 9 Acetylation Obstructs ATM Activation and Promotes Ionizing Radiation Sensitivity in Normal Stem Cells

“…H3K9ac has been shown to be downregulated locally around the DNA damage sites in a fraction of murine ES cells (Bártová et al., 2011). We tested whether in ES such downregulation occurs around DSB sites despite the significantly high global H3K9 acetylation in contrast to the ED.…”

“…This study supports previous observations and additionally demonstrates an increased constitutive H3K9ac along with distinctly reduced H3K9me3 in stem cells, in contrast to the isogenic non-stem ED cells in culture as well as in vivo. Local downregulation of H3K9ac has been reported in ESCs, accompanying recruitment of the transcription factor OCT4 to DNA lesions (Bártová et al., 2011). In the same study, a downregulation of H3K9 acetylation was not observed in one-third of the analyzed stem cells, which is in agreement with our finding of a local decrease of H3K9ac in 40% of the stem cells in culture.…”

“…Recruitment of Suv39h1 enabling H3K9me3 and assembly of binding partners HP1 and KAP1 is rapid and transient, observed for few minutes after DSB induction. Interestingly, acetylation on the same amino acid residue (H3K9ac) is locally downregulated at the DNA damage sites (Bártová et al., 2011). …”

Three steps to the immortality of cancer cells: senescence, polyploidy and self-renewal

“…In accordance with this observation, PML protein was reported to be required for activating chromatin remodelling of the Oct4 promoter in stem cells [90], while Bartova and colleagues showed in ESC that OCT4 becomes recruited to chromatin at sites of DNA damage [91]. Oct-4 was further shown to be critical for the survival/apoptosis-resistance of murine ES cells subjected to stress through interactions with Stat3 and survivin [92].…”