11 research outputs found
Recruitment of Oct4 Protein to UV-Damaged Chromatin in Embryonic Stem Cells
Background: Oct4 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 Findings: We 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 (cH2AX). Moreover, we observed an ESC-specific nuclear distribution of cH2AX 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/Significance: Our 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 unwante
Differentiation-Independent Fluctuation of Pluripotency-Related Transcription Factors and Other Epigenetic Markers in Embryonic Stem Cell Colonies
GFP-Oct4 at UV-damaged chromatin and inhibition of transcription.
<p><b>A</b>, GFP-Oct4 levels in GOWT1 mESCs after the inhibition of transcription by actinomycin D treatment are shown. Treatment with actinomycin D reduced the level of Oct4 (panels <b>a</b> and <b>d</b>) in regions of laser-generated DNA lesions, which were γH2AX-positive (red) (panels <b>b</b> and <b>e</b> show γH2AX; panels <b>c</b> and <b>f</b> show the overlay of γH2AX with all DNA content). Protein levels of Oct4 (green) and γH2AX (red) were quantified using LEICA LAS AF (version 2.1.2.) software, and the results are shown in panel <b>g</b>. <b>Ba</b>, Panels show the possibilities of GFP-Oct4 (green) levels in UV-induced DNA lesions after actinomycin D treatment. <b>Bb</b>, H3K9 acetylation (red) at irradiated chromatin of actinomycin D-treated cells was slightly reduced or not changed in comparison with that in nonirradiated areas of the same cell.</p
Accumulation of γH2AX and appearance of 53PB1-positive regions in spontaneously occurring or induced DNA lesions.
<p><b>A</b>, Appearance of 53BP1-positive foci (red) in heterochromatin (accumulated blue) and euchromatin (dispersed blue) nuclear regions of control, γ-irradiated, and CPT or ETOP-treated cells. <b>B</b>, Spontaneously occurring γH2AX foci (red) that overlap with GFP-Oct4-positive regions (green) in GOWT1 mESC nuclei. Quantification in the graph was performed using LEICA LAS AF software (version 2.1.2.). <b>Ca</b>, GFP-Oct4 and 53BP1 nuclear patterns in control nonirradiated cells, <b>γ</b>-irradiated cells, and CPT or ETOP-treated cells. <b>Cb</b>, As an example, 53BP1-positive IRIF (red) and analysis of Oct4 and 53BP1 colocalization are shown. The level of colocalization was evaluated using a quantification tool from LEICA LAS AF software (version 2.1.2.), and the results are shown in the bar chart as mean±S.E. <b>Scale bars</b>: in each panel, the scale bar is shown with the relevant value.</p
ATP depletion and Oct4 levels at UV-damaged chromatin.
<p><b>A</b>, In comparison with control (<b>a</b>), ATP depletion reduced the diameter of mitochondria (<b>b</b>). <b>c</b>, Panel shows the comparison of the average diameter of mitochondria in control cells and ATP-depleted cells. <b>d</b>, Mitochondria staining in control cells. <b>e</b>, Mitochondria staining after ATP depletion. <b>B</b>, In comparison with control nonirradiated cells (<b>a</b>), GFP-Oct4 become focally distributed after ATP depletion (<b>b</b>). <b>C</b>, DNA lesions were induced in GOWT1 mESCs after ATP depletion, but the absence of GFP-Oct4 accumulation was observed. γH2AX positivity was low and homogeneous within entire cells. <b>D</b>, H3K9 acetylation levels were low in UV-irradiated chromatin after ATP depletion. <b>Scale bars</b>: in each panel, the scale bar is shown with the relevant value.</p
Recruitment of selected proteins to UV-damaged chromatin.
<p>Recruitment of selected proteins to UV-damaged chromatin.</p
Oct4 kinetics in irradiated and nonirradiated regions.
<p><b>A</b>, FRAP was used to study Oct4 kinetics in nonirradiated mESCs and GFP-Oct4 recovery in UV-damaged chromatin. <b>B</b>, GFP-Oct4 recovery after photobleaching was studied in control nonirradiated cell nuclei (blue line) and compared with that in TSA-treated (magenta line) and actinomycin D-treated (green line) cells. <b>C</b>, FRAP was performed in UV-irradiated regions with pronounced Oct4 accumulation (control, blue line) and with unchanged levels of Oct4 after TSA treatment (magenta line). <b>D</b>, FRAP data for Oct4 in nonirradiated cells undergoing ATP depletion compared to that in nontreated control cells (blue line). After ATP depletion, Oct4 recovery was measured in the nucleoplasm when Oct4 did not accumulate into foci (magenta line) and when GFP-Oct4 was accumulated into foci (green line). One (two) asterisk(s) show(s) statistically significant differences at p≤0.05 (p≤0.01).</p
Detection of Nanog and c-MYC at UV-irradiated regions.
<p>The cells with accumulated GFP-Oct4 (green) had relatively stable levels of Nanog (red, <b>A</b>) and c-MYC protein (red, <b>B</b>) at UV-irradiated chromatin. Quantification of the protein levels in the graphs of panel A was performed using LEICA LAS AF software (version 2.1.2.). <b>Scale bars</b>: in each panel, the scale bar is shown with the relevant value.</p
Oct4 recruitment to DNA lesions and acetylation events.
<p><b>A</b>, <b>B</b>, Panels show the possibilities of how GFP-Oct4 recognizes UV-damaged chromatin after treatment of cells with the HDACi TSA. <b>C</b>, Oct4 and γH2AX levels in UV-irradiated ROIs of TSA-treated cells. <b>D</b>, Oct4 and γH2AX levels in UV-irradiated ROIs of SAHA-treated cells. <b>E</b>, Oct4 and HDAC1 levels in UV-irradiated ROIs of nontreated control cells. <b>F</b>, Oct4 and H3K9 acetylation levels in UV-irradiated ROIs of nontreated control cells. <b>G</b>, Oct4 and HDAC1 levels in UV-irradiated ROIs of TSA-treated cells. <b>H</b>, Oct4 and H3K9 acetylation levels in UV-irradiated ROIs of TSA-treated cells. <b>I</b>, Oct4 and HDAC1 levels in UV-irradiated ROIs of SAHA-treated cells. <b>Scale bars</b>: in each panel, the scale bar is shown with the relevant value.</p
Model of Oct4 accumulation at UV-damaged chromatin.
<p><b>A</b>, Oct4 was significantly accumulated at chromatin with laser-induced DNA lesions. In these regions, the levels of Nanog and c-MYC were not changed. This event was accompanied by γH2AX accumulation at DNA lesions. <b>B</b>, TSA-induced changes in acetylation stopped the increased accumulation of Oct4 at UV-damaged chromatin. Phosphorylation of H2AX was not observed in the irradiated regions of TSA-treated cells, but high levels of γH2AX were found in nonirradiated regions of these cells. <b>Ca</b>, High levels of γH2AX were observed after actinomycin D treatment in both irradiated chromatin and the entire genome. These levels were accompanied by an absence of Oct4 at UV-irradiated chromatin, and there were subtle levels of H3K9 acetylation. <b>Cb</b>, ATP depletion did not induce pronounced Oct4 accumulation at irradiated chromatin, and in many cases, there was an absence of Oct4 in UV-damaged genomic regions. After ATP depletion, the H3K9 acetylation level was very low, and γH2AX slightly appeared throughout the entire genome.</p