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Dose-dependent alcohol-induced alterations in chromatin structure persist beyond the window of exposure and correlate with fetal alcohol syndrome birth defects.

Veazey KJ, Parnell SE, Miranda RC, Golding MC - Epigenetics Chromatin (2015)

Bottom Line: Unexpectedly, we do not observe a correlation between alcohol-induced changes in chromatin structure and alterations in transcription.Interestingly, the majority of epigenetic changes observed occur in marks associated with repressive chromatin structure, and we identify correlative disruptions in transcripts encoding Dnmt1, Eed, Ehmt2 (G9a), EzH2, Kdm1a, Kdm4c, Setdb1, Sod3, Tet1 and Uhrf1.Collectively, our results indicate that alcohol-induced modifications to chromatin structure persist beyond the window of exposure, and likely contribute to the development of fetal alcohol syndrome-associated congenital abnormalities.

View Article: PubMed Central - PubMed

Affiliation: Room 338 VMA, 4466 TAMU, Department of Veterinary Physiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843-4466 USA.

ABSTRACT

Background: In recent years, we have come to recognize that a multitude of in utero exposures have the capacity to induce the development of congenital and metabolic defects. As most of these encounters manifest their effects beyond the window of exposure, deciphering the mechanisms of teratogenesis is incredibly difficult. For many agents, altered epigenetic programming has become suspect in transmitting the lasting signature of exposure leading to dysgenesis. However, while several chemicals can perturb chromatin structure acutely, for many agents (particularly alcohol) it remains unclear if these modifications represent transient responses to exposure or heritable lesions leading to pathology.

Results: Here, we report that mice encountering an acute exposure to alcohol on gestational Day-7 exhibit significant alterations in chromatin structure (histone 3 lysine 9 dimethylation, lysine 9 acetylation, and lysine 27 trimethylation) at Day-17, and that these changes strongly correlate with the development of craniofacial and central nervous system defects. Using a neural cortical stem cell model, we find that the epigenetic changes arising as a consequence of alcohol exposure are heavily dependent on the gene under investigation, the dose of alcohol encountered, and that the signatures arising acutely differ significantly from those observed after a 4-day recovery period. Importantly, the changes observed post-recovery are consistent with those modeled in vivo, and associate with alterations in transcripts encoding multiple homeobox genes directing neurogenesis. Unexpectedly, we do not observe a correlation between alcohol-induced changes in chromatin structure and alterations in transcription. Interestingly, the majority of epigenetic changes observed occur in marks associated with repressive chromatin structure, and we identify correlative disruptions in transcripts encoding Dnmt1, Eed, Ehmt2 (G9a), EzH2, Kdm1a, Kdm4c, Setdb1, Sod3, Tet1 and Uhrf1.

Conclusions: These observations suggest that the immediate and long-term impacts of alcohol exposure on chromatin structure are distinct, and hint at the existence of a possible coordinated epigenetic response to ethanol during development. Collectively, our results indicate that alcohol-induced modifications to chromatin structure persist beyond the window of exposure, and likely contribute to the development of fetal alcohol syndrome-associated congenital abnormalities.

No MeSH data available.


Related in: MedlinePlus

EtOH exposure in vitro alters levels of transcripts encoding Sod3 and Tet1, but does not impact measures of cell death or oxidative stress. a Measurements of transcripts encoding proteins involved in the metabolic processing of alcohol and oxidative stress response pathways. Primary neuroepithelial stem cells were cultured in the presence of 160 or 240 mg/dL EtOH for 3 days, followed by a 4-day recovery in media lacking EtOH. Samples were harvested at days-3 and 7, and transcript levels determined by RT-qPCR. Graphs represent three independent biological replicates (N = 3), with two independent RT reactions and three independent qPCR measurements for each RT. Significance was measured using a one-way ANOVA, error bars represent SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. b–e Measures of cellular stress and apoptosis in primary neuroepithelial stem cells exposed to alcohol. Cells were cultured in the presence of 80–240 mg/dL EtOH for 3 days, then assayed for markers of b and c apoptosis, d oxidative stress and e cellular stress. Differences were measured using a one-way ANOVA, error bars represent SEM. Graphs represent three separate biological replicates (N = 3)
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Fig2: EtOH exposure in vitro alters levels of transcripts encoding Sod3 and Tet1, but does not impact measures of cell death or oxidative stress. a Measurements of transcripts encoding proteins involved in the metabolic processing of alcohol and oxidative stress response pathways. Primary neuroepithelial stem cells were cultured in the presence of 160 or 240 mg/dL EtOH for 3 days, followed by a 4-day recovery in media lacking EtOH. Samples were harvested at days-3 and 7, and transcript levels determined by RT-qPCR. Graphs represent three independent biological replicates (N = 3), with two independent RT reactions and three independent qPCR measurements for each RT. Significance was measured using a one-way ANOVA, error bars represent SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. b–e Measures of cellular stress and apoptosis in primary neuroepithelial stem cells exposed to alcohol. Cells were cultured in the presence of 80–240 mg/dL EtOH for 3 days, then assayed for markers of b and c apoptosis, d oxidative stress and e cellular stress. Differences were measured using a one-way ANOVA, error bars represent SEM. Graphs represent three separate biological replicates (N = 3)

Mentions: Given the observed increases in ROS following alcohol exposure, researchers have speculated that some of the teratogenicity associated with EtOH exposure is linked to oxidative stress [15, 44]. Recently, a link between components of the oxidative stress pathways and enzymes controlling chromatin structure has been identified [21, 45]. To examine a potential link between mobilization of the oxidative stress response, and the observed alterations in chromatin structure, we began by quantifying the transcript levels of 23 well characterized candidate genes involved in either the metabolic processing of alcohol or the oxidative stress response pathway [44]. Of these 23 candidates, transcripts encoding Cyp2e1, Gpx2 and Gsta2 could not be detected in RNA samples isolated from our neurosphere cultures. Surprisingly, of the remaining 20 candidates, the majority of genes exhibited a down-regulation at the Day-3 time point and no significant alterations at Day-7 (Fig. 2a). The two notable exceptions to this trend were Sod3, and Tet1, which measured a 1.7-fold increase over the control at the Day-7 time point.Fig. 2


Dose-dependent alcohol-induced alterations in chromatin structure persist beyond the window of exposure and correlate with fetal alcohol syndrome birth defects.

Veazey KJ, Parnell SE, Miranda RC, Golding MC - Epigenetics Chromatin (2015)

EtOH exposure in vitro alters levels of transcripts encoding Sod3 and Tet1, but does not impact measures of cell death or oxidative stress. a Measurements of transcripts encoding proteins involved in the metabolic processing of alcohol and oxidative stress response pathways. Primary neuroepithelial stem cells were cultured in the presence of 160 or 240 mg/dL EtOH for 3 days, followed by a 4-day recovery in media lacking EtOH. Samples were harvested at days-3 and 7, and transcript levels determined by RT-qPCR. Graphs represent three independent biological replicates (N = 3), with two independent RT reactions and three independent qPCR measurements for each RT. Significance was measured using a one-way ANOVA, error bars represent SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. b–e Measures of cellular stress and apoptosis in primary neuroepithelial stem cells exposed to alcohol. Cells were cultured in the presence of 80–240 mg/dL EtOH for 3 days, then assayed for markers of b and c apoptosis, d oxidative stress and e cellular stress. Differences were measured using a one-way ANOVA, error bars represent SEM. Graphs represent three separate biological replicates (N = 3)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4587584&req=5

Fig2: EtOH exposure in vitro alters levels of transcripts encoding Sod3 and Tet1, but does not impact measures of cell death or oxidative stress. a Measurements of transcripts encoding proteins involved in the metabolic processing of alcohol and oxidative stress response pathways. Primary neuroepithelial stem cells were cultured in the presence of 160 or 240 mg/dL EtOH for 3 days, followed by a 4-day recovery in media lacking EtOH. Samples were harvested at days-3 and 7, and transcript levels determined by RT-qPCR. Graphs represent three independent biological replicates (N = 3), with two independent RT reactions and three independent qPCR measurements for each RT. Significance was measured using a one-way ANOVA, error bars represent SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. b–e Measures of cellular stress and apoptosis in primary neuroepithelial stem cells exposed to alcohol. Cells were cultured in the presence of 80–240 mg/dL EtOH for 3 days, then assayed for markers of b and c apoptosis, d oxidative stress and e cellular stress. Differences were measured using a one-way ANOVA, error bars represent SEM. Graphs represent three separate biological replicates (N = 3)
Mentions: Given the observed increases in ROS following alcohol exposure, researchers have speculated that some of the teratogenicity associated with EtOH exposure is linked to oxidative stress [15, 44]. Recently, a link between components of the oxidative stress pathways and enzymes controlling chromatin structure has been identified [21, 45]. To examine a potential link between mobilization of the oxidative stress response, and the observed alterations in chromatin structure, we began by quantifying the transcript levels of 23 well characterized candidate genes involved in either the metabolic processing of alcohol or the oxidative stress response pathway [44]. Of these 23 candidates, transcripts encoding Cyp2e1, Gpx2 and Gsta2 could not be detected in RNA samples isolated from our neurosphere cultures. Surprisingly, of the remaining 20 candidates, the majority of genes exhibited a down-regulation at the Day-3 time point and no significant alterations at Day-7 (Fig. 2a). The two notable exceptions to this trend were Sod3, and Tet1, which measured a 1.7-fold increase over the control at the Day-7 time point.Fig. 2

Bottom Line: Unexpectedly, we do not observe a correlation between alcohol-induced changes in chromatin structure and alterations in transcription.Interestingly, the majority of epigenetic changes observed occur in marks associated with repressive chromatin structure, and we identify correlative disruptions in transcripts encoding Dnmt1, Eed, Ehmt2 (G9a), EzH2, Kdm1a, Kdm4c, Setdb1, Sod3, Tet1 and Uhrf1.Collectively, our results indicate that alcohol-induced modifications to chromatin structure persist beyond the window of exposure, and likely contribute to the development of fetal alcohol syndrome-associated congenital abnormalities.

View Article: PubMed Central - PubMed

Affiliation: Room 338 VMA, 4466 TAMU, Department of Veterinary Physiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843-4466 USA.

ABSTRACT

Background: In recent years, we have come to recognize that a multitude of in utero exposures have the capacity to induce the development of congenital and metabolic defects. As most of these encounters manifest their effects beyond the window of exposure, deciphering the mechanisms of teratogenesis is incredibly difficult. For many agents, altered epigenetic programming has become suspect in transmitting the lasting signature of exposure leading to dysgenesis. However, while several chemicals can perturb chromatin structure acutely, for many agents (particularly alcohol) it remains unclear if these modifications represent transient responses to exposure or heritable lesions leading to pathology.

Results: Here, we report that mice encountering an acute exposure to alcohol on gestational Day-7 exhibit significant alterations in chromatin structure (histone 3 lysine 9 dimethylation, lysine 9 acetylation, and lysine 27 trimethylation) at Day-17, and that these changes strongly correlate with the development of craniofacial and central nervous system defects. Using a neural cortical stem cell model, we find that the epigenetic changes arising as a consequence of alcohol exposure are heavily dependent on the gene under investigation, the dose of alcohol encountered, and that the signatures arising acutely differ significantly from those observed after a 4-day recovery period. Importantly, the changes observed post-recovery are consistent with those modeled in vivo, and associate with alterations in transcripts encoding multiple homeobox genes directing neurogenesis. Unexpectedly, we do not observe a correlation between alcohol-induced changes in chromatin structure and alterations in transcription. Interestingly, the majority of epigenetic changes observed occur in marks associated with repressive chromatin structure, and we identify correlative disruptions in transcripts encoding Dnmt1, Eed, Ehmt2 (G9a), EzH2, Kdm1a, Kdm4c, Setdb1, Sod3, Tet1 and Uhrf1.

Conclusions: These observations suggest that the immediate and long-term impacts of alcohol exposure on chromatin structure are distinct, and hint at the existence of a possible coordinated epigenetic response to ethanol during development. Collectively, our results indicate that alcohol-induced modifications to chromatin structure persist beyond the window of exposure, and likely contribute to the development of fetal alcohol syndrome-associated congenital abnormalities.

No MeSH data available.


Related in: MedlinePlus