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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

Distinct alterations in homeobox gene transcription arising during and after the window of EtOH exposure. Neural 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. Cells were harvested at days-3 and 7, and transcript levels measured using RT-qPCR. Graphs represent three independent replicates (N = 3), with two independent RT reactions and three qPCR measurements for each RT. Differences were measured using a one-way ANOVA, error bars represent SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
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Fig5: Distinct alterations in homeobox gene transcription arising during and after the window of EtOH exposure. Neural 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. Cells were harvested at days-3 and 7, and transcript levels measured using RT-qPCR. Graphs represent three independent replicates (N = 3), with two independent RT reactions and three qPCR measurements for each RT. Differences were measured using a one-way ANOVA, error bars represent SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Mentions: Published reports examining acute EtOH exposure have been unable to demonstrate consistent correlations between alterations in H3K4 me3; a histone mark enriched at the promoter regions of actively transcribed genes [66], and changes in transcription [10, 67, 68]. We were therefore curious to determine if the candidate genes demonstrating changes in any of the measured chromatin marks either before or after the recovery period would display alterations in gene transcription. To this end, RNA was isolated from all treatment groups and gene expression measured using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Of the 22 candidate genes examined in Fig. 1, transcripts encoding Ascl1, Dlx2, Dlx3, Pax6, Nkx2.2, Sox1, Sox2, Sox17, and Tbx2 could be detected in our cultures (Fig. 5). Similar to our previous studies [10], only a very small number (20 %) of candidate genes (Ascl1 and Sox2) displayed altered expression profiles arising as a consequence of EtOH exposure at Day-3. In contrast, eight of the nine detected candidates (~88 %) displayed significant alterations in transcript levels on Day-7, across both concentrations of EtOH tested. These candidate homeobox genes sit at the hub of multiple transcriptional pathways controlling cellular identity and proliferation. We therefore assayed RNA samples for alterations in the expression of known markers of both cellular growth (Ki67, cMyc, and Rb1) and neural stem cell proliferation/identity (Fabp7, Gfap, Gli3, Nestin, Olig2 and Tuj1). These analyses revealed changes in a small number of candidates within the 240 mg/dL treatments on Day-3 (Ki67, Fabp7, and Tuj1), while the larger impact was again observed on Day-7, with eight of the nine candidates demonstrating alterations in transcription (Fig. 6). These observations indicate the larger impact of EtOH exposure on the developmental program may arise beyond the initial period of exposure.Fig. 5


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)

Distinct alterations in homeobox gene transcription arising during and after the window of EtOH exposure. Neural 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. Cells were harvested at days-3 and 7, and transcript levels measured using RT-qPCR. Graphs represent three independent replicates (N = 3), with two independent RT reactions and three qPCR measurements for each RT. Differences were measured using a one-way ANOVA, error bars represent SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4587584&req=5

Fig5: Distinct alterations in homeobox gene transcription arising during and after the window of EtOH exposure. Neural 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. Cells were harvested at days-3 and 7, and transcript levels measured using RT-qPCR. Graphs represent three independent replicates (N = 3), with two independent RT reactions and three qPCR measurements for each RT. Differences were measured using a one-way ANOVA, error bars represent SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Mentions: Published reports examining acute EtOH exposure have been unable to demonstrate consistent correlations between alterations in H3K4 me3; a histone mark enriched at the promoter regions of actively transcribed genes [66], and changes in transcription [10, 67, 68]. We were therefore curious to determine if the candidate genes demonstrating changes in any of the measured chromatin marks either before or after the recovery period would display alterations in gene transcription. To this end, RNA was isolated from all treatment groups and gene expression measured using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Of the 22 candidate genes examined in Fig. 1, transcripts encoding Ascl1, Dlx2, Dlx3, Pax6, Nkx2.2, Sox1, Sox2, Sox17, and Tbx2 could be detected in our cultures (Fig. 5). Similar to our previous studies [10], only a very small number (20 %) of candidate genes (Ascl1 and Sox2) displayed altered expression profiles arising as a consequence of EtOH exposure at Day-3. In contrast, eight of the nine detected candidates (~88 %) displayed significant alterations in transcript levels on Day-7, across both concentrations of EtOH tested. These candidate homeobox genes sit at the hub of multiple transcriptional pathways controlling cellular identity and proliferation. We therefore assayed RNA samples for alterations in the expression of known markers of both cellular growth (Ki67, cMyc, and Rb1) and neural stem cell proliferation/identity (Fabp7, Gfap, Gli3, Nestin, Olig2 and Tuj1). These analyses revealed changes in a small number of candidates within the 240 mg/dL treatments on Day-3 (Ki67, Fabp7, and Tuj1), while the larger impact was again observed on Day-7, with eight of the nine candidates demonstrating alterations in transcription (Fig. 6). These observations indicate the larger impact of EtOH exposure on the developmental program may arise beyond the initial period of exposure.Fig. 5

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