Early moderate prenatal alcohol exposure and maternal diet impact offspring DNA methylation across species
- Telethon Kids Institute, The University of Western Australia, Australia
- Respiratory Environmental Health, Wal-yan Respiratory Research Centre, Telethon Kids Institute, Australia
- Occupation, Environment and Safety, School of Population Health, Curtin University, Australia
- Telethon Kids Institute, Australia
- School of Psychological Sciences, University of Western Australia, Australia
- University of Sydney, Faculty of Medicine and Health, Specialty of Child and Adolescent Health, Australia
- Sydney Children’s Hospitals Network (Westmead) and Kids Research, Australia
- Deakin University, IMPACT – the Institute for Mental and Physical Health and Clinical Translation, School of Medicine, Australia
- Murdoch Children’s Research Institute, Royal Children’s Hospital, Australia
- Department of Paediatrics, University of Melbourne, Royal Children’s Hospital, Australia
- Reproductive Epidemiology, Murdoch Children's Research Institute, Australia
- Deakin University, School of Psychology, Faculty of Health, Australia
- Murdoch Children’s Research Institute, Centre for Adolescent Health, Royal Children’s Hospital, Australia
- University New South Wales, National Drug and Alcohol Research Centre, Australia
- Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, The University of Western Australia, Australia
- ARC Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, Australia
- National Drug Research Institute, enAble Institute, Curtin University, Australia
- Wal-yan Respiratory Research Centre, Telethon Kids Institute, Australia
Figures
Figure 1
Overview of prenatal alcohol exposure (PAE) model.
A schematic representation of the experiment design is shown in the figure. Fifteen dams were allocated to each treatment group. PAE mice were exposed to ethanol (10% vol/vol in non-acidified …
Figure 2
PAE and HMD effects on dam characteristics.
(a) Dam weight progression was significantly affected by HMD but not PAE by quadratic mixed effects model without interaction. (b) Trajectory of liquid consumption across pregnancy was affected by …
Figure 3
Prenatal alcohol exposure (PAE) and high methyl donor (HMD) effects on dam characteristics.
There was no significant difference in the average gain of weight in dams between (a) days 1–17 and (b) days 1–19 by treatment group. Both timepoints were included due to some pregnancies ending by …
Figure 4
No evidence for global disruption of methylation by prenatal alcohol exposure (PAE).
The figure shows methylation levels averaged across cytosine-guanines (CpGs) in different regulatory genomic contexts. Neither brain tissue (a and b) nor liver tissue (c and d) were grossly affected …
Figure 5
Prenatal alcohol exposure (PAE) was associated with site-specific differences in offspring DNA methylation.
The majority of differentially methylated regions (DMRs) lost methylation with PAE in (a) brain and (b) liver of mice given normal chow (NC). Each point represents one DMR. Point colour indicates …
Figure 6
High methyl donor (HMD) partially mitigated effects of prenatal alcohol exposure (PAE) on offspring DNA methylation.
Average DNA methylation effect sizes above 30% with PAE were observed in some (a) brain and (b) liver differentially methylated regions (DMRs) in normal chow (NC) mice. Mean absolute difference in …
Figure 7
PAE had no significant effect on other assessed behavioural outcomes.
PAE and HMD had no significant effect on anxiety as evident by no significant difference by unpaired t-test in the (a) percent time in the inner zone in the open field test (N=104) and (b) percent …
Figure 8
HMD was associated with increased locomotor activity.
HMD was associated with increased locomotor activity compared to NC, indicated by significantly greater total distance travelled in the (a) open field test (N=104), (b) object recognition test …
Figure 9
Seven prenatal alcohol exposure (PAE) differentially methylated regions (DMRs) identified in the murine model were successfully replicated in the Lussier et al., 2018 human fetal alcohol spectrum disorder (FASD) cohort.
Examples of two PAE DMRs that had significantly lower DNA methylation with a clinical diagnosis of FASD in the Lussier et al., 2018 cohort (a and c), while their mouse liftover DMR also had …
Tables
Table 1
Table of differentially methylated regions (DMRs) identified in the intronic regions of genes that contained DMRs in both the brain and liver.
Δmeth indicates the percentage change in average methylation level within the DMR with prenatal alcohol exposure (PAE) compared to non-PAE mice.
| Gene | Tissue | Intronic DMR | Width | No. CpGs | Δmeth | p-Value |
|---|---|---|---|---|---|---|
| Auts2 | Brain | chr5:131510296–131510465 | 170 | 5 | –23.5% | <0.05 |
| Auts2 | Liver | chr5:131621828–131621999 | 172 | 4 | –22.5% | <0.05 |
| Adgb | Brain | chr10:10455557–10455883 | 327 | 4 | –25.0% | <0.05 |
| Adgb | Liver | chr10:10353338–10353613 | 276 | 4 | –25.9% | <0.05 |
| Rbfox1 | Brain | chr16:6813039–6813217 | 179 | 5 | –24.3% | <0.05 |
| Rbfox1 | Liver | chr16:6781985–6782330 | 346 | 5 | –22.6% | <0.05 |
Table 2
Number and percentage of brain and liver differentially methylated regions (DMRs) that overlap with tissue-specific regulatory regions.
ATAC-seq, H3K4me1, and H3K27ac regions were obtained at 0 days postnatal from the ENCODE database. p-Values for permutation testing using a randomisation strategy.
| Assay type | Brain DMRs | Brain randomised regions | Liver DMRs | Liver randomised regions |
|---|---|---|---|---|
| ATAC-seq | 21/78 (26.92%), p=0.01 | 1/78 (1.28%), p=0.16 | 53/759 (6.98%) p=0.01 | 22/759 (2.90%) p=0.31 |
| H3K4me1 | 4/78 (5.13%) p=0.03 | 2/78 (2.56%) p=0.18 | 38/759 (5.01%) p=0.05 | 35/759 (4.61%) p=0.32 |
| H3K27ac | 9/78 (11.54%) p=0.01 | 2/78 (2.56%) p=0.74 | 48/759 (6.32%) p=0.01 | 19/759 (2.50%) p=0.26 |
Table 3
Differentially methylated regions (DMRs) identified in the murine model that were validated in the Lussier et al., 2018 human case-control cohort for a clinical diagnosis of fetal alcohol spectrum disorder (FASD).
The upper section describes properties of Lussier et al., 2018 human DMRs. The lower section describes properties of the equivalent murine model DMRs.
| DMR | Organism | Tissue | Chr | Start | End | Width | No. CpGs | FDR | Meandiff | Gene |
|---|---|---|---|---|---|---|---|---|---|---|
| 1 | Human | Buccal | 1 | 68151571 | 68152310 | 740 | 5 | 0.028636 | –0.00497 | GADD45A |
| 2 | Human | Buccal | 19 | 13000782 | 13002357 | 1576 | 11 | 0.000197 | –0.00203 | GCDH |
| 3 | Human | Buccal | 7 | 33148815 | 33149316 | 502 | 11 | 0.001149 | –0.00011 | RP9 |
| 4 | Human | Buccal | 17 | 33905444 | 33905888 | 445 | 14 | 0.000171 | –0.00359 | AP2B1, PEX12 |
| 5 | Human | Buccal | 17 | 27181503 | 27182342 | 840 | 11 | 0.018536 | –0.00246 | ERAL1, FAM222B |
| 6 | Human | Buccal | 19 | 12992181 | 12992479 | 299 | 9 | 0.037431 | –0.00179 | CTD-2265O21.7, DNASE2 |
| 7 | Human | Buccal | 19 | 11849531 | 11850013 | 483 | 9 | 0.022724 | –0.00244 | ZNF823 |
| 1 | Mouse | Liver | 6 | 67034885 | 67035082 | 197 | 4 | <0.05 | –0.220833 | E230016M11Rik |
| 2 | Mouse | Liver | 8 | 84901298 | 84901544 | 246 | 5 | <0.05 | –0.234457 | Klf1 |
| 3 | Mouse | Liver | 9 | 22453836 | 22453893 | 57 | 5 | <0.05 | –0.226427 | Rp9 |
| 4 | Mouse | Brain | 14 | 21403570 | 21403622 | 52 | 4 | <0.05 | –0.234193 | Adk |
| 5 | Mouse | Liver | 11 | 78069463 | 78070002 | 539 | 9 | <0.05 | –0.255864 | Mir144, Mir451a |
| 6 | Mouse | Liver | 11 | 78072079 | 78072313 | 234 | 4 | <0.05 | –0.215227 | Mir144, Mir451a |
| 7 | Mouse | Liver | 2 | 177091927 | 177092945 | 1018 | 5 | <0.05 | –0.224354 | Intergenic |
Additional files
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Supplementary file 1
Statistical results from MWA studies.
(a) Table of brain differentially methylated regions (DMRs) identified by DSS and annotated with annotatr. (b) Table of liver DMRs identified by DSS and annotated with annotatr. (c) Table of gene set enrichment analysis (GSEA) ontology results from genes associated with liver DMRs. (d) GSEA Ontology Gene/Gene Set Overlap Matrix for liver DMRs. (e) List of genes included in candidate gene analysis. (f) Table of regions assessed in candidate genes analysis. (g) Table of brain DMRs having differences to DNA methylation with prenatal alcohol exposure (PAE) being rescued by dietary supplementation. (h) Table of liver DMRs having differences to DNA methylation with PAE being rescued by dietary supplementation. (i) Table of FDR-significant brain DMLs from candidate gene regions in regular diet mice. (j) Table of FDR-significant liver DMLs from candidate gene regions in regular diet mice. (k) Sequencing statistics and bioinformatic quality control.
- https://cdn.elifesciences.org/articles/92135/elife-92135-supp1-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/92135/elife-92135-mdarchecklist1-v1.docx
