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Sexual dimorphism in epigenomic responses of stem cells to extreme fetal growth.

Delahaye F, Wijetunga NA, Heo HJ, Tozour JN, Zhao YM, Greally JM, Einstein FH - Nat Commun (2014)

Bottom Line: Extreme fetal growth is associated with increased susceptibility to a range of adult diseases through an unknown mechanism of cellular memory.We tested whether heritable epigenetic processes in long-lived CD34(+) haematopoietic stem/progenitor cells showed evidence for re-programming associated with the extremes of fetal growth.Here we show that both fetal growth restriction and over-growth are associated with global shifts towards DNA hypermethylation, targeting cis-regulatory elements in proximity to genes involved in glucose homeostasis and stem cell function.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics &Gynecology and Women's Health, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Block Building, Room 631, Bronx, New York 10461, USA.

ABSTRACT
Extreme fetal growth is associated with increased susceptibility to a range of adult diseases through an unknown mechanism of cellular memory. We tested whether heritable epigenetic processes in long-lived CD34(+) haematopoietic stem/progenitor cells showed evidence for re-programming associated with the extremes of fetal growth. Here we show that both fetal growth restriction and over-growth are associated with global shifts towards DNA hypermethylation, targeting cis-regulatory elements in proximity to genes involved in glucose homeostasis and stem cell function. We find a sexually dimorphic response; intrauterine growth restriction is associated with substantially greater epigenetic dysregulation in males, whereas large for gestational age growth predominantly affects females. The findings are consistent with extreme fetal growth interacting with variable fetal susceptibility to influence cellular ageing and metabolic characteristics through epigenetic mechanisms, potentially generating biomarkers that could identify infants at higher risk for chronic disease later in life.

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Biological validationValidation of significant loci of interest by targeted bisulphite sequencing (TBS) in Cohort 2 for loci at the (a) WNT6 and (b) PTCH1 genes. Candidate differentially-methylated loci are shown as the HpaII sites within the amplicon regions (gray boxes), with results of DNA methylation distributions for controls and cases (IUGR and LGA combined) from HELP-tagging (orange) and TBS (white) depicted as violin plots (mean shown in red, 1st and 3rd quartile are depicted by the thick black bar). The results show concordance for similar types of changes between HELP-tagging and TBS results at these loci with significant results (p<0.05, t-test) for TBS marked with asterisks.
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Figure 5: Biological validationValidation of significant loci of interest by targeted bisulphite sequencing (TBS) in Cohort 2 for loci at the (a) WNT6 and (b) PTCH1 genes. Candidate differentially-methylated loci are shown as the HpaII sites within the amplicon regions (gray boxes), with results of DNA methylation distributions for controls and cases (IUGR and LGA combined) from HELP-tagging (orange) and TBS (white) depicted as violin plots (mean shown in red, 1st and 3rd quartile are depicted by the thick black bar). The results show concordance for similar types of changes between HELP-tagging and TBS results at these loci with significant results (p<0.05, t-test) for TBS marked with asterisks.

Mentions: To test the robustness of our genome-wide technique, we assess the reproducibility of DNA methylation differences at our candidate differentially-methylated loci using single locus quantitative validation studies. We first perform verification studies on samples from Cohort 1, on whom the genome-wide studies had been performed, testing 4 loci selected for differing levels of DNA methylation in 24 subjects. A strong correlation between bisulphite MassArray and HELP-tagging is found (R2 = 0.98, Supplementary Fig. 4). In a second, independent set of CD34+ HSPC samples (Cohort 2) consisting of 8 new subjects/group (control, IUGR, LGA) with equal numbers of males and females in each group, we perform a targeted bisulphite sequencing (TBS) assay, using bisulphite treatment, targeted PCR and massively-parallel sequencing to measure DNA methylation at 72 loci in the 24 subjects (see Methods and Supplementary Table 3). The correlations between HELP-tagging with MassArray in Cohort 1 (R2=0.98, Supplementary Fig. 4) and with TBS in Cohort 2 (R2=0.72, Supplementary Fig. 5 and Supplementary Table 3) are both strong. These highly quantitative verification and validation studies demonstrate the technical robustness of the genome-wide HELP-tagging assay, as well as the potential to validate DNA methylation differences, even when using a new cohort of subjects. Of the 54 candidate differentially-methylated loci from the HELP-tagging group comparisons, we focus on loci implicated by our GOseq-normalized GSEA results, using primers for candidate differentially-methylated loci proximal to WNT6 (Fig. 5a) and PTCH1 (Fig. 5b) from the Hedgehog (HH)signaling pathway and MAFA from the Maturity onset diabetes of the young pathway (Supplementary Fig. 6). We find the direction of DNA methylation changes to be concordant between genome-wide and targeted assays for all three loci, with statistically significant differences demonstrable for TBS data from WNT6 (p=0.023) and PTCH1 (p=0.014) (Supplementary Table 4). We also show the PTCH1 and WNT6 genes to have increased DNA methylation by TBS at local cis-regulatory elements in cases (IUGR and LGA) compared to controls (Supplementary Table 4, p<0.05). Finally, we interrogate loci associated with genes that are differentially methylated on average between cases (IUGR plus LGA) and controls in Cohort 1 and previously found to have epigenetic alterations related to metabolic syndrome and type 2 diabetes mellitus, IGF234,35 and RXRA36. A positive correlation is seen between the HELP-tagging and TBS DNA methylation levels, but the TBS DNA methylation differences between cases and controls are of insufficient magnitude for statistical significance to be attributed (Supplementary Table 4). The number of new samples in Cohort 2 is limited and thus these validation studies are likely to confirm loci of major effect only. Overall, the TBS data are concordant with the genome-wide data, indicating that the conclusions based on the genome-wide results are tenable.


Sexual dimorphism in epigenomic responses of stem cells to extreme fetal growth.

Delahaye F, Wijetunga NA, Heo HJ, Tozour JN, Zhao YM, Greally JM, Einstein FH - Nat Commun (2014)

Biological validationValidation of significant loci of interest by targeted bisulphite sequencing (TBS) in Cohort 2 for loci at the (a) WNT6 and (b) PTCH1 genes. Candidate differentially-methylated loci are shown as the HpaII sites within the amplicon regions (gray boxes), with results of DNA methylation distributions for controls and cases (IUGR and LGA combined) from HELP-tagging (orange) and TBS (white) depicted as violin plots (mean shown in red, 1st and 3rd quartile are depicted by the thick black bar). The results show concordance for similar types of changes between HELP-tagging and TBS results at these loci with significant results (p<0.05, t-test) for TBS marked with asterisks.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4197137&req=5

Figure 5: Biological validationValidation of significant loci of interest by targeted bisulphite sequencing (TBS) in Cohort 2 for loci at the (a) WNT6 and (b) PTCH1 genes. Candidate differentially-methylated loci are shown as the HpaII sites within the amplicon regions (gray boxes), with results of DNA methylation distributions for controls and cases (IUGR and LGA combined) from HELP-tagging (orange) and TBS (white) depicted as violin plots (mean shown in red, 1st and 3rd quartile are depicted by the thick black bar). The results show concordance for similar types of changes between HELP-tagging and TBS results at these loci with significant results (p<0.05, t-test) for TBS marked with asterisks.
Mentions: To test the robustness of our genome-wide technique, we assess the reproducibility of DNA methylation differences at our candidate differentially-methylated loci using single locus quantitative validation studies. We first perform verification studies on samples from Cohort 1, on whom the genome-wide studies had been performed, testing 4 loci selected for differing levels of DNA methylation in 24 subjects. A strong correlation between bisulphite MassArray and HELP-tagging is found (R2 = 0.98, Supplementary Fig. 4). In a second, independent set of CD34+ HSPC samples (Cohort 2) consisting of 8 new subjects/group (control, IUGR, LGA) with equal numbers of males and females in each group, we perform a targeted bisulphite sequencing (TBS) assay, using bisulphite treatment, targeted PCR and massively-parallel sequencing to measure DNA methylation at 72 loci in the 24 subjects (see Methods and Supplementary Table 3). The correlations between HELP-tagging with MassArray in Cohort 1 (R2=0.98, Supplementary Fig. 4) and with TBS in Cohort 2 (R2=0.72, Supplementary Fig. 5 and Supplementary Table 3) are both strong. These highly quantitative verification and validation studies demonstrate the technical robustness of the genome-wide HELP-tagging assay, as well as the potential to validate DNA methylation differences, even when using a new cohort of subjects. Of the 54 candidate differentially-methylated loci from the HELP-tagging group comparisons, we focus on loci implicated by our GOseq-normalized GSEA results, using primers for candidate differentially-methylated loci proximal to WNT6 (Fig. 5a) and PTCH1 (Fig. 5b) from the Hedgehog (HH)signaling pathway and MAFA from the Maturity onset diabetes of the young pathway (Supplementary Fig. 6). We find the direction of DNA methylation changes to be concordant between genome-wide and targeted assays for all three loci, with statistically significant differences demonstrable for TBS data from WNT6 (p=0.023) and PTCH1 (p=0.014) (Supplementary Table 4). We also show the PTCH1 and WNT6 genes to have increased DNA methylation by TBS at local cis-regulatory elements in cases (IUGR and LGA) compared to controls (Supplementary Table 4, p<0.05). Finally, we interrogate loci associated with genes that are differentially methylated on average between cases (IUGR plus LGA) and controls in Cohort 1 and previously found to have epigenetic alterations related to metabolic syndrome and type 2 diabetes mellitus, IGF234,35 and RXRA36. A positive correlation is seen between the HELP-tagging and TBS DNA methylation levels, but the TBS DNA methylation differences between cases and controls are of insufficient magnitude for statistical significance to be attributed (Supplementary Table 4). The number of new samples in Cohort 2 is limited and thus these validation studies are likely to confirm loci of major effect only. Overall, the TBS data are concordant with the genome-wide data, indicating that the conclusions based on the genome-wide results are tenable.

Bottom Line: Extreme fetal growth is associated with increased susceptibility to a range of adult diseases through an unknown mechanism of cellular memory.We tested whether heritable epigenetic processes in long-lived CD34(+) haematopoietic stem/progenitor cells showed evidence for re-programming associated with the extremes of fetal growth.Here we show that both fetal growth restriction and over-growth are associated with global shifts towards DNA hypermethylation, targeting cis-regulatory elements in proximity to genes involved in glucose homeostasis and stem cell function.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics &Gynecology and Women's Health, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Block Building, Room 631, Bronx, New York 10461, USA.

ABSTRACT
Extreme fetal growth is associated with increased susceptibility to a range of adult diseases through an unknown mechanism of cellular memory. We tested whether heritable epigenetic processes in long-lived CD34(+) haematopoietic stem/progenitor cells showed evidence for re-programming associated with the extremes of fetal growth. Here we show that both fetal growth restriction and over-growth are associated with global shifts towards DNA hypermethylation, targeting cis-regulatory elements in proximity to genes involved in glucose homeostasis and stem cell function. We find a sexually dimorphic response; intrauterine growth restriction is associated with substantially greater epigenetic dysregulation in males, whereas large for gestational age growth predominantly affects females. The findings are consistent with extreme fetal growth interacting with variable fetal susceptibility to influence cellular ageing and metabolic characteristics through epigenetic mechanisms, potentially generating biomarkers that could identify infants at higher risk for chronic disease later in life.

Show MeSH
Related in: MedlinePlus