Birthweight is associated with DNA promoter methylation of the glucocorticoid receptor in human placenta

doi:10.4161/epi.6.5.15236

Epigenetics. 2011 May; 6(5): 566–572.

Published online 2011 May. doi: 10.4161/epi.6.5.15236

PMCID: PMC3121971

Birthweight is associated with DNA promoter methylation of the glucocorticoid receptor in human placenta

Amanda C Filiberto,^#¹ Matthew A Maccani,^#¹ Devin Koestler,² Charlotte Wilhelm-Benartzi,² Michele Avissar-Whiting,¹ Carolyn E Banister,¹ Luc A Gagne,¹ and Carmen J Marsit^1,²

¹Department of Pathology and Laboratory Medicine; Brown University; Providence, RI USA

²Center for Environmental Health and Technology; Department of Community Health; Brown University; Providence, RI USA

Corresponding author.

^#Contributed equally.

Correspondence to: Carmen J. Marsit; Email: Carmen_Marsit/at/brown.edu

Received December 15, 2010; Accepted February 18, 2011.

This article has been cited by other articles in PMC.

Abstract

Birthweight has been associated with a number of health outcomes throughout life. Crucial to proper infant growth and development is the placenta, and alterations to placental gene function may reflect differences in the intrauterine environment which functionally contribute to infant growth and may ultimately affect the child's health. To examine if epigenetic alteration to the glucocorticoid receptor (GR) gene was linked to infant growth, we analyzed 480 human placentas for differential methylation of the GR gene exon 1F and examined how this variation in methylation extent was associated with fetal growth. Multivariable linear regression revealed a significant association (p < 0.0001) between differential methylation of the GR gene and large for gestational age (LGA) status. Our work is one of the first to link infant growth as a measure of the intrauterine environment and epigenetic alterations to the GR and suggests that DNA methylation may be a critical determinant of placental function.

Key words: DNA methylation, placenta, fetal development, birthweight, epigenetics

Introduction

The period of intrauterine development represents a sensitive period when disruption or modification of the intrauterine environment can influence fetal development as well as lead to programming of health throughout the life course.¹^,² In particular, environmental exposures during intrauterine development have been shown to be associated with several chronic diseases in adulthood,³ and adverse intrauterine conditions have been shown to associate with fetal growth outcomes.²^,⁴^–⁶ There is growing interest in determining the functional molecular basis of this fetal programming.

The metabolic and endocrine activity and ability to transport nutrients, water, gas and waste products marks the placenta as a vital organ for the growing fetus.⁷ Dysregulated placental gene expression may result from in utero exposures and thus represent a record of the intrauterine environment during pregnancy. These alterations may also play a critical role in establishing a fetal program that can influence health outcomes throughout life.⁷ Investigating epigenetic alterations in the placenta, a functional indicator of intrauterine environment,⁸ may add to our understanding of the molecular mechanisms behind many developmental outcomes that may be influenced by adverse intrauterine conditions.

Low birthweight has been associated with increased infant morbidity and mortality and with an increased risk for certain diseases later in life, particularly those comprising the metabolic syndrome.⁹ High birthweight, as well, has been shown to have serious adverse health outcomes in both developing children and later in life.¹⁰ Previous studies have shown high birthweight as a risk factor for insulin resistance,¹¹ obesity¹² and cancers such as leukemia and breast, prostate and colon cancers.¹³ Studies to determine the potential risk factors for mothers of large for gestational age (LGA) babies are ongoing¹⁰ and more research is needed to identify potential risk factors behind this developmental outcome.

The glucocorticoid receptor (GR), a known mediator of glucocorticoid signaling, has been shown to be regulated, in part, by epigenetic mechanisms, specifically DNA methylation.¹⁴^–¹⁹ Both rat and human studies have shown an association between methylation of the hippocampal GR gene and early postnatal outcomes.¹⁶ Early maternal care of rat pups leads to altered methylation status of the NGFI-A consensus binding site within the promoter of the GR gene in the hippocampus, and these changes have been linked to altered stress responses later in life, as well as to growth restriction in utero.¹⁴^,¹⁵^,²⁰^–²² Oberlander and colleagues suggested a potential association between methylation status of the analogous human NGFI-A binding site in promoter region 1F of the GR gene in infant cord blood and maternal mood.¹⁸^,²⁰

As birthweight is known to be associated with a variety of later life health outcomes and aberrant methylation of the GR gene has been linked with adverse developmental outcomes in animal models, we sought to investigate the associations between birthweight and GR promoter methylation in human placenta.

Results

We examined the variation in the extent of methylation of the GR gene promoter 1F region in a large series of human placenta samples obtained from term infants of various birthweights. Table 1 shows the demographics of the study population in total (n = 480) and by birthweight grouping: small for gestational age (SGA, <10th percentile of birthweight for gestational age, n = 102), appropriate for gestational age (AGA, 10–90th percentile of birthweight for gestational age, n = 343), and large for gestational age (LGA, >90th percentile of weight for gestational age, n = 35). Distributions of gestational age, infant gender, maternal race, prenatal vitamin use and maternal age were not significantly different across birthweight groupings. As expected, maternal tobacco use during pregnancy was more common amongst SGA infants, and LGA infants were more often delivered through Caesarean section. There was a low prevalence of recreational drug use, alcohol use and maternal gestational diabetes across all groups.

Table 1

Demographics of the study population

Figure 1 depicts the difference in the extent of methylation of the 13 CpG sites examined in the GR exon 1F region in SGA, AGA and LGA placentas. Differential methylation was noted across groups at all CpG sites in the GR exon 1F but only CpG site 7 and the mean across all CpG sites showed a significantly different extent of methylation across the groups following a Bonferroni correction for multiple comparisons (p < 0.0036).

Figure 1

Comparison of the mean extent of methylation of the 13 CpG sites in the GR exon 1F promoter region in SGA (n = 102), AGA (n = 343) and LGA (n = 35) placentas determined by bisulfite pyrosequencing. Error bars represent standard error. * indicates p < (more ...)

As these sites are in close proximity, and as methylation likely occurs throughout the region in a coordinated fashion, we examined the correlation between the extent of methylation at the individual CpG sites, using pairwise correlations for methylation extent between each of the 13 CpG sites (Fig. 2). Of note, there is a moderate to strong correlation in methylation between many of the 13 CpG sites. Moreover, the correlation in methylation between CpG sites appears to be a function of the distance between sites, consistent with the findings of Nautiyal and colleagues.²³ This relatively high degree of correlation suggests that use of the mean may be a reliable marker of the extent of methylation across this region.

Figure 2

Correlation matrix describing pair wise Pearson correlations of methylation status among the 13 CpG sites within the exon 1F promoter region of the GR in all placenta samples (n = 480). Numbers in parentheses indicate bp distance of specific GR Position (more ...)

Examination of the correlation of each of the covariates listed in Table 1 with log mean GR methylation extent was conducted (Sup. Table 1). This examination revealed that birthweight was significantly correlated with mean GR methylation extent (r = 0.16, p = 0.0004). None of the other demographic or clinical characteristics of the subjects was significantly correlated with GR methylation. To further investigate the association between birthweight category and mean GR methylation, and controlling for potential confounders, we performed multivariable linear regression (Table 2). Analysis revealed that in LGA infants, the log mean percent methylation of the GR increased significantly by 0.40 (p < 0.0001), controlled for gestational age, maternal age, maternal ethnicity, tobacco use during pregnancy, alcohol use during pregnancy, recreational drug use during pregnancy, prenatal vitamin use, delivery method, maternal gestational diabetes and infant gender. Being SGA was not a significant predictor of GR methylation extent, nor was any of the covariates included as confounders in the model, consistent with the univariate findings.

Table 2

Multivariable linear regression model of log-transformed mean GR methylation extent

To demonstrate the functional role of promoter methylation on GR gene expression, the placenta choriocarcinoma cell line JEG3 was treated with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-azaC). Pyrosequencing of bisulfite modified DNA from JEG-3 cells treated with 5-azaC showed decreased methylation with increasing doses of 5-azaC across all 13 CpG sites in the GR (Fig. 3A). The mean methylation across this region demonstrated an almost 20% decrease in cells treated with 1.25 µM 5-azaC, and a 50% decrease in methylation in cells treated with 2.5 µM 5-azaC, compared to cells treated with DMSO alone. GR gene expression, measured by qRT-PCR, also showed a significant (p < 0.004) dose-dependent increase with increasing exposure to 5-azaC (Fig. 3B). Our cell culture data suggests that JEG-3 cells do not express GR unless treated with 5-azaC, and increasing doses of 5-azaC resulted in a dose-dependent increase in GR gene expression.

Figure 3

Examination of the effects of 5-aza-2′deoxycytidine (5-azaC) on placenta choriocarcinoma cell line JEG-3. (A) Extent of methylation at CpG sites in GR exon 1F region examined by bisulfite pyrosequencing with increasing doses of 5-azaC. (B) Expression (more ...)

Discussion

Perturbations to placental gene expression may result in the altered metabolic and endocrine function of the placenta and may also affect the placenta's ability to transport water, gas, nutrients and waste products crucial for the proper growth and survival of the fetus.⁷ Previous findings have reported that DNA methylation marks are stable and accessible for measurement in placenta,²⁴ and that the GR gene is expressed in placenta.²⁵ Taken collectively, our data combined with this previous work suggests that DNA methylation of the GR gene promoter may be important in regulating GR gene expression, and this epigenetic alteration is linked to infant birthweight. Specifically, we have demonstrated that infants considered LGA have significantly higher GR methylation and that birthweight classification as LGA explains a great deal of the variability in methylation of this promoter region. While there is a difference in the percent methylation observed in the placental choriocarcinoma cell lines versus that in tissue, the levels of GR methylation that we observed in tissue are consistent with previous findings in other primary human tissues.¹⁸ As birthweight is a multifactorial outcome as well as a predictor of later life health and disease, this finding begins to provide a mechanistic link between the intrauterine environment and health throughout life.

Previous work has shown that altered methylation status of the NGF1-A consensus binding site within the rat exon 1–7 and analogous human exon 1F of the GR gene can lead to reduced NGF1-A binding, and reduced expression.¹⁴^,¹⁵^,¹⁹^,²⁶ Our work is an extension of the previous work focused on the 13 CpG sites in exon 1F,¹⁵^,¹⁸ driven by the hypothesis that the environment may be playing a critical role in determining the DNA methylation status of this gene regulatory region. The NGFI-A binding site occurs at CpG sites 3 and 4. Our results suggest elevated methylation at these sites in LGA compared to SGA and AGA infants, although the differences were not significant. Methylation at these sites in infant cord blood was previously associated with maternal mood and stress.¹⁸ Although we could not examine these associations in this population, our results suggest that the environment represented by birthweight may have greater impacts at other CpG sites in the 1F promoter than at the NGF1A sites. This difference may be related to differences in the environment influencing methylation, or may be related to differences in susceptibility of the tissues examined. Specific examinations of potential environmental influences on methylation of this region in placenta and other tissues are warranted to better explain this difference. There are also several alternative first exons of the GR involved in gene expression regulation in a tissue specific manner. Future studies aimed at exploring and more completely characterizing the complex regulatory mechanisms controlling GR gene expression are needed in the placenta and in other primary human tissues.

Our in vitro work in human placenta choriocarcinoma cells suggests that methylation of this region of the gene is also associated with reduced gene expression in this cell type, thereby suggesting a relationship between the methylation of this region in human placentas. Our cell culture data suggests that JEG-3 cells do not express GR unless treated with 5-azaC, and increasing doses of 5-azaC resulted in a dose-dependent increase in GR gene expression. Because 5-azaC acts as a non-specific DNA methyltransferase inhibitor, we cannot exclude the possibility that its effects were not directly related to changes in exon 1F, but these results are consistent with previous work performed in other cell types.²⁶ Future follow-up studies can be performed to more completely characterize the molecular mechanisms underlying control of GR gene expression.

Rich maternal forecasts represented by LGA status can prove to be incorrect if the child is born into a nutrient poor environment. Moreover, high birthweight, especially birthweights that would categorize babies as LGA, is associated with an increased risk for disease in both children and adults. Previous work has shown high birthweight as a risk factor for insulin resistance¹¹ and obesity,¹² as well as for a number of cancers,¹³ and it is clear that glucocorticoid signaling can play a role in these pathologic processes. While normal physiological levels of glucocorticoids and GR are essential for metabolic control, altered glucocorticoid action has also been shown to be associated with a variety of metabolic diseases such as obesity and type 2 diabetes.²⁷ Altered methylation of the GR in the placenta and subsequent dysregulated expression of the GR (and thereby altered glucocorticoid signaling) may lead to specific adult phenotypes, such as glucocorticoid resistance, as well as to increased risks of metabolic disorders, such as Type 2 diabetes and obesity.²⁸^–³⁰ To date, associations between placental GR methylation and adult metabolic outcomes have yet to be elucidated. Ke and colleagues demonstrated an increase in expression of some forms of GR in the hippocampus of IUGR rats.¹⁷ Our data suggest an increase in DNA methylation of the exon 1F of the GR associated with LGA status, which would be consistent with the findings of Ke and colleagues. More research is needed to identify risk factors that may lead to the developmental outcome of high birthweight, as well as further elucidation of the role of the glucocorticoid pathways and other critical molecular mediators leading to this phenotype and the associated later onset disease risk including metabolic disorders.

In summary, our study is one of the first to show an association between the methylation status of the promoter of the GR gene in human placenta and LGA status. This may serve as an early epigenetic marker of maternal exposures and may be involved in the pathway leading to adverse health outcomes associated with increased birthweight. More work is necessary to further elucidate the mechanisms involved in this complex and multifactorial pathway and will be crucial for better understanding the developmental origins of health and disease.

Materials and Methods

Placenta samples.

480 placenta samples were collected at Women and Infants Hospital in Providence, Rhode Island, in accordance with protocols approved by the Institutional Review Boards of both Women and Infants Hospital and Brown University. Samples were collected from women in good physical health between the ages of 18–42, whose infants were at term and viable with no known genetic disorders. An approximately 1 g biopsy of placenta was excised, free of maternal decidua, from the maternal side of the placenta 2 cm from the umbilical cord insertion site within 4 h of delivery, using a protocol designed to reduce potential sample degradation. The sample was immediately placed in RNAlater (Applied Biosystems, Inc.,) and stored at 4°C. At least 72 h later, placenta samples were removed from the RNAlater, blotted dry and stored at −80°C until further analysis. Medical information, such as infant's birth weight, length, gender, mode of delivery, gestational age and maternal demographics was recorded from patient medical charts using a structured chart review. Birthweight percentiles were calculated using the method of Fenton (2003).³¹

DNA extraction and modification.

DNA was extracted from the placenta samples using the QIAmp DNA Mini Kit (Qiagen, Inc.,) following manufacturer's protocols. Purified DNA was quantified using a ND-1000 spectrophotometer (Nanodrop, Wilmington, DE), and DNA samples (1 µg) were bisulfite-modified using the EZ DNA Methylation Kit (Zymo Research, CA) and stored at −20°C.

Bisulfite pyrosequencing DNA methylation analysis.

Pyrosequencing was performed on PCR product amplified from bisulfite-modified DNA as described previously in reference ¹⁸. Bisulfite conversion was performed using individual columns using the EZ DNA Methylation Kit (Zymo Research) and manufacturer's protocol. In order for a sample's methylation extent to be called, it must exhibit at least a 93% bisulfite conversion rate, as assessed by pyrosequencing, and all samples examined exhibited a rate >95%. To prevent batch effects from bisulfite treatments interfering with the analysis, samples were randomized across batches. 10% of the samples were repeated independently and the R² for repeats is 0.98. The region of interest, exon 1F of the human GR (NR3C1) gene, has 13 CpG sites, and is considered the human homologue of the rat NR3C1 gene, exon 1–7 previously shown to have differential methylation in response to maternal exposures.¹⁹^,³²

In brief, HotStar Taq DNA Polymerase (Qiagen) and the following forward and biotinylated reverse primers were used: PMHumGCCRF, 5′-TTT TTT TTT TGA AGT TTT TTT A-3′ and PMHumGCCRR, 5′-CCC CCA ACT CCC CAA AAA-3′ (IDT Inc., Coralville, IA). Cycling conditions were 94°C for 15 min followed by 50 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 1 min with a final extension of 10 min at 72°C. PCR products were sequenced using a PyroMark MD system and the two following sequencing primers (IDT): PMHumGCCRS2, 5′-GAG TGG GTT TGG AGT-3′ and PMHumGCCRS3, 5′-AGA AAA GAA TTG GAG AAA TT-3′. The first sequencing primer was designed to sequence the first five CpG sites, and the second sequencing primer was designed to sequence the following eight CpG sites for a total of thirteen sites sequenced. The dispensation orders for the two assays were GTC TGT CGA GTA GTC GGT CGA GAG CTA TGT CGA G for the first assay and ATC GTG TTG ATC TGT CGC TTA GAG AGA CTA TGT CAG TTC TGT CGT AGT CTG TCG TA for the second. The percent methylation at each CpG site was quantified using the Pyro Q-CpG software, version 1.0.9 (Qiagen). A >99% success rate of the pyrosequencing reaction was observed for the samples examined.

RNA extraction.

RNA was isolated from cultured cells using the miRvana miRNA Isolation Kit (Ambion, Inc., Austin, TX) following the manufacturer protocol. Extracted RNA was quantified using a ND-1000 spectrophotometer and stored at −80°C.

Quantitative RT-PCR (qRT-PCR).

Gene expression was measured using commercially available TaqMan Gene Expression Assays (Applied Biosystems, Valencia, CA) on an Applied Biosystems 7500 Real Time PCR system and analyzed with 7500 System Software. All reactions were run in triplicate, with GAPDH serving as a referent. In addition, a no-RT control was run with each plate.

Cell culture.

The human choriocarcinoma cell line JEG-3 was cultured in Eagle's minimal essential medium (MEM) (ATTC, Manassas, VA), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. JEG-3 cells were treated with the inhibitor of DNA methylation, 5-aza-2′deoxycytidine (Sigma-Aldrich), using doses of 1.25 µM and 2.5 µM in DMSO for seven days, based on previously published studies in reference ³³. Media containing DMSO vehicle only was used as the mock treatment. All experimental and mock exposures were done in biological triplicate.

Statistical analysis.

Differences in covariates between birthweight groups (SGA, AGA and LGA) were assessed by ANOVA for continuous variables and chi-square test for categorical variables. Pearson correlation coefficients and corresponding p-values were used to examine the correlation of each of the covariates with log GR mean methylation extent. Univariate associations between birthweight status and methylation extent were assessed using the nonparametric Kruskal-Wallis test, with Bonferroni correction to control for multiple comparisons. Controlling for potential confounders, multivariable linear regression modeling was used to examine the association between DNA methylation (log-transformed to fit the normality assumptions of the model) and birthweight. Covariates were added to the model because of their potential as confounders of birthweight. Two-tailed t-tests were used to analyze gene expression of 5-aza-2′deoxycytidine exposed cells compared to control cells. Data were analyzed by SAS 9.1 and R.

Acknowledgements

Many thanks to Gilda Ferro, Joyce Lee and Keila Veiga for placenta sample and medical history collection and processing.

Financial Support

This work was supported by NIH grants from the COBRE for Perinatal Biology P20 RR018728.

Supplementary Material

Supplementary Material

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