Strategy for the generation and analysis of RNA from F1 hybrid mice.

<p>F1 hybrid progeny were generated from reciprocal matings between C57BL/6J and <i>Mus musculus castaneus</i> mice. Tissues were isolated from fetal and adult brain of the F1 hybrid mice and mRNA-seq was performed. Using SNPs (asterisks) that were identified in the parental DNA, biases in transcription of parental alleles can be assessed.</p

BPA exposure altered total mRNA expression of imprinted genes relative to reference genes.

<p>Samples from control, lower dose and upper dose exposure groups were analyzed for total expression of the imprinted (A) <i>Snrpn</i>, (B) <i>Igf2</i>, (C) <i>Kcnq1ot1</i>, (D) <i>Cdkn1c</i> and (E and F) <i>Ube3a</i> genes with sample sizes (n) indicated. Assayed samples included embryos (<i>Igf2</i>), placentas (<i>Snrpn, Kcnq1ot1, Cdkn1c</i>, and <i>Ube3a</i>) or brains (<i>Ube3a</i>) showing normal, monoallelic (black circle) or biallelic expression (red circle). Average total expression in each exposure group is indicated with a black horizontal line; a = P<0.05; b = P<0.01 and c = P<0.001.</p

Bisphenol A Exposure Disrupts Genomic Imprinting in the Mouse

<div><p>Exposure to endocrine disruptors is associated with developmental defects. One compound of concern, to which humans are widely exposed, is bisphenol A (BPA). In model organisms, BPA exposure is linked to metabolic disorders, infertility, cancer, and behavior anomalies. Recently, BPA exposure has been linked to DNA methylation changes, indicating that epigenetic mechanisms may be relevant. We investigated effects of exposure on genomic imprinting in the mouse as imprinted genes are regulated by differential DNA methylation and aberrant imprinting disrupts fetal, placental, and postnatal development. Through allele-specific and quantitative real-time PCR analysis, we demonstrated that maternal BPA exposure during late stages of oocyte development and early stages of embryonic development significantly disrupted imprinted gene expression in embryonic day (E) 9.5 and 12.5 embryos and placentas. The affected genes included <i>Snrpn, Ube3a, Igf2, Kcnq1ot1, Cdkn1c</i>, and <i>Ascl2</i>; mutations and aberrant regulation of these genes are associated with imprinting disorders in humans. Furthermore, the majority of affected genes were expressed abnormally in the placenta. DNA methylation studies showed that BPA exposure significantly altered the methylation levels of differentially methylated regions (DMRs) including the <i>Snrpn</i> imprinting control region (ICR) and <i>Igf2</i> DMR1. Moreover, exposure significantly reduced genome-wide methylation levels in the placenta, but not the embryo. Histological and immunohistochemical examinations revealed that these epigenetic defects were associated with abnormal placental development. In contrast to this early exposure paradigm, exposure outside of the epigenetic reprogramming window did not cause significant imprinting perturbations. Our data suggest that early exposure to common environmental compounds has the potential to disrupt fetal and postnatal health through epigenetic changes in the embryo and abnormal development of the placenta.</p> </div

Impact of BPA exposure is shown as number of genes exhibiting imprinting perturbations.

<p>Effects of BPA exposure were more significant in the (B) placentas compared to (A) embryos. For each exposure group (control [blue], lower dose [pink] or upper dose [red]), percentage of tissues showing LOI (Y-axis) of at least 1 gene (≥1), 2 genes (≥2) or 3 genes (≥3) is indicated; a = P<0.05; b = P<0.01; c = P<0.001.</p

Representative LC-SRM/MS chromatograms demonstrating significantly higher BPA level in serum from treated mice.

<p>Top panels represent the chromatograms for unlabeled BPA and lower panels labeled BPA ([¹³C₁₂]-BPA) spiked prior to sample extraction to quantify BPA levels in serum from representative control (B) and upper dose treated mice (C). Blank sample used as control for storage and extraction is indicated in (A). Y-axis represents relative abundance of signal intensity and X-axis retention time in minute. Differences in BPA concentrations were determined based on peak heights (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003401#s4" target="_blank">Materials and Methods</a>). NL = normalized levels of intensity; <i>m/z = </i>mass to charge ratio.</p

Allele-specific expression studies showed increased proportion of tissues with loss of imprinting following BPA exposure.

<p>(A–D) Each black circle represents an individual E9.5 embryo (<i>Igf2</i>) or placenta (<i>Snrpn</i>, <i>Kcnq1ot1</i> and <i>Ascl2</i>). Y-axis indicates percentage of total mRNA expression derived from the repressed allele. Loss of imprinting (LOI) or biallelic expression was called when the repressed allele exhibited ≥10% of total expression. Data from control (n = 23), lower dose (n = 22) and upper dose (n = 28) exposure groups are shown for (A) <i>Snrpn</i>, (B) <i>Igf2</i>, (C) <i>Kcnq1ot1</i> and (D) <i>Ascl2</i>; a = P<0.05; b = P<0.01 and c = P<0.001. Number of tissues (out of total examined) showing LOI is indicated below each exposure group. In (E), percentages of placentas (<i>Snrpn and Kcnq1ot1</i>) and embryos (<i>Igf2</i>) showing LOI at E9.5 and E12.5 are compared.</p

Map of mouse chromosome 7 showing imprinted domains analyzed in the current work is illustrated.

<p>Black = paternally expressed genes; dark grey = maternally expressed genes; and light grey = differentially methylated regions (DMRs). Arrows indicated direction of gene transcription. C = centromere and T = telomere. Not drawn to scale.</p

BPA exposure altered DNA methylation at the <i>Igf2</i> DMR1 and <i>H19/Igf2</i> ICR in the embryos.

<p>A. Pyrosequencing assays tested methylation of 4 and 6 CpGs (highlighted in bold) in 457 bp and 221 bp genomic regions at the <i>Igf2</i> DMR1 and <i>H19/Igf2</i> ICR, respectively. Methylation at the (B) <i>Igf2</i> DMR1 and (C) <i>H19/Igf2</i> ICR was analyzed in embryos from controls, lower dose and upper dose exposure groups. Analysis in the upper dose exposure group included embryos that showed monoallelic (black circles) or biallelic (red circles) expression of the <i>Igf2</i> gene. Sample sizes analyzed in each exposure group are indicated with a = P<0.05 and b = P<0.01. Black horizontal line indicates average methylation level in each exposure group.</p

BPA exposure reduced genome-wide DNA methylation in the placenta but not the embryo.

<p>Results of LUMA studies showed genome-wide DNA methylation levels in the (A) embryos and (B) placentas from control, lower dose and upper dose BPA exposure groups. Black circles indicate tissues with normal, monoallelic expression of all imprinted genes tested; red circles are tissues that showed LOI of at least 1 imprinted gene. Black horizontal lines represent average total methylation in each exposure group; a = P<0.05.</p

Differential Methylation of Genes Associated with Cell Adhesion in Preeclamptic Placentas

<div><p>Preeclampsia (PE), a hypertensive disorder of pregnancy, is hypothesized to be associated with, if not mechanistically related to abnormal placental function. However, the exact mechanisms regulating the pathogenesis of PE remain unclear. While many studies have investigated changes in gene expression in the PE placenta, the role of epigenetics in PE associated placental dysfunction remains unclear. Using the genome-wide Illumina Infinium Methylation 450 BeadChip array, we analyzed gene-specific alterations in DNA methylation in placental biopsies collected from normal pregnant women delivering at term (n = 14), with term PE (≥37 weeks; n = 19) or with preterm PE (<37 weeks, n = 12). Of the 485,582 gene loci on the array, compared to controls, 229 loci were differentially methylated in PE placentas and 3411 loci were differentially methylated in preterm PE (step up p-value <0.05 and >5% methylation difference). Functional annotation of the differentially methylated genes in preterm PE placentas revealed a 32 gene cluster in the cadherin and cell adhesion functional groups (Benjamini p<0.00001). Hypermethylation of CDH11 (p = 0.0143), COL5A1 (p = 0.0127) and TNF (p = 0.0098) and hypomethylation of NCAM1 (p = 0.0158) was associated with altered mRNA expression in preterm PE placentas. Demethylation of first trimester extravillous trophoblast cells resulted in altered CDH11 (p = 0.0087), COL5A1 (p = 0.0043), NCAM1 (p = 0.0260) and TNF (p = 0.0022) mRNA expression. These studies demonstrate aberrant methylation, correlating with disease severity, in PE placentas. Furthermore, we provide evidence that disruption of gene-specific methylation in preterm PE placentas and first trimester trophoblasts is significantly associated with altered gene expression demonstrating that epigenetic modifications early in pregnancy can have effects on trophoblast function contributing to PE.</p></div