Cynthia Shope

Maternal tobacco use modestly alters correlated epigenome-wide placental DNA methylation and gene expression

Several studies linking alterations in differential placental methylation with pregnancy disorders have implicated (de)regulation of the placental epigenome with fetal programming and later-in-life disease. We have previously demonstrated that maternal tobacco use is associated with alterations in promoter methylation of placental CYP1A1 and that these changes are correlated with CYP1A1 gene expression and fetal growth restriction. In this study we sought to expand our analysis of promoter methylation by correlating it to gene expression on a genome-wide scale. Employing side-by-side IlluminaHG-12 gene transcription with Infinium27K methylation arrays, we interrogated correlative changes in placental gene expression and DNA methylation associated with maternal tobacco smoke exposure at an epigenome-wide level and in consideration of signature gene pathways. We observed that the expression of 623 genes and the methylation of 1024 CpG dinucleotides are significantly altered among smokers, with only 38 CpGs showing significant differential methylation (differing by a methylation level of ≥ 10%). We identified a significant Pearson correlation (≥ 0.7 or ≤ -0.7) between placental transcriptional regulation and differential CpG methylation in only 25 genes among non-smokers but in 438 genes among smokers (18-fold increase, p < 0.0001), with a dominant effect among oxidative stress pathways. Differential methylation at as few as 6 sites was attributed to maternal smoking-mediated birth weight reduction in linear regression models with Bonferroni correction (p < 1.8x10-6). These studies suggest that a common perinatal exposure (such as maternal smoking) deregulates placental methylation in a CpG site-specific manner that correlates with meaningful alterations in gene expression along signature pathways.

IN UTERO TOBACCO EXPOSURE EPIGENETICALLY MODIFIES PLACENTAL CYP1A1 EXPRESSION

The metabolic pathways used by higher-eukaryotic organisms to deal with potentially carcinogenic xenobiotic compounds from tobacco smoke have been well characterized. Carcinogenic compounds such as polycyclic aromatic hydrocarbons are metabolized sequentially in 2 phases: in phase I, CYP1A1 catalyzes conversion into harmful hydrophilic DNA adducts, whereas in phase II, GSTT1 enables excretion via conjugation into polar electrophiles. In an effort to understand susceptibility to in utero tobacco exposure, we previously characterized known metabolic functional polymorphisms and demonstrated that although deletion of fetal GSTT1 significantly modified birth weight in smokers, no polymorphism fully accounted for fetal growth restriction. Because smoking up-regulates CYP1A1 expression, we hypothesized that nonallelic (epigenetic) dysregulation of placental CYP1A1 expression via alterations in DNA methylation (meCpG) may further modify fetal growth. In the present article, we compared placental expression of multiple CYP family members among gravidae and observed significantly increased CYP1A1 expression among smokers relative to controls (4.4-fold, P < .05). To fully characterize CYP1A1 meCpG status, bisulfite modification and sequencing of the entire proximal 1-kilobase promoter (containing 59 CpG sites) were performed. CpG sites immediately proximal to the 5′-xenobiotic response element transcription factor binding element were significantly hypomethylated among smokers (55.6% vs 45.9% meCpG, P = .027), a finding that uniquely correlated with placental gene expression (r = 0.737, P = .007). Thus, in utero tobacco exposure significantly increases placental CYP1A1 expression in association with differential methylation at a critical xenobiotic response element.

Epigenomics: maternal high-fat diet exposure in utero disrupts peripheral circadian gene expression in nonhuman primates

The effect of in utero exposure to a maternal high‐fat diet on the peripheral circadian system of the fetus is unknown. Using mRNA copy number analysis, we report that the components of the peripheral circadian machinery are transcribed in the nonhuman primate fetal liver in an intact phase‐antiphase fashion and that Npas2, a paralog of the Clock transcription factor, serves as the rate‐limiting transcript by virtue of its relative low abundance (10‐ to 1000‐fold lower). We show that exposure to a maternal high‐fat diet in utero significantly alters the expression of fetal hepatic Npas2 (up to 7.1‐fold, P<0.001) compared with that in control diet‐exposed animals and is reversible in fetal offspring from obese dams reversed to a control diet (1.3‐fold, P>0.05). Although the Npas2 promoter remains largely unmethylated, differential Npas2 promoter occupancy of acetylation of fetal his‐tone H3 at lysine 14 (H3K14ac) occurs in response to maternal high‐fat diet exposure compared with control diet‐exposed animals. Furthermore, we find that disruption of Npas2 is consistent with high‐fat diet exposure in juvenile animals, regardless of in utero diet exposure. In summary, the data suggest that peripheral Npas2 expression is uniquely vulnerable to diet exposure.—Suter, M., Bocock, P., Showalter, L., Hu, M., Shope, C., McKnight, R., Grove, K., Lane, R., Aagaard‐Tillery, K. Epigenomics: maternal high‐fat diet exposure in utero disrupts peripheral circadian gene expression in nonhuman primates. FASEB J. 25, 714–726 (2011). www.fasebj.org

Transcriptome profiling of microRNA by Next-Gen deep sequencing reveals known and novel miRNA species in the lipid fraction of human breast milk.

While breast milk has unique health advantages for infants, the mechanisms by which it regulates the physiology of newborns are incompletely understood. miRNAs have been described as functioning transcellularly, and have been previously isolated in cell-free and exosomal form from bodily liquids (serum, saliva, urine) and tissues, including mammary tissue. We hypothesized that breast milk in general, and milk fat globules in particular, contain significant numbers of known and limited novel miRNA species detectable with massively parallel sequencing. Extracted RNA from lactating mothers before and following short-term treatment with recombinant human growth hormone (rhGH) was smRNA-enriched. smRNA-Seq was performed to generate 124,110,646 36-nt reads. Of these, 31,102,927 (25%) exactly matched known human miRNAs; with relaxing of stringency, 74,716,151 (60%) matched known miRNAs including 308 of the 1018 (29%) mature miRNAs (miRBase 16.0). These miRNAs are predicted to target 9074 genes; the 10 most abundant of these predicted to target 2691 genes with enrichment for transcriptional regulation of metabolic and immune responses. We identified 21 putative novel miRNAs, of which 12 were confirmed in a large validation set that included cohorts of lactating women consuming enriched diets. Of particular interest, we observed that expression of several novel miRNAs were altered by the perturbed maternal diet, notably following a high-fat intake (p<0.05). Our findings suggest that known and novel miRNAs are enriched in breast milk fat globules, and expression of several novel miRNA species is regulated by maternal diet. Based on robust pathway mapping, our data supports the notion that these maternally secreted miRNAs (stable in the milk fat globules) play a regulatory role in the infant and account in part for the health benefits of breast milk. We further speculate that regulation of these miRNA by a high fat maternal diet enables modulation of fetal metabolism to accommodate significant dietary challenges.

Dyslipidemia and Auditory Function

Abstract: The relationship between dyslipidemia and hearing is unclear. This study was conducted to investigate whether elevated serum lipid levels impact auditory function in humans and in guinea pigs. In the human study, a cross-sectional study of 40 volunteers with dyslipidemia was conducted. Pure tone thresholds, distortion product otoacoustic emissions, and lipid profiles were analyzed. When controlled for patient age and sex, we found that elevated triglycerides were associated with reduced hearing. In the guinea pig study, a prospective study of animals fed a high-fat diet for 14 weeks was conducted. Although the high-fat diet led to a dramatic elevation in the average weight and total cholesterol in all animals (from 61 to 589 mg/dl), there were no meaningful changes in distortion product otoacoustic emission magnitudes. These results suggest that whereas chronic dyslipidemia associated with elevated triglycerides may reduce auditory function, short-term dietary changes may not.

Maternal High-Fat Diet Modulates the Fetal Thyroid Axis and Thyroid Gene Expression in a Nonhuman Primate Model

Thyroid hormone (TH) is an essential regulator of both fetal development and energy homeostasis. Although the association between subclinical hypothyroidism and obesity has been well studied, a causal relationship has yet to be established. Using our well-characterized nonhuman primate model of excess nutrition, we sought to investigate whether maternal high-fat diet (HFD)-induced changes in TH homeostasis may underlie later in life development of metabolic disorders and obesity. Here, we show that in utero exposure to a maternal HFD is associated with alterations of the fetal thyroid axis. At the beginning of the third trimester, fetal free T(4) levels are significantly decreased with HFD exposure compared with those of control diet-exposed offspring. Furthermore, transcription of the deiodinase, iodothyronine (DIO) genes, which help maintain thyroid homeostasis, are significantly (P < 0.05) disrupted in the fetal liver, thyroid, and hypothalamus. Genes involved in TH production are decreased (TRH, TSHR, TG, TPO, and SLC5A5) in hypothalamus and thyroid gland. In experiments designed to investigate the molecular underpinnings of these observations, we observe that the TH nuclear receptors and their downstream regulators are disrupted with maternal HFD exposure. In fetal liver, the expression of TH receptor β (THRB) is increased 1.9-fold (P = 0.012). Thorough analysis of the THRB promoter reveals a maternal diet-induced alteration in the fetal THRB histone code, alongside differential promoter occupancy of corepressors and coactivators. We speculate that maternal HFD exposure in utero may set the stage for later in life obesity through epigenomic modifications to the histone code, which modulates the fetal thyroid axis.

MATERNAL TOBACCO USE IS ASSOCIATED WITH INCREASED MARKERS OF OXIDATIVE STRESS IN THE PLACENTA

OBJECTIVE We sought to extend our prior observations and histopathologically characterize key metabolic enzymes (CYP1A1) with markers of oxidative damage in the placental sections from smokers. STUDY DESIGN Placental specimens were collected from term singleton deliveries from smokers (n = 10) and nonsmokers (n = 10) and subjected to a detailed histopathological examination. To quantify the extent of oxidative damage, masked score-graded (0-6) histopathology against 4-hydroxy-2-nonenal (4-HNE) and 8-hydroxydeoxyguanisine (8-OHdG) was performed. Minimal significance (P < .05) was determined with a Fishers exact and a 2-tailed Student t test as appropriate. RESULTS We observed a significant increase in the presence of syncytial knots in placentas from smokers (70% vs 10%, P = .02). These gross observations were accompanied by a significant aberrant placental aromatic hydrocarbon metabolism (increased CYP1A1, 4.4 vs 2.1, P = .002) in addition to evidence of oxidative damage (4-HNE 3.4 vs 1.1, P = .00005; 8-OHdG 4.9 vs 3.1, P = .0038). CONCLUSION We observed a strong association between maternal tobacco use and aberrant placental metabolism, syncytial knot formation, and multiple markers of oxidative damage.

Administration of antenatal glucocorticoids and postnatal surfactant ameliorates respiratory distress syndrome–associated neonatal lethality in Erk3 −/− mouse pups

Background:Respiratory distress syndrome (RDS) persists as a prevalent cause of infant morbidity and mortality. We have previously demonstrated that deletion of Erk3 results in pulmonary immaturity and neonatal lethality. Using RNA sequencing, we identified corticotrophin releasing hormone (CRH) and surfactant protein B (SFTPB) as potential molecular mediators of Erk3-dependent lung maturation. In this study, we characterized the impact of antenatal glucocorticoids and postnatal surfactant on neonatal survival of Erk3 null mice.Methods:In a double crossover design, we administered dexamethasone (dex) or saline to pregnant dams during the saccular stage of lung development, followed by postnatal surfactant or saline via inhalation intubation. Survival was recorded, and detailed lung histological analysis and staining for CRH and SFTPB protein expression were performed.Results:Without treatment, Erk3 null pups die within 6 h of birth with reduced aerated space, impaired thinning of the alveolar septa, and abundant glycogen stores, as described in human RDS. The administration of dex and surfactant improved RDS-associated lethality of Erk3−/− pups and partially restored functional fetal lung maturation by accelerating the downregulation of pulmonary CRH and partially rescuing the production of SFTPB.Conclusion:These findings emphasize that Erk3 is integral to terminal differentiation of type II cells, SFTPB production, and fetal pulmonary maturity.

From the Field to the Laboratory: Air Pollutant-Induced Genomic Effects in Lung Cells

Current in vitro studies do not typically assess cellular impacts in relation to real-world atmospheric mixtures of gases. In this study, we set out to examine the feasibility of measuring biological responses at the level of gene expression in human lung cells upon direct exposures to air in the field. This study describes the successful deployment of lung cells in the heavily industrialized Houston Ship Channel. By examining messenger RNA (mRNA) levels from exposed lung cells, we identified changes in genes that play a role as inflammatory responders in the cell. The results show anticipated responses from negative and positive controls, confirming the integrity of the experimental protocol and the successful deployment of the in vitro instrument. Furthermore, exposures to ambient conditions displayed robust changes in gene expression. These results demonstrate a methodology that can produce gas-phase toxicity data in the field.

Conditional postnatal deletion of the neonatal murine hepatic circadian gene, Npas2, alters the gut microbiome following restricted feeding

BACKGROUND: We have recently shown in both non‐human primates and in rodents that fetal and neonatal hepatic expression of the circadian transcription factor, Npas2, is modulated by a high fat maternal diet and plays a critical role in establishing life‐long metabolic homeostasis. Similarly, we and others have also established the importance of the maternal and early postnatal diet on establishment of the early gut microbiome. OBJECTIVE: We hypothesized that altered circadian gene expression solely in the neonatal liver would result in gut microbiome dysbiosis, especially with diet‐induced metabolic stress (ie, restricted feeding). Using a murine model in which we conditionally knock out Npas2 in the neonatal liver, we aimed to determine the role of the circadian machinery in gut dysbiosis with restricted feeding. STUDY DESIGN: We collected fecal samples from liver Npas2 conditional knockout (n = 11) and wild‐type (n = 13) reproductive‐aged mice before (study day 0) and after the restricted feeding study (study day 17). Extracted DNA was sequenced using the MiSeq Illumina platform using primers specific for the V4 region of the 16S ribosomal DNA gene. The resulting sequences were quality filtered, aligned, and assigned taxonomy. Principal coordinate analysis was performed on unweighted and weighted UniFrac distances between samples with a permutation analysis of variance to assess clustering significance between groups. Microbial taxa that significantly differ between groups of interest was determined using linear discriminate analysis effect size and randomForrest. RESULTS: Principal coordinate analysis performed on weighted UniFrac distances between male conditional knockout and wild‐type cohorts revealed that the gut microbiome of the mice did not differ by genotype at the start of the restricted feeding study but did differ by virtue of genotype at the end of the study (P = .001). Moreover, these differences could be at least partially attributed to restricted feeding–associated alterations in relative abundance of the Bacteroides genus, which has been implicated as crucial to establishing a healthy gut microbiome early in development. CONCLUSION: Here we have provided an initial key insight into the interplay between neonatal establishment of the peripheral circadian clock in the liver and the ability of the gut microbiome to respond to dietary and metabolic stress. Because Npas2 expression in the liver is a target of maternal high‐fat diet–induced metabolic perturbations during fetal development, we speculate that these findings have potential implications in the long‐term metabolic health of their offspring.