Robert A. McKnight

Developmental origins of disease and determinants of chromatin structure: maternal diet modifies the primate fetal epigenome

Chromatin structure is epigenetically altered via covalent modifications of histones to allow for heritable gene regulation without altering the nucleotide sequence. Multiple lines of evidence from rodents have established a role for epigenetic remodeling in regulating gene transcription in response to an altered gestational milieu. However, to date, it is unknown whether variations in the intrauterine environment in primates similarly induce changes in key determinants of hepatic chromatin structure. We hypothesized that a maternal high-fat diet would alter the epigenomic profile of the developing offspring, which would result in alterations in fetal gene expression. Age- and weight-matched adult female Japanese macaques were placed on control (13% fat) or high-fat (35% fat) breeder diets and mated annually over a 4-year interval. Fetuses in successive years were delivered near term (e130 of 167 days) and underwent necropsy with tissue harvest. Fetal histones were acid extracted for characterization of H3 modification and chromatin immunoprecipitation (ChIP) with differential display PCR; fetal RNA, DNA, and cytoplasmic and nuclear protein extracts were similarly extracted for comparison. Chronic consumption of a maternal high-fat diet results in a threefold increase in fetal liver triglycerides and histologic correlates of non-alcoholic fatty liver disease. These gross changes in the fetal liver are accompanied by a statistically significant hyperacetylation of fetal hepatic tissue at H3K14 (199.85+/-9.64 vs 88.8+/-45.4; P=0.038) with a trend towards the increased acetylation at H3K9 (140.9+/-38.7 vs 46.6+/-6.53; P=0.097) and at H3K18 (69.0+/-3.54 vs 58.0+/-4.04; P=0.096). However, epigenetic modifications on fetal hepatic H3 associated with gene repression were absent or subtle (P>0.05). Subsequent characterization of key epigenetic determinants associated with H3 acetylation marks revealed similar significant alterations in association with a high-fat maternal diet (e.g., relative fetal histone deacetylase 1 (HDAC1) gene expression 0.61+/-0.25; P=0.011). Consistent with our mRNA expression profile, fetal nuclear extracts from offspring of high-fat diet animals were observed to be significantly relatively deplete of HDAC1 protein (36.07+/-6.73 vs 83.18+/-7.51; P=0.006) and in vitro HDAC functional activity (0.252+/-0.03 vs 0.698+/-0.02; P<0.001). We employ these observations in ChIP differential display PCR to attempt to identify potential fetal genes whose expression is reprogramed under conditions of a high-fat maternal diet. We quantitatively confirm a minimum of a 40% alteration in the expression of several genes of interest: glutamic pyruvate transaminase (alanine aminotransferase) 2 (GPT2) (1.59+/-0.23-fold; P=0.08), DNAJA2 (1.36+/-0.21; P=0.09), and Rdh12 (1.88+/-0.15; P=0.01) are appreciably increased in fetal hepatic tissue from maternal caloric-dense diet animals when compared with control while Npas2, a peripheral circadian regulator, was significantly downmodulated in the offspring of high-fat diet animals (0.66+/-0.08; P=0.03). In this study, we show that a current significant in utero exposure (caloric-dense high-fat maternal diet) induces site-specific alterations in fetal hepatic H3 acetylation. Employing ChIP, we extend these observations to link modifications of H3 acetylation with alterations in gene-specific expression. These results suggest that a caloric-dense maternal diet leading to obesity epigenetically alters fetal chromatin structure in primates via covalent modifications of histones and hence lends a molecular basis to the fetal origins of adult disease hypothesis.

Epigenetics: intrauterine growth retardation (IUGR) modifies the histone code along the rat hepatic IGF-1 gene

Intrauterine growth restriction (IUGR) decreases serum insulin growth factor‐1 (IGF‐1) levels. IGF‐1 is an epigenetically regulated gene that has two promoters, alternative exon 5 splicing, and multiple termination sites. The regulation of gene expression involves the whole gene, as evidenced by the aforementioned IGF‐1 paradigm. We hypothesized that IUGR in the rat would affect hepatic IGF‐1 expression and alter the epigenetic characteristics of the IGF‐1 gene along its length. IUGR was induced through a bilateral uterine artery ligation of the pregnant rat, a well‐characterized model of IUGR. Pups from anesthesia and shamoperated dams were used as controls. Real‐time RT‐ PCR and ELISA was used to measure expression at day of life (DOL) 0 and 21. Bisulfite sequencing and chromatin immunoprecipitation (ChIP) quantified IGF‐1 epigenetic characteristics. A nontranscribed intergenic control was used for ChIP studies. IUGR decreased hepatic and serum IGF‐1. Concurrently, IUGR modified epigenetic characteristics, particularly the histone code, along the length of the hepatic IGF‐1 gene. Many changes persisted postnatally, and the postnatal effect of IUGR on the histone code was gender‐specific. We conclude that IUGR modifies epigenetic characteristics of the rat hepatic IGF‐1 gene along the length of the whole gene.— Fu, Q.,Yu, X., Callaway, C. W., Lane, R. H., McKnight, R. A. Epigenetics: intrauterine growth retardation (IUGR) modifies the histone code along the rat hepatic IGF‐1 gene. FASEBJ. 23, 2438–2449 (2009)

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

Epigenetics of programmed obesity: alteration in IUGR rat hepatic IGF1 mRNA expression and histone structure in rapid vs. delayed postnatal catch-up growth

Maternal food restriction (FR) during pregnancy results in intrauterine growth-restricted (IUGR) offspring that show rapid catch-up growth and develop metabolic syndrome and adult obesity. However, continued nutrient restriction during nursing delays catch-up growth and prevents development of obesity. Epigenetic regulation of IGF1, which modulates growth and is synthesized and secreted by the liver, may play a role in the development of these morbidities. Control (AdLib) pregnant rats received ad libitum food through gestation and lactation, and FR dams were exposed to 50% food restriction from days 10 to 21. FR pups were nursed by either ad libitum-fed control dams (FR/AdLib) or FR dams (FR/FR). All pups were weaned to ad libitum feed. Maternal FR resulted in IUGR newborns with significantly lower liver weight and, with the use of chromatin immunoprecipitation, decreased dimethylation at H3K4 in the IGF1 region was observed. Obese adult FR/AdLib males had decreased dimethylation and increased trimethylation of H3K4 in the IGF1 region. This corresponded to an increase in mRNA expression of IGF1-A (134 ± 5%), IGF1-B (165 ± 6%), IGF1 exon 1 (149 ± 6%), and IGF1 exon 2 (146 ± 7%) in the FR/AdLib compared with the AdLib/AdLib control group. In contrast, nonobese FR/FR had significantly higher IGF1-B mRNA levels (147 ± 19%) than controls with no difference in IGF1-A, exon 1 or exon 2. Modulation of the rate of IUGR newborn catch-up growth may thus protect against IGF1 epigenetic modifications and, consequently, obesity and associated metabolic abnormalities.

Growth retardation alters the epigenetic characteristics of hepatic dual specificity phosphatase 5

ABSTRACT Uteroplacental insufficiency leads to intrauterine growth retardation (IUGR) and adult onset insulin resistance in both humans and rats. IUGR rat liver is characterized by persistent changes in histone 3 lysine 9 and lysine 14 acetylation, which may induce postnatal changes in gene expression. We hypothesized that it would be possible to identify hepatic genes whose epigenetic characteristics and mRNA levels are altered due to IUGR using chromatin immunoprecipitation (ChIP) coupled with random primed differential display polymerase chain reaction (PCR). One of the isolated sequences identified contained exon 2 of the dual specificity phosphatase‐5 gene (DUSP5). IUGR affected hepatic DUSP5 mRNA levels and exon 2 DNA methylation into adulthood in the rat. DUSP5 dephosphorylates Erk1 and Erk2 within the MAPK signaling cascade, which in turn affects serine 612 phosphorylation of insulin receptor substrate − 1 (p612 IRS‐1). In adult rat liver, IUGR increased Erk1/Erk2 phosphorylation and p612 IRS‐1 phosphorylation. Increased serine phosphorylation of hepatic IRS‐1 may contribute to the insulin resistance that characterizes these animals. We conclude that intrauterine growth retardation induced by uteroplacental insufficiency 1) affects the hepatic epigenetic characteristics and mRNA of the DUSP‐5 and 2) increases hepatic insulin receptor substrate‐1 phosphorylation at serine 612 in adult rats.—Fu, Q., McKnight, R. A., Yu, X., Callaway, C. W., Lane, R. H. Growth retardation alters the epigenetic characteristics of hepatic dual specificity phosphatase 5. FASEB J. 20, E1441–E1450 (2006)

Fetal growth restriction alters transcription factor binding and epigenetic mechanisms of renal 11β-hydroxysteroid dehydrogenase type 2 in a sex-specific manner

Intrauterine growth restriction (IUGR) increases the risk of serious adult morbidities such as hypertension. In an IUGR rat model of hypertension, we reported a persistent decrease in kidney 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) mRNA and protein levels from birth through postnatal (P) day 21. This enzyme deficiency can lead to hypertension by limiting renal glucocorticoid deactivation. In the present study, we hypothesized that IUGR affects renal 11beta-HSD2 epigenetic determinants of chromatin structure and alters key transcription factor binding to the 11beta-HSD2 promoter in association with persistent downregulation of its mRNA expression. To test this hypothesis, we performed bilateral uterine artery ligation on embryonic day 19.5 pregnant rats and harvested kidneys at day 0 (P0) and P21. Key transcription factors that can affect 11beta-HSD2 expression include transcriptional enhancers specificity protein 1 (SP1) and NF-kappaB p65 and transcriptional repressors early growth response factor (Egr-1) and NF-kappaB p50. Our most important findings were as follows: 1) IUGR significantly decreased SP1 and NF-kappaB (p65) binding to the 11beta-HSD2 promoter in males, while it increased Egr-1 binding in females and NF-kappaB (p50) binding in males; 2) IUGR increased CpG methylation status, as well as modified the pattern of methylation in several CpG sites of 11beta-HSD2 promoter at P0 also in a sex-specific manner; and 3) IUGR decreased trimethylation of H3K36 in exon 5 of 11beta-HSD2 at P0 and P21 in both genders. We conclude that IUGR is associated with altered transcriptional repressor/activator binding in connection with increased methylation in the 11beta-HSD2 promoter region in a sex-specific manner, possibly leading to decreased transcriptional activity. Furthermore, IUGR decreased trimethylation of H3K36 of the 11beta-HSD2 gene in both genders, which is associated with decreased transcriptional elongation. We speculate that alterations in transcription factor binding and chromatin structure play a role in in utero reprogramming.

IUGR decreases PPARγ and SETD8 Expression in neonatal rat lung and these effects are ameliorated by maternal DHA supplementation

Intrauterine growth restriction (IUGR) is associated with altered lung development in human and rat. The transcription factor PPARγ, is thought to contribute to lung development. PPARγ is activated by docosahexanoic acid (DHA). One contribution of PPARγ to lung development may be its direct regulation of chromatin modifying enzymes, such as Setd8. In this study, we hypothesized that IUGR would result in a gender-specific reduction in PPARγ, Setd8 and associated H4K20Me levels in the neonatal rat lung. Because DHA activates PPARγ, we also hypothesized that maternal DHA supplementation would normalize PPARγ, Setd8, and H4K20Me levels in the IUGR rat lung. We found that IUGR decreased PPARγ levels, with an associated decrease in Setd8 levels in both male and female rat lungs. Levels of the Setd8-dependent histone modification, H4K20Me, were reduced on the PPARγ gene in both males and females while whole lung H4K20Me was only reduced in male lung. Maternal DHA supplementation ameliorated these effects in offspring. We conclude that IUGR decreases lung PPARγ, Setd8 and PPARγ H4K20Me independent of gender, while decreasing whole lung H4K20Me in males only. These outcomes are offset by maternal DHA. We speculate that maintenance of the epigenetic milieu may be one role of PPARγ in the lung and suggests a novel benefit of maternal DHA supplementation in IUGR.

Uteroplacental Insufficiency Increases Visceral Adiposity and Visceral Adipose PPARγ2 Expression in Male Rat Offspring Prior to the Onset of Obesity

Uteroplacental insufficiency (UPI) induced intrauterine growth restriction (IUGR) predisposes individuals to adult onset metabolic morbidities, including insulin resistance and cardiovascular disease. An underlying component of the development of these morbidities is adipose dysfunction; specifically a disproportionately abundant visceral adipose tissue. We hypothesize that IUGR will increase rats visceral adiposity and visceral expression of PPARgamma, a key regulator of adipogenesis. To test this hypothesis we employed a well described UPI induced IUGR rat model. Subcutaneous and visceral adipose levels were measured in adolescent control and IUGR rats using MRI. Expression of PPARgamma mRNA and protein, as well as PPARgamma target genes, was measured in neonatal, adolescent and adult rats. UPI induced IUGR increases the relative amount of visceral adipose tissue in male, but not female, adolescent rats in conjunction with an increase in PPARgamma2mRNA and protein in male visceral adipose. Importantly, these effects are seen prior to the onset of overt obesity. We conclude that increased PPARgamma2 expression in VAT of IUGR males is associated with increased visceral adiposity. We speculate that the increase in visceral adiposity may contribute to the metabolic morbidities experienced by this population.

Glucose transporter GLUT8 translocation in neurons is not insulin responsive

We examined the subcellular distribution of a novel glucose transporter isoform (GLUT8) in murine N2A neuroblastoma cells. Exogenous expression of GLUT8‐green fluorescent protein (GFP) DNA constructs mimicked the endogenous GLUT8 localization to intracellular vesicles and minimally to the Giantin‐positive Golgi. This distribution was unlike the distributions of endogenous GLUT1 and GLUT3 (predominant neuronal isoform), which were limited predominantly to the plasma membrane and minimal in the cytoplasm. Although GLUT4‐GFP (insulin responsive isoform) was expressed transiently, no endogenous GLUT4 was detected in N2A cells. By employing stable transfectants that expressed GLUT8‐GFP, the effect of insulin and insulin‐like growth factor‐I, potassium chloride (depolarized state), and 3% oxygen on translocation of GLUT8 to the plasma membrane of N2A cells was examined immunohistochemically and by subfractionation, followed by Western blot analysis. None of these agents translocated GLUT8 to the plasma membrane. However, when the internalization dileucine motif (L12,13) of GLUT8 was mutated to a dialanine motif (A12,13), GLUT8 colocalized with GLUT3 in the plasma membrane. We conclude that GLUT8 translocation to the N2A cellular plasma membrane is not observed secondary to the various stimuli investigated. Mutation of the N‐terminal dileucine motif led to constitutive GLUT8 localization in the plasma membrane. The endogenous stimulus required for translocating neuronal GLUT8 is unknown. This stimulus, which is necessary for uncoupling the “cytoplasmic vesicular anchor” of GLUT8, would be crucial for its glucose‐transporting function.

Nasal Ventilation Alters Mesenchymal Cell Turnover and Improves Alveolarization in Preterm Lambs

RATIONALE Bronchopulmonary dysplasia (BPD) is a frequent cause of morbidity in preterm infants that is characterized by prolonged need for ventilatory support in an intensive care environment. BPD is characterized histopathologically by persistently thick, cellular distal airspace walls. In normally developing lungs, by comparison, remodeling of the immature parenchymal architecture is characterized by thinning of the future alveolar walls, a process predicated on cell loss through apoptosis. OBJECTIVES We hypothesized that minimizing lung injury, using high-frequency nasal ventilation to provide positive distending pressure with minimal assisted tidal volume displacement, would increase apoptosis and decrease proliferation among mesenchymal cells in the distal airspace walls compared with a conventional mode of support (intermittent mandatory ventilation). METHODS Accordingly, we compared two groups of preterm lambs: one group managed by high-frequency nasal ventilation and a second group managed by intermittent mandatory ventilation. Each group was maintained for 3 days. MEASUREMENTS AND MAIN RESULTS Oxygenation and ventilation targets were sustained with lower airway pressures and less supplemental oxygen in the high-frequency nasal ventilation group, in which alveolarization progressed. Thinning of the distal airspace walls was accompanied by more apoptosis, and less proliferation, among mesenchymal cells of the high-frequency nasal ventilation group, based on morphometric, protein abundance, and mRNA expression indices of apoptosis and proliferation. CONCLUSIONS Our study shows that high-frequency nasal ventilation preserves the balance between mesenchymal cell apoptosis and proliferation in the distal airspace walls, such that alveolarization progresses.