Christopher W. Callaway

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)

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.

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.

Uteroplacental insufficiency affects kidney VEGF expression in a model of IUGR with compensatory glomerular hypertrophy and hypertension

Low nephron endowment secondary to intrauterine growth restriction (IUGR) results in compensatory hypertrophy of the remaining glomeruli, which in turn is associated with hypertension. However, gender differences exist in the response of the kidney to injury, and IUGR female offspring seems protected from an unfavorable outcome. We previously reported differences in gender-specific gene expression in the IUGR kidney as well as increased circulating corticosterone levels following uteroplacental insufficiency (UPI). Vascular endothelial growth factor (VEGF), which is critical for renal development, is an important candidate in the IUGR kidney since its expression can be regulated by sex-steroids and glucocorticoids. We hypothesize that IUGR leads to altered kidney VEGF expression in a gender-specific manner. Following uterine ligation in the pregnant rat, UPI decreases renal VEGF levels in male and female IUGR animals at birth and through postnatal day 21. However, by day 120 of life, IUGR females have increased kidney VEGF expression, not present in the IUGR males. In addition, IUGR males exhibit increased serum testosterone levels as well as proteinuria. These findings are intriguing in light of the difference in glomerular hypertrophy observed: IUGR males show increased glomerular area when compared to IUGR females. In this model characterized by decreased nephron number and adult onset hypertension, UPI decreases renal VEGF expression during nephrogenesis. Our most intriguing finding is the increased renal VEGF levels in adult IUGR females, associated with a more benign phenotype. We suggest that the mechanisms underlying renal disease in response to IUGR are most likely regulated in a gender specific manner.

IUGR differentially alters MeCP2 expression and H3K9Me3 of the PPARγ gene in male and female rat lungs during alveolarization

Intrauterine growth restriction (IUGR) increases the risk of postnatal lung disease, with males more affected. In rat lungs, IUGR impairs alveolarization in conjunction with altered expression of peroxisome proliferator-activated receptor gamma (PPARγ). In non-lung cells, PPARγ transcription is regulated in part by the epigenetic modifying enzyme, and the methyl CpG binding protein 2 (MeCP2). However, it is unknown if IUGR affects MeCP2 expression or its interaction with PPARγ in the rat lung during alveolarization. In this study, we hypothesized that the rat lung would be characterized by the presence of MeCP2 short and long mRNA transcripts, MeCP2 protein isoforms, and the MeCP2 regulatory micro RNA, miR132. We further hypothesized that IUGR would, in a gender-specific manner, alter the levels of MeCP2 components in association with changes in PPARγ mRNA, MeCP2 occupancy at the PPARγ promoters, and PPARγ histone 3 lysine 9 trimethylation (H3K9Me3). To test these hypotheses, we used a well-characterized rat model of uteroplacental insufficiency-induced IUGR. We demonstrated the presence of MeCP2 mRNA, protein, and miR132 in the rat lung throughout alveolarization. We also demonstrated that IUGR alters MeCP2 expression and its interaction with PPARγ in a gender-divergent manner. We conclude that IUGR induces gender-specific alterations in the epigenetic milieu in the rat lung. We speculate that in the IUGR rat lung, this altered epigenetic milieu may predispose to gender-specific alterations in alveolarization.

IUGR decreases elastin mRNA expression in the developing rat lung and alters elastin content and lung compliance in the mature rat lung

Complications of intrauterine growth restriction (IUGR) include increased pulmonary morbidities and impaired alveolar development. Normal alveolar development depends upon elastin expression and processing, as well as the formation and deposition of elastic fibers. This is true of the human and rat. In this study, we hypothesized that uteroplacental insufficiency (UPI)-induced IUGR decreases mRNA levels of elastin and genes required for elastin fiber synthesis and assembly, at birth (prealveolarization) and postnatal day 7 (midalveolarization) in the rat. We further hypothesized that this would be accompanied by reduced elastic fiber deposition and increased static compliance at postnatal day 21 (mature lung). We used a well characterized rat model of IUGR to test these hypotheses. IUGR decreases mRNA transcript levels of genes essential for elastic fiber formation, including elastin, at birth and day 7. In the day 21 lung, IUGR decreases elastic fiber deposition and increases static lung compliance. We conclude that IUGR decreases mRNA transcript levels of elastic fiber synthesis genes, before and during alveolarization leading to a reduced elastic fiber density and increased static lung compliance in the mature lung. We speculate that the mechanism by which IUGR predisposes to pulmonary disease may be via decreased lung elastic fiber deposition.