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Prenatal smoke exposure (PSE) is associated with reduced birth weight, impaired fetal development, and increased risk for diseases later in life. Changes in DNA methylation may be involved, as multiple large-scale epigenome-wide association studies showed that PSE is robustly associated with DNA methylation changes in blood among offspring in early life. Insulin-like growth factor-1 (IGF1) is important in growth, differentiation, and repair processes after injury. However, no studies investigated the organ-specific persistence of PSE-induced methylation change of Igf1 into adulthood. Based on our previous studies on the PSE effect on Igf1 promoter methylation in fetal and neonatal mouse offspring, we now have extended our studies to adulthood. Our data show that basal Igf1 promoter methylation generally increased in the lung but decreased in the liver (except for 2 persistent CpG sites in both organs) across three different developmental stages. PSE changed Igf1 promoter methylation in all three developmental stages, which was organ and sex specific. The PSE effect was less pronounced in adult offspring compared with the fetal and neonatal stages. In addition, the PSE effect in the adult stage was more pronounced in the lung compared with the liver. For most CpG sites, an inverse correlation was found for promoter methylation and mRNA expression when the data of all three stages were combined. This was more prominent in the liver. Our findings provide additional evidence for sex- and organ-dependent prenatal programming, which supports the developmental origins of health and disease (DOHaD) hypothesis.

Prenatal smoke exposure (PreSE) is a risk factor for nicotine dependence, which is further enhanced by postnatal smoke exposure (PostSE). One susceptibility gene to nicotine dependence is Cytochrome P450 (CYP) 2A6, an enzyme responsible for the conversion of nicotine to cotinine in the liver. Higher CYP2A6 activity is associated with nicotine dependence and could be regulated through DNA methylation. In this study we investigated whether PostSE further impaired PreSE-induced effects on nicotine metabolism, along with Cyp2a5, orthologue of CYP2A6, mRNA expression and DNA methylation. Using a mouse model where prenatally smoke-exposed adult offspring were exposed to cigarette smoke for 3 months, enzyme activity, mRNA levels, and promoter methylation of hepatic Cyp2a5 were evaluated. We found that in male offspring, PostSE increased PreSE-induced cotinine levels and Cyp2a5 mRNA expression. In addition, both PostSE and PreSE changed Cyp2a5 DNA methylation in male groups. PreSE however decreased cotinine levels whereas it had no effect on Cyp2a5 mRNA expression or methylation. These adverse outcomes of PreSE and PostSE were most prominent in males. When considered in the context of the human health aspects, the combined effect of prenatal and adolescent smoke exposure could lead to an accelerated risk for nicotine dependence later in life.

Prenatal smoke exposure (PreSE) is a risk factor for nicotine dependence, which is further enhanced by postnatal smoke exposure (PostSE). One susceptibility gene to nicotine dependence is Cytochrome P450 (CYP) 2A6, an enzyme responsible for the conversion of nicotine to cotinine in the liver. Higher CYP2A6 activity is associated with nicotine dependence and could be regulated through DNA methylation. In this study we investigated whether PostSE further impaired PreSE-induced effects on nicotine metabolism, along with Cyp2a5, orthologue of CYP2A6, mRNA expression and DNA methylation. Using a mouse model where prenatally smoke-exposed adult offspring were exposed to cigarette smoke for 3 months, enzyme activity, mRNA levels, and promoter methylation of hepatic Cyp2a5 were evaluated. We found that in male offspring, PostSE increased PreSE-induced cotinine levels and Cyp2a5 mRNA expression. In addition, both PostSE and PreSE changed Cyp2a5 DNA methylation in male groups. PreSE however decreased cotinine levels whereas it had no effect on Cyp2a5 mRNA expression or methylation. These adverse outcomes of PreSE and PostSE were most prominent in males. When considered in the context of the human health aspects, the combined effect of prenatal and adolescent smoke exposure could lead to an accelerated risk for nicotine dependence later in life.

Identification of target genes that mediate required functions downstream of transcription factors is hampered by the large number of genes whose expression changes when the factor is removed from a specific tissue and the numerous binding sites for the factor in the genome. Retinoic acid (RA) regulates transcription via RA receptors bound to RA response elements (RAREs) of which there are thousands in vertebrate genomes. Here, we combined chromatin immunoprecipitation sequencing (ChIP-seq) for epigenetic marks and RNA-seq on trunk tissue from wild-type and Aldh1a2-/- embryos lacking RA synthesis that exhibit body axis and forelimb defects. We identified a relatively small number of genes with altered expression when RA is missing that also have nearby RA-regulated deposition of histone H3 K27 acetylation (H3K27ac) (gene activation mark) or histone H3 K27 trimethylation (H3K27me3) (gene repression mark) associated with conserved RAREs, suggesting these genes function downstream of RA. RA-regulated epigenetic marks were identified near RA target genes already known to be required for body axis and limb formation, thus validating our approach; plus, many other candidate RA target genes were found. Nuclear receptor 2f1 (Nr2f1) and nuclear receptor 2f2 (Nr2f2) in addition to Meis homeobox 1 (Meis1) and Meis homeobox 2 (Meis2) gene family members were identified by our approach, and double knockouts of each family demonstrated previously unknown requirements for body axis and/or limb formation. A similar epigenetic approach can be used to determine the target genes for any transcriptional regulator for which a knockout is available., Identification of the target genes required to implement the downstream functions of transcription factors is challenging. This study uses ChIP-seq of retinoic acid-regulated epigenetic marks combined with RNA-seq to identify a small number of important retinoic acid target genes; disruption of several genes reveals required functions in body axis and/or limb development.

Prenatal smoke exposure (PSE) is associated with reduced birth weight, impaired fetal development, and increased risk for diseases later in life. Changes in DNA methylation may be involved, as multiple large-scale epigenome-wide association studies showed that PSE is robustly associated with DNA methylation changes in blood among offspring in early life. Insulin-like growth factor-1 (IGF1) is important in growth, differentiation, and repair processes after injury. However, no studies investigated the organ-specific persistence of PSE-induced methylation change of

Identification of target genes that mediate required functions downstream of transcription factors is hampered by the large number of genes whose expression changes when the factor is removed from a specific tissue and the numerous binding sites for the factor in the genome. Retinoic acid (RA) regulates transcription via RA receptors bound to RA response elements (RAREs) of which there are thousands in vertebrate genomes. Here, we combined ChIP-seq for epigenetic marks and RNA-seq on trunk tissue from wild-type andAldh1a2-/-embryos lacking RA synthesis that exhibit body axis and forelimb defects. We identified a relatively small number of genes with altered expression when RA is missing that also have nearby RA-regulated deposition of H3K27ac (gene activation mark) or H3K27me3 (gene repression mark) associated with conserved RAREs, suggesting they have important downstream functions. RA-regulated epigenetic marks were identified near RA target genes already known to be required for body axis and limb formation, thus validating our approach, plus many other candidate RA target genes were found.Nr2f1,Nr2f2,Meis1, andMeis2gene family members were identified by our approach, and double knockouts of each family demonstrated previously unknown requirements for body axis and/or limb formation. These findings demonstrate that our method for identifying RA-regulated epigenetic marks can be used to discover genes important for development.

Background: Prenatal smoke exposure (PSE) is a risk factor for COPD. One susceptibility gene for COPD is Cytochrome P450 (CYP) 2A6, an enzyme responsible for the conversion of nicotine to cotinine and nicotine clearance in the liver. Higher CYP2A6 activity is associated nicotine dependence. However, no research has addressed the PSE effects on CYP2A6, or CYP2A5 in the mouse.Aim: To investigate the PSE effect on Cyp2a5 gene expression and promoter methylation in fetal and neonatal offspring.Methods: C57BL/6 mice were exposed to fresh air or smoke from 3 weeks before conception until delivery. From liver and lung tissue, DNA and RNA were isolated for gene expression and promoter methylation analysis of Cyp2a5 in fetuses (E17.5) and neonates (D3).Results: Cyp2a5 mRNA expression was increased in male neonatal liver from PSE offspring. In contrast, in the lung, Cyp2a5 expression was reduced in both fetal and neonatal male PSE offspring. Promoter methylation was sex-dependently induced in both fetal and neonatal liver from PSE offspring when compared to their corresponding control groups. In the fetal lung, methylation was downregulated by PSE. Promoter methylation of several CpG sites (inversely) correlated with mRNA expression in liver and lung in both fetal and neonatal offspring.Conclusions: This study presents a PSE effect on Cyp2a5 expression, which is opposite in liver and lung and associated with promoter methylation. Induced Cyp2a5 gene expression in the neonatal liver may indicate a higher nicotine metabolism which, when translated to the human situation, could lead to an increased risk for COPD and higher nicotine dependence later in life.

Background: Prenatal smoke exposure (PSE) causes aberrant fetal development and is a risk factor for the offspring9s future health. DNA methylation changes have been shown in children from smoking mothers, but evidence for sex-dependent smoke-induced effects is limited. Insulin like growth factor (IGF) signaling plays a critical role in prenatal tissue growth and maturation. We hypothesized that PSE causes fetal programming of the IGF network. Aim: To assess the effects of PSE on promoter methylation patterns as well as mRNA levels of Igf1r and Igf1 . Methods: Lungs and livers of prenatally smoke exposed fetuses (E17.5) and three day old (D3) offspring were collected and subjected to DNA methylation analysis as well as qPCR. These analyses were followed by correlation analysis to identify possible links between PSE-induced differential methylation status and mRNA levels. Results: Our results suggest that CpG-site specific aberrant DNA methylation patterns of Igf1r and Igf1 vary per sex, organ and time point. Similarly, smoking during pregnancy reduces gene expression of Igf1r and Igf1 in lungs and liver, dependent on sex and developmental stage. Conclusions: We show that PSE sex-dependently alters promoter methylation of Igf1r and Igf1 and also deregulates their gene expression. These findings provide evidence that PSE can cause tissue specific rather than systemic alterations on fetal programming, exemplified by methylation and expression of IGF network representatives. A sex-specific link between PSE, epigenetic modifications and gene expression may be used to identify future targets for therapeutic intervention.

The impact of prenatal smoke exposure (PSE) on DNA methylation has been demonstrated in blood samples from children of smoking mothers, but evidence for sex-dependent smoke-induced effects is limited. As the identified differentially methylated genes can be associated with developmental processes, and insulin-like growth factors (IGFs) play a critical role in prenatal tissue growth, we hypothesized that PSE induces fetal programming of Igf1r and Igf1. Using a mouse model of smoking during pregnancy, we show that PSE alters promoter methylation of Igf1r and Igf1 and deregulates their gene expression in lung and liver of fetal (E17.5) and neonatal (D3) mouse offspring. By further comparing female versus male, lung versus liver, or fetal versus neonatal time point, our results demonstrate that CpG site-specific aberrant methylation patterns sex-dependently vary per organ and time point. Moreover, PSE reduces gene expression of Igf1r and Igf1, dependent on organ, sex, and offspring's age. Our results indicate that PSE may be a source of organ-specific rather than general systemic fetal programming. This is exemplified here by gene promoter methylation and mRNA levels of Igf1r and Igf1, together with a sex- and organ-specific naturally established correlation of both parameters that is affected by prenatal smoke exposure. Moreover, the comparison of fetuses with neonates suggests a CpG site-dependent reversibility/persistence of PSE-induced differential methylation patterns.

Background: Airway mucus hyperproduction contributes to the morbidity and mortality in asthma and COPD. Reducing mucus production is crucial for improving patients9 quality of life. The transcription factor SPDEF plays a critical role in the regulation of mucus production, and therefore could be a potential target. Here, we present an innovative approach to repress SPDEF expression. Objective: To reduce mucus production by targeted silencing of SPDEF expression with a zinc finger (ZF)-based approach. Methods: Four ZFs were designed to target the SPDEF promoter. They were fused to the Kruppel Associated Box (SKD) repressor domain and ZFs-SKD were lentivirally delivered into the mucoglycoprotein-producing A549 airway epithelial cells. Gene expressions of SPDEF, as well as other mucus-related genes MUC5AC and AGR2 were detected by qRT-PCR. Protein level of SPDEF was investigated by western blot and MUC5AC by immunochemistry staining. Results: Three ZFs-SKD were able to effectively suppress SPDEF expression (up to 96% repression efficiencies) at both mRNA and protein levels. Reduced SPDEF expression resulted in decreased mRNA expression of MUC5AC and AGR2 (both up to 90% repression efficiencies), and also decreased protein expression of MUC5AC. Conclusions: Engineered ZFs-SKD can efficiently reduce SPDEF and consequently, MUC5AC and AGR2 expression. This opens up an innovative avenue to inhibit mucus production by long-lasting epigenetic reprogramming. We currently investigate the effects of ZFs linked to epigenetic editing domains (DNMT3a, G9a) to induce DNA methylation or histone modifications (H3K9me2), in A549 cells and in primary bronchial epithelial cells.

Evidence is now emerging that early life environment can have lifelong effects on metabolic, cardiovascular, and pulmonary function in offspring, a concept also known as fetal or developmental programming. In mammals, developmental programming is thought to occur mainly via epigenetic mechanisms, which include DNA methylation, histone modifications, and expression of non-coding RNAs. The effects of developmental programming can be induced by the intrauterine environment, leading to intergenerational epigenetic effects from one generation to the next. Transgenerational epigenetic inheritance may be considered when developmental programming is transmitted across generations that were not exposed to the initial environment which triggered the change. So far, inter- and transgenerational programming has been mainly described for cardiovascular and metabolic disease risk. In this review, we discuss available evidence that epigenetic inheritance also occurs in respiratory diseases, using asthma and chronic obstructive pulmonary disease (COPD) as examples. While multiple epidemiological as well as animal studies demonstrate effects of 'toxic' intrauterine exposure on various asthma-related phenotypes in the offspring, only few studies link epigenetic marks to the observed phenotypes. As epigenetic marks may distinguish individuals most at risk of later disease at early age, it will enable early intervention strategies to reduce such risks. To achieve this goal further, well designed experimental and human studies are needed.