The NADPH oxidase (NOX) family of enzymes, which catalyze the reduction of O2 to form reactive oxygen species (ROS), have increased in number during eukaryotic evolution1,2. Seven isoforms of the NOX gene family have been identified in mammals; however, specific roles of NOX enzymes in mammalian physiology and pathophysiology have not been fully elucidated3,4. The best established physiological role of NOX enzymes is in host defense against pathogen invasion in diverse species, including plants5,6. The prototypical member of this family, NOX2 (gp91phox), is expressed in phagocytic cells and mediates microbicidal activities7,8. Here, we report a role for the NOX4 isoform in tissue repair functions of myofibroblasts and fibrogenesis. Transforming growth factor-β1 (TGF-β1) induces NOX4 expression in lung mesenchymal cells by a SMAD3-dependent mechanism. NOX4-dependent generation of hydrogen peroxide (H2O2) is required for TGF-β1-induced myofibroblast differentiation, extracellular matrix (ECM) production, and contractility. NOX4 is upregulated in lungs of mice subjected to non-infectious injury and in human idiopathic pulmonary fibrosis (IPF). Genetic or pharmacologic targeting of NOX4 abrogates fibrogenesis in two different murine models of lung injury. These studies support a novel function for NOX4 in tissue fibrogenesis and provide proof-of-concept for therapeutic targeting of NOX4 in recalcitrant fibrotic disorders.

Previous studies have established that a subset of head and neck tumors contains human papillomavirus (HPV) sequences and that HPV-driven head and neck cancers display distinct biological and clinical features. HPV is known to drive cancer by the actions of the E6 and E7 oncoproteins, but the molecular architecture of HPV infection and its interaction with the host genome in head and neck cancers have not been comprehensively described. We profiled a cohort of 279 head and neck cancers with next generation RNA and DNA sequencing and show that 35 (12.5%) tumors displayed evidence of high-risk HPV types 16, 33, or 35. Twentyfive cases had integration of the viral genome into one or more locations in the human genome with statistical enrichment for genic regions. Integrations had a marked impact on the human genome and were associated with alterations in DNA copy number, mRNA transcript abundance and splicing, and both inter-and intrachromosomal rearrangements. Many of these events involved genes with documented roles in cancer. Cancers with integrated vs. nonintegrated HPV displayed different patterns of DNA methylation and both human and viral gene expressions. Together, these data provide insight into the mechanisms by which HPV interacts with the human genome beyond expression of viral oncoproteins and suggest that specific integration events are an integral component of viral oncogenesis.cancer | head and neck | papilloma virus | genome rearrangement | integration sites H ead and neck cancer (HNC) is a heterogeneous group of tumors characterized by a common anatomic origin, and most such tumors develop from within the mucosa and are classified as head and neck squamous cell carcinomas (HNSCCs) (1). HNSCC, the sixth most common cancer diagnosed worldwide and the eighth most common cause of cancer death (2), is frequently associated with human papillomavirus (HPV) infection (3, 4). Depending on the anatomic site of the tumor, HPV prevalence is estimated at 23-36% (5). HPV-positive HNSCCs form a distinct subset of HNCs that differs from HPV-negative HNSCCs in tumor biology and clinical characteristics, including superior clinical outcomes (6-9).The molecular pathogenesis of HPV-driven HNSCC also seems distinct from HPV-negative tumors, with previous studies showing a divergent spectrum of alterations in gene expression, mutations, amplifications, and deletions as well as distinct epigenome alterations (10-15). HPV is known to drive tumorigenesis through the actions of its major oncoproteins E6 and E7, which target numerous cellular pathways, including inactivation of p53 and the retinoblastoma (Rb) protein (16-18). Together with E5, they also play an important role in immune evasion, being involved in both innate and adaptive immunity (19,20).Initially after infection, HPV is identified in circular extrachromosomal particles or episomes. A critical step in progression to cancer is the integration of viral DNA into the host cell Significance A significant proportion of head and neck cancer is driven by human papil...

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Animal studies have linked perinatal bisphenol A (BPA) exposure to altered DNA methylation, but little attention is given to analyzing multiple physiologically relevant doses. Utilizing the viable yellow agouti (Avy) mouse, we examine the effects of developmental exposure through maternal diet to 50 ng BPA/kg (n = 14 litters), 50 μg BPA/kg (n = 9 litters), or 50 mg BPA/kg (n = 13 litters) on global and candidate gene methylation at postnatal day 22. Global methylation analysis reveals hypermethylation in tail tissue of a/a and Avy/a offspring across all dose groups compared with controls (n = 11 litters; P < 0.02). Analysis of coat color phenotype replicates previous work showing that the distribution of 50 mg BPA/kg Avy/a offspring shifts toward yellow (P = 0.006) by decreasing DNA methylation in the retrotransposon upstream of the Agouti gene (P = 0.03). Maternal exposure to 50 μg or 50 ng BPA/kg, however, results in altered coat color distributions in comparison with control (P = 0.04 and 0.02), but no DNA methylation effects at the Agouti gene are noted. DNA methylation at the CDK5 activator-binding protein (CabpIAP) metastable epiallele shows hypermethylation in the 50 μg BPA/kg offspring, compared with controls (P = 0.02). Comparison of exposed mouse liver BPA levels to human fetal liver BPA levels indicates that the three experimental exposures are physiologically relevant. Thus, perinatal BPA exposure affects offspring phenotype and epigenetic regulation across multiple doses, indicating the need to evaluate dose effects in human clinical and population studies.

Piwi-interacting RNAs (piRNAs) are small non-coding RNAs that associate with PIWI proteins for transposon silencing via DNA methylation and are highly expressed and extensively studied in the germline. Mature germline piRNAs typically consist of 24-32 nucleotides, with a strong preference for a 5ʹ uridine signature, an adenosine signature at position 10, and a 2ʹ-O-methylation signature at the 3ʹ end. piRNA presence in somatic tissues, however, is not well characterized and requires further systematic evaluation. In the current study, we identified piRNAs and associated machinery from mouse somatic tissues representing the three germ layers. piRNA specificity was improved by combining small RNA size selection, sodium periodate treatment enrichment for piRNA over other small RNA, and small RNA next-generation sequencing. We identify PIWIL1, PIWIL2, and PIWIL4 expression in brain, liver, kidney, and heart. Of note, somatic piRNAs are shorter in length and tissuespecific, with increased occurrence of unique piRNAs in hippocampus and liver, compared to the germline. Hippocampus contains 5,494 piRNA-like peaks, the highest expression among all tested somatic tissues, followed by cortex (1,963), kidney (580), and liver (406). The study identifies 26 piRNA sequence species and 40 piRNA locations exclusive to all examined somatic tissues. Although piRNA expression has long been considered exclusive to the germline, our results support that piRNAs are expressed in several somatic tissues that may influence piRNA functions in the soma. Once confirmed, the PIWI/piRNA system may serve as a potential tool for future research in epigenome editing to improve human health by manipulating DNA methylation.

The ability of environmental factors to shape health and disease involves epigenetic mechanisms that mediate gene-environment interactions. Metastable epiallele genes are variably expressed in genetically identical individuals due to epigenetic modifications established during early development. DNA methylation within metastable epialleles is stochastic due to probabilistic reprogramming of epigenetic marks during embryogenesis. Maternal nutrition and environment have been shown to affect metastable epiallele methylation patterns and subsequent adult phenotype. Little is known, however, about the role of histone modifications in influencing metastable epiallele expression and phenotypic variation. Utilizing chromatin immunoprecipitation followed by qPCR, we observe variable histone patterns in the 5′ long terminal repeat (LTR) of the murine viable yellow agouti (Avy) metastable epiallele. This region contains 6 CpG sites, which are variably methylated in isogenic Avy/a offspring. Yellow mice, which are hypomethylated at the Avy LTR and exhibit constitutive ectopic expression of Agouti (a), also display enrichment of H3 and H4 di-acetylation (p = 0.08 and 0.09, respectively). Pseudoagouti mice, in which Avy hypermethylation is thought to silence ectopic expression, exhibit enrichment of H4K20 tri-methylation (p = 0.01). No differences are observed for H3K4 tri-methylation (p = 0.7), a modification often enriched in the promoter of active genes. These results show for the first time the presence of variable histone modifications at a metastable epiallele, indicating that DNA methylation acts in concert with histone modifications to affect inter-individual variation of metastable epiallele expression. Therefore, the potential for environmental factors to influence histone modifications, in addition to DNA methylation, should be addressed in environmental epigenomic studies.

BackgroundBirth by cesarean delivery (CD) as opposed to vaginal delivery (VD) is associated with altered health outcomes later in life, including respiratory disorders, allergies and risk of developing type I diabetes. Epigenetic gene regulation is a proposed mechanism by which early life exposures affect later health outcomes. Previously, type of delivery has been found to be associated with differences in global methylation levels, but the sample sizes have been small. We measured global methylation in a large birth cohort to identify whether type of delivery is associated with epigenetic changes.MethodsDNA was isolated from cord blood collected from the University of Michigan Women’s & Children Hospital and bisulfite-converted. The Luminometric Methylation Assay (LUMA) and LINE-1 methylation assay were run on all samples in duplicate.ResultsGlobal methylation data at CCGG sites throughout the genome, as measured by LUMA, were available from 392 births (52% male; 65% CD), and quantitative methylation levels at LINE-1 repetitive elements were available for 407 births (52% male; 64% CD). LUMA and LINE-1 methylation measurements were negatively correlated in this population (Spearman’s r = −0.13, p =0.01). LUMA measurements were significantly lower for total CD and planned CD, but not emergency CD when compared to VD (median VD = 74.8, median total CD = 74.4, p = 0.03; median planned CD = 74.2, p = 0.02; median emergency CD = 75.3, p = 0.39). However, this association did not persist when adjusting for maternal age, maternal smoking and infant gender. Furthermore, total CD deliveries, planned CD and emergency CD deliveries were not associated with LINE-1 measurements as compared to VD (median VD = 82.2, median total CD = 81.9, p = 0.19; median planned CD = 81.9, p = 0.19; median emergency CD = 82.1, p = 0.52). This lack of association held when adjusting for maternal age, maternal smoking and infant gender in a multivariable model.ConclusionsType of delivery was not associated with global methylation in our population, even after adjustment for maternal age, maternal smoking, and infant gender. While type of birth may be associated with later health outcomes, our data suggest that it does not do so through changes in global genomic methylation.

BackgroundEnvironmental factors during perinatal development may influence developmental plasticity and disease susceptibility via alterations to the epigenome. Developmental exposure to the endocrine active compound, bisphenol A (BPA), has previously been associated with altered methylation at candidate gene loci. Here, we undertake the first genome-wide characterization of DNA methylation profiles in the liver of murine offspring exposed perinatally to multiple doses of BPA through the maternal diet.ResultsUsing a tiered focusing approach, our strategy proceeds from unbiased broad DNA methylation analysis using methylation-based next generation sequencing technology to in-depth quantitative site-specific CpG methylation determination using the Sequenom EpiTYPER MassARRAY platform to profile liver DNA methylation patterns in offspring maternally exposed to BPA during gestation and lactation to doses ranging from 0 BPA/kg (Ctr), 50 μg BPA/kg (UG), or 50 mg BPA/kg (MG) diet (N = 4 per group). Genome-wide analyses indicate non-monotonic effects of DNA methylation patterns following perinatal exposure to BPA, corroborating previous studies using multiple doses of BPA with non-monotonic outcomes. We observed enrichment of regions of altered methylation (RAMs) within CpG island (CGI) shores, but little evidence of RAM enrichment in CGIs. An analysis of promoter regions identified several hundred novel BPA-associated methylation events, and methylation alterations in the Myh7b and Slc22a12 gene promoters were validated. Using the Comparative Toxicogenomics Database, a number of candidate genes that have previously been associated with BPA-related gene expression changes were identified, and gene set enrichment testing identified epigenetically dysregulated pathways involved in metabolism and stimulus response.ConclusionsIn this study, non-monotonic dose dependent alterations in DNA methylation among BPA-exposed mouse liver samples and their relevant pathways were identified and validated. The comprehensive methylome map presented here provides candidate loci underlying the role of early BPA exposure and later in life health and disease status.